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User Manual - CALM - Confocal and Advanced Light Microscopy

1. Sigma Defines the Sigma filter The Sigma filter is for the filtering out of statistical noise Individual pixels of gray values deviating greatly from their surrounding area will however not be affected by the Sigma filter The Sigma filter assumes that statistical noise in an image is distributed in a Gaus sian shape This means that for a single color image with a great deal of noise the frequency of the gray values to the left and right of the mean value drop in accor dance with a Gaussian curve This can be corroborated using the histogram of a noise image Under this condition 95 5 of all pixel values are located within the double Sigma width around the mean value The algorithm replaces the central pixel of a pixel environment with a new gray value For a specified Sigma o a gray value area G 20 G 2o0 around the central gray value G is calculated Then the amount of all pixels of the surrounding area which are within this gray value area is determined All gray values outside this gray value area very likely belong to another set of object information and are therefore ignored The central pixel is replaced with the mean gray value of all pixels comprising the 20 area If the number of pixels comprising the defined surrounding area is less than or equal to the value 236 The Process menu Define Filter Number of neighbors 2 the central pixel will be replaced b
2. H 2D Object Ce Gray Value Max Density panne pe Angle l UH Gray Value Mear Density eee i Group of points Ce Gray Value Min Density ee Line CU Gray Value StdDev Density Belek a Point 1 Gray Value Variance Density O Fe U Green Density o OH Hue Density Help g at Density Density oe Selected Measurements Court Group of points Point 3 Poirit Poirit Point Length Line Value Angle The integral image intensity is the sum of all the intensities of a particle multiplied by the pixel area For a particle containing regions of highly differing density this method will produce a better estimation than Integral Intensity Gray Value Mean az long az the variation i not caused by noize or By the imaging process All measurements are divided into five different types points point groups lines angles and 2D objects Each type has defaults for various measurement parameters which are offered as defaults for a measurement A circle for example is a 2D object By default the area and perimeter are exported as a measurement result Please note that the measurement parameters for all types of measurement objects are shown in the Selected Measuremenis list Only the measurement parameters which are relevant to the measured object however are shown in the tree view of the measurement display An angle value will not be exported for a 2D object You can change the sequence of the active measurement paramete
3. cccccceesececeeeeeeeeaeeeaees 336 DEING IMAJGOS irienna E a E uubavedss 336 The Modi of the Stage Navigator cccccceccccscecseeeeeeeeceeeeseseeceeeesseeessueeneeseneass 336 Change Stage Navigator Properties ccccssceseeceececeeeceeeceeeeceeesaeesueseueenaeess 338 Acquire parts of the overview image with a higher magnification 006 340 ZOOMMIMG MOD AT AGS sili ssa suet Needs coeuevunien tosh EEE 341 Adding Stage Navigator positions cccccccccccececeeeceeeeeeeeseeeeeeeseeeseeesseeeeeeeaeeeees 342 Moving to Stage Navigator positions and editing them ccceecceeeceeeeeeeeees 342 TROUDIG SMOOUAG aieiaiee E T O ss 343 Before you begin Before you begin Before you begin Warning S Software Protection The software is protect ed by a dongle The USB dongle is delivered as a Standard It is illus trated on the left and the LPT dongle used with the parallel port on the right The software package you have chosen was created by Soft Imaging System You have thus entered into the worldwide user community of our image analysis systems Welcome The broad range of functions for digital image acquisition image processing analysis database archiving and results documentation are all at your disposal We think you ll find working with cell a tremendously satisfying experience Your image analysis software is available in a great variety of expansion versions and
4. Image 2 List Gall ie M File name aua Files of type Tage Optimizing display 1 Press Ctrl Alt T to generate a test image e The image window contains a button bar with which you can quickly alter the appearance of the images in the image window Press Ctrl Alt T to pee generate atest image It eg ea La LE will have a color image overlay which displays les fioo a al Mal current monitor resolu tion and other informa tion Press Ctrl Alt Shift T to generate a color test image The test image will auto matically be the same size as the active view port The test image will always be displayed at 100 zoom The header shows the number of the image s image buffer 2 the image name Test and the current zoom factor 100 First Steps Saving GUI configuration 2 Click the Arrange Viewports button to redefine the number and arrangement of viewports Select a 1x2 arrangement gt The image window will be divided up into two viewports The test image is in the left viewport Image buffers will be reassigned Zoom factors will be set to Auto Although somewhat reduced in size the entire test image will be shown 3 Click the Single View button to display just one image in the image window the active viewport image gt The viewport arrangement and what image buffers are shown in which viewports remain unchanged 4 Select one of the predefined options in th
5. gt 20 4 FS mal ulel alal Expres sDatabase 01 Ye ExpressDataba Folder 1 has been created dy ExpressD stabasel1 apl Field name Field value L Folder 1 File Hame ExpressDatabasel1 Fath F ExpressD atabaselll ExpressD atabasell apl Created 01 12 2003 14 34 08 Last access 01 12 2003 Modified 01 12 2003 14 34 18 Opened exclusively yes Read only no Database identifier 4ANSTSecw Converted from mdb no Database version 1 02 101 ExpressD atabasell Set 1 O0 kB Z 65 Archiving Images Define organizational fields Define organizational fields Background Information Predefined fields Predefined fields are provided by your image analysis program and are saved with each record automatically They contain the image attributes and data which the system assigns on input into the database They cannot be edited or deleted The only exception is the Record Name field which you can edit when inserting an image or editing a record as long as you have not defined otherwise in the Database Settings dialog box User defined data User defined database fields can be set up according to your requirements e g base fields User Project Comment or Instrument You can define the data type of the entry for each field You can also define whether during the insertion of images e an entry in the field is required a picklist with possible entries is to be offered the en
6. 250 The Process menu Define Processing Minimum The images in the Source and Source 2 image buffers will be compared with each other The resulting image contains the smaller of the two gray values The resulting image will thus have minimum intensity Multiplication The active image Source 1 will be multiplied with a Constant or with the Source 2 image set Overflow clipping will be performed at 65535 for 16 bit data and at 255 for 8 bit data OR Pixel values in binary form are compared bit by bit If a bit is 1 for either of the sources the result will be 1 Example 8 bit Source 1 170 10101010 Source 2 15 00001111 Result 10101111 175 Subtraction A Constant or the Source 2 image set will be subtracted from the active image Source 1 Negative values will be set to 0 XOR exclusive OR Pixel values in binary form are compared bit by bit If the bits differ the result is 1 If the bits are identical the result is O Example 8 bit Source 1 170 10101010 Source 2 15 00001111 Result 10100101 165 Define Processing About the Process Automator Use this command to create a completely automatic application for your own require ments The heart of the automator is the Define Processing dialog box in which you completely define the automatic process This dialog box is opened with the Process gt Define Processing command With this one dialog box you completely automate yo
7. Original Size F Cut Margins Cancel Background Egualze Help The image area shows the result of the automatic image alignment The size of the dialog box is automatically adjusted to the size of your monitor so that you can observe the mounted image at optimum resolution You can shift the individual images manually in order to correct possible errors that have occurred when aligning the images This feature is especially important when some images could not be aligned with the prescribed correlation Color images are displayed as gray value images In order to change the image position manually move the mouse cursor within the image When the image is selected the mouse cursor changes into a hand and a red rectangle indicates the image border Leftclick to shift the selected image with the mouse button depressed The maximum possible shift of an image is half of one image side because the arrangement of the images cannot be changed Release the left mouse button and the selected image will be inserted into the overview image The overlap area is displayed according to the method selected from the Overlap Area list Note An overlap of 100 pixels is sufficient for the automatic image alignment The overlap region of the images should be not too large Otherwise you may run into problems when positioning the images manually because the maximum possible shift is not sufficient From the Overlap Area list you select how you want to dis
8. 155 The Image menu Adjust Display Click the Linear button to delete the active mapping function Then a linear mapping function will be applied instead of a mapping function that you have defined 16 bit gray value images A 16 bit gray value image can include up to 65535 gray values On the monitor only 256 gray values can be displayed By using the functions of this dialog box you can determine which range of 256 gray values is to be reproduced on your monitor In this way you define an LUT lookup table between the 16 bit image and the monitor This LUT extends linearly between the minimum value that is the smallest gray value that is to be displayed and the maximum gray value Adjustment Automatic Manual Overflow Lett aso Ee Right 0 50 Kz 29 0000 2689 0000 Cancel Help Automatic In the Adjustment group select the Automatic option to have the image s complete gray value range displayed on your monitor The program calculates the minimum and maximum gray values in the image and displays the gray value range Min Max on your monitor For the automatic display the Overflow group is at your disposal The overflow s Left and Right values are taken into consideration when the minimum and maximum values are calculated For example a value of 3 means that the darkest in the case of the left overflow respectively the brightest pixels right overflow will not be linearly displayed They wi
9. 293 The Measure menu Grid Select the HS option to have the following color components color value saturation and intensity displayed in the matrix Select the RGB option for the color compo nents red green and blue Grid Draws a measurement grid into the overlay A dialog box is opened by this command for determining the size of the grid frames and the color of the grid lines Grid lines are drawn into the overlay Any change of settings can be seen directly in the image Size and position of grid frames can be determined interactively The image is thus divided up into equal segments useful for e g counting the particles in the individual segments Imagesize 705 00 Fisel x 518 00 Pixel Gridsize O K Horizontal f o Pinel ance Vertical f on 5 Fixel Draw Options Use starting point Interactive W Label fields W Automatic gridsize Help minimum of WE fields The Imagesize field contains the size of the active image in the calibration unit Determine the size of grid frames in the Gridsize group Enter the width and height of the grid frames in the Horizontal and Vertical fields in the unit of image calibration Values between a minimum of 10 pixels and a maximum of the image size can be selected The minimum value of 10 pixels will be calculated into the unit of image calibration Gridsize is displayed in the upper left corner of the overlay as long as the Label fields
10. Acquiring images Extended Focal Imaging Select the Append check box to avoid overwriting any existent EFI accumulative image when you start image acquisition You can use this option for situations where your EFI image still has areas that are not in focus By acquiring additional images later and then having them computed into the EFI image you can further improve your results Click on the Start button to begin image acquisition The button will then be changed to the Stop button As soon as you re satisfied with the accumulatively computed EFI image click this button EFI Focus Control Note Use this command to start up the fully automated method of EFI image computation The fully automated method means that EFI will acquire a focus series according to your settings without your having to do a thing the images will then be placed in successive image buffers a height map and the EFI image will then be computed based on these images The computation done here is just like the standard method s When using remote controlled stage and lens be careful not to damage the lens by colliding into the specimen with it After initiating this command the EFl Focus control dialog box will be opened EFI Focus Control Positians Close AoE Execute E attor po Help Z Distance 0 00 mm Invert height map Enter the total number of individual images you intend to acquire in the Z Positions gt Count field Cli
11. In the Command group list the internal function groups are listed Select the check boxes next to an internal function group to record the commands belonging to this group All commands and functions can be classified into specific groups These command groups are located in all dialog boxes for definition of user configurations e g in the Edit Button Bars dialog box Select the Record commands check box to start recording the processing steps Only the commands belonging to the selected groups will be scripted Clear the Record commands check box to stop the recording mode Click the Select all button to check all check boxes included in the Command group Click the Remove all button to clear all check boxes included in the Command group Click the Default button to get a preset standard selection Image Information Displays an overview of all image data The General tab The General tab contains general image information This tab is accessible for any image loaded The kind of information you get will depend on where you got the image from and its file format 193 The Image menu The General tab Image Information General Dimensions Image name EE File name C Archive LifeScienceD emo 4X560858_Doc Image buffer Frame 1 1 Memoru size a22 AB File size 8336 KB Created 26 04 2003 16 00 Channel F View Trigger Fa Magnification B x Resolution 1376 1032 16 Width 153 7 pm Height 115 2 um
12. Selecting the fluorochrome You only need to carry out this step once You should select those fluorochromes that you use from the list of all of the fluorochromes In this way the steps that follow will be much more clear to you and will take less time 1 Use the Acquire gt mFIP gt Define Fluorochromes command 2 Select several check boxes in the Dye list all of your microscope s contrast procedures e g lt Brightfield gt lt Phase Contrast gt lt DIC gt and the fluores cence markers that you use with your samples e g for your cellular tissue sample DAPI Bodipy FL and MitroTracker Red 3 Exit the dialog box with OK It can take some time before the dialog box will close since a number of files will need to be updated Setting fluorescence You only need to carry out this step once When you acquire multi channel images at a later date you can fall back on this setting 1 Only with the motorized microscopes of the BX51 61 type or the IX71 81 type do you register yourself with the microscope 2 Use the Acquire gt mFIP gt Define Fluorescences command 3 Click on the New button In the Fluorescence group select the fluorochrome the corresponding target structure and all of the necessary microscope settings Only with the motorized microscopes of the BX51 61 type or the IX71 81 type do you select the microscope s observation method instead Click the Apply button 115 4
13. Step by step This is how you measure distances and areas You want to make various distance and area measurements on an image 1 Load the image to be measured or acquire an image Warning i A correct image calibration is a requirement for a correct measurement 2 You can use the Alt 4 key stroke or the Measure gt Measurements Bar menu command to switch to the measurements environment gt A button bar with all the measurement functions is shown next to the image manager e The image manager alters its appearance The Measurements tab serves as the measurement display and is activated The measurement display is still empty 3 Select the measurement function you would like to use from the measurement button bar Click the Arbitrary line button to measure any distance gt The pointer moves to the image window 4 Position the pointer to the starting point of the distance to be measured and depress the left mouse button gt Two edges appear in the image s overlay which are always vertical to the distance to be measured Use the edges as positioning helpers for the distance to be measured 5 Position the pointer to the end point of the distance to be measured and depress the left mouse button gt The measured distance is shown in the image overlay gt The measured length is written to the measurement display 6 Repeat the last two steps for all distances to be measured 7 You end the distance measurement b
14. Target structure Fluorochrome Filter position optional Acquiring images Multiple Fluorescences In the same way you can define other fluorescences for all of the fluorescence markers and for all of the contrast procedures Example settings You will need to adjust these settings to fit your samples MitoTracker Bodipy FL Overview2 Red DAPI Mitochondria Cytosceleton lt Brightfield gt lt DIC gt C A E Filter block optional U MNU U MWG U MWB BF Transmitted DIC Trans mitted Objective position 5 optional Objective optional 5 E e E a Exit the dialog box with OK Setting the microscope 1 2 Lay the object under your microscope Bring the appropriate objective into po sition In the brightfield mode and or in the fluorescence mode choose an ap propriate part of the object and focus on it if you can Use the beam splitter to check that light is reaching the camera On your microscope switch to the fluorescence mode Defining the acquisition sequence 1 2 3 4 5 ke 6 7 8 Use the Acquire gt mFIP gt Define Fluorescence Acquisition command Click the Add Fluorescence button Select all of the fluorescences keeping the Ctrl key depressed that your acquisition sequence is to contain e g DAPI Kern MitoTracker Red mitochondria Bodipy FL cytosceleton lt Brightfield gt Overview1 and click the OK button Use the two arrow buttons to
15. Time Frames From ff E to Jeo Step WES All copy non processed data OF Cancel Help Depending on the structure of the multi channel data set one or more of these groups will be active Select the check box prior to the name of a color channel in the Color Channels group to select this channel in the Kinetic process Clear the check box to have the color channel exempted from the calculation In the Z Layers and or Time Frames group use the fields From to and Step to define the frames to be considered Click the All button to undo any constraint and select all frames of the appropriate group Phase Color Coding Colors image regions according to the threshold settings This command is only available for 8 or 16 bit gray value images and for binary images as well not for 24 bit true color images It is available for 8 bit false color images only then if the Special gt Preferences gt Image gt Allow operations on false color images check box within the Image tab is selected Phase Color Coding is a false color display of homogeneous gray value or color areas Image areas you wish to have displayed in color must first be defined You define the image areas by setting threshold values in the image s histogram The gray value respectively the intensity range between the threshold values defines a phase Objects in gray value images are defined with the Process gt Set Thresholds command Up to eig
16. when you just want to see exactly where images are positioned on a page Defining multiple image page layouts for printing out 1 Select the File gt Define Page Layout command 2 Select the Single Image tab to define header and footer position e Define borders in cm in the Border group Have a look at the illustration previous page to see what the various fields are for e Both headers and footers may have multiple lines of text If the text is too long it will not be completely printed out i e it will be cut off when the page is printed 12 First Steps E mailing images r Es Bd ied Be F F BI e Select the Fixed image ratio check box to maintain the image s original length width ratio when printed out 3 Select the first Header Footer tab the first one on the left going left to right to define headers and footers for the whole page e Enter the text desired in the Header and Footer fields e g page Page date Today to have the page number and current date printed on the page 4 Select the second Header Footer tab to define a different caption for each image e Enter e g 6 Name in the Footer field to have the image s name printed beneath the image automatically e Select the Print scale bar check box to have a scale bar printed beneath each image e Select the Print page header footer check box to have the page headers and footers defined in step 3 also printed when you print out
17. 5 Use the Previous record in history button to return to the record which was a marked before the query was carried out 6 Mark the Query results database folder in the tree structure 7 Mark one of the images that has been found Click the right mouse button and select the Goto Record command from the context menu gt The marking in the tree structure jumps to the database folder which contains the selected record 8 Mark the Query results database folder again Use the Del key to delete the search result Load data Background Information Load stored data If you have already archived part of your data on removable data media the system searches for a data medium with a fixed name when loading This explicit data medium name is automatically assigned by the system during filing You will be asked to label the data medium with the name To load the data insert the required data medium Then select the DocumentFiles directory or click the Query button Integrate images in Use the report generator to produce professional multiple page reports File gt Report reports gt New It offers layout options for the highest standard of reports Images docu ments and database fields can be easily integrated directly You can also import values from previously specified table boxes into a report Export images You can copy database images and documents as files onto any network drive at any time Database gt Export record
18. Division Minimum Maximum Align Divides the graph in source with source 2 The contents of each channel of the graph in the source graph buffer is divided by the contents of the corresponding channel in the source 2 graph The resulting new graph is written to the destination graph buffer Compares the graph in source with source 2 and takes the minimum as result The resulting new graph is written to the destination graph buffer Compares the graph in source with source 2 and takes the maximum as result The resulting new graph is written to the destination graph buffer Aligns two graphs Use the Align command to merge two graphs with differing but overlapping x ranges The graphs have to have the same channel width for the alignment to be carried out Set the graphs to be aligned as source 1 and source 2 respectively The resulting new graph will be written into the destination graph buffer The channels in the overlapping areas will be averaged The Y scaling of the graph covering the lower X range will be adapted accordingly Graph Information Defines and shows graph parameter Use this dialog box to change the name of the active graph buffer to enter a graph comment and or to have graph information displayed 325 Graph Define Graph History Graph Information General Title Histogram of EFI M amp F Graph data Date 23 07 04 Graph start ao hra Graph end Ne iii Channel width Us
19. If you have selected the table view the gallery in the upper right of the database window is replaced by a sheet All the field information that you have selected using the Arrange Fields command for the Table View is displayed To switch to the table view select the Database gt View gt Choose View command The button bar in the database window provides fast access to frequently used data base commands The status bar in the database window contains the names of the current record and the number of marked records The context menu contains frequently used commands You can open the menu by clicking in the database window with the right mouse button 85 1 The button shown on the left change the data base view The data base window is divided into four sections in the standard full view button 1 1 3 Button 1 remains depress ed in full view You can alternate between gallery view left and table view right by clicking the Next View button 3 Archiving Images Choose View Preview window LifeScienceDemoS TAI C4 ES fe Gallery Ae x l la e a 2 8 ny 8 GFP_ YFPLony GFP YFP Unrmixing 2 Query results H CA FISH A Time Lapse fast QB Autofluorescence Antibody Labeling Unlimited Depth of Histochemistry CH Brightfield Microscop gy HeLa cells with Fura Unmising Deconvolution Urnmising 29 06 2004 10 10 Experiment Mame Date
20. In gray value images gray value distribution is measured and along with integration used to compute a cumulative histogram On the Y axis of the histogram the number of pixels whose gray values are less than or equal to the current gray value will be plotted against the gray value represented on the X axis The value range of this number of pixels will be normed to the image s maximum possible value range 8 bit gray value images have a value range of 0 255 16 bit gray value images of 0 65535 The normed histogram function is used as a mapping function in order to assign the gray values of the original image to the gray values of the resulting image Intervals having gray values of frequent occurrence will be spread in comparison to the gray value distribution of the original image Intervals with gray values of rarer occurrence will be compressed This means that the resulting image will have a gray value distribution where all gray values occur at the same frequency An image that is quite dark and is weak in contrast will become brighter as a whole contrast will be enhanced An image with too much contrast will have its contrast reduced 8 bit false color image are processed just like 8 bit gray value images False color images are in fact gray value images that are displayed on the monitor in color via a look up table LUT To look up the LUT used for the display of the active image use e g the Image gt Edit Image command Th
21. The Differentiate Y filter is one of the derivative class of filters If you wish only edges on the X axis to be filtered out of the image then select the Differentiate X filter If you wish to extract all edges of an object then select either the Laplace or Laplace filter Performs a gradient filter for edge detection with a light weighted Laplace filter Filter Matrix 0 1 0 1 4 1 0 1 0 The Laplace filter differentiates the image on both X and Y axes This results in edges and smaller image structures being accentuated The Laplace filter does however enhance noise as well as genuine image information The Laplace filter is one of the derivative class of filters If you wish to have image edges more greatly accentuated then select the Laplace II filter If you wish to differ entiate on the X axis only then select the Differentiate X filter for the Y axis the Differ entiate Y filter Performs a gradient filter for edge detection with a heavily weighted Laplace filter 243 The Process menu Mean Filter Matrix ae en 1 8 1 1 1 1 The Laplace II filter differentiates the image on both X and Y axes and in the diago nals This emphasizes edges and smaller image structures independent of axis orientation against the background The Laplace II filter does however enhance noise as well as genuine image information The Laplace II filter is one of the derivative class of filters If you wish to have edges
22. The Process menu Image Geometry Image Geometry Resize Resizes image in source image buffer and stores result into destination image buffer Properties Dimensions Mew Size n 1 EA ar E Size a7 A jes FI jai 6B Voxel Ratio Zoom axes homogeneously Interpolation o Wearest Weiahbour IRTE amp Triinear EYZ Bicubic xY Linear 7 OF Cancel Help Select the Image size desired either as a zoom Factor or as an absolute value in Pixels The Interpolation group is where you select whether an image is to be bilinearly or bicubically interpolated or not interpolated at all when you zoom the image This command is only available for false color images if the Special gt Preferences gt Images gt Allow operations on false color images check box has been selected Select None if you do not want the image interpolated when you zoom it Select Bilinear to smooth gray color transitions The new gray color values will remain within the value range of the transitions within the original image This inter polation is thus a good one for measuring gray color values interactively or automat ically Select Bicubic to obtain an image that has the best visual appearance because its transitional areas are smoothest Overshooting may occur along sharp edges i e gray color values which are outside the value ranges of the transitional areas as in the original image This method is thus n
23. Time Expenment Comment LifeScienceDemoS TAR 4 Un i Tree structure Sel 1 OkB Zz os apma ea a4 amp LifeScienceDemoS TAR apl Mle E3 S900 BRE A E File Name 4X5608 4x5608 4x5608 Unmixing GFP_ony Unmixing LifeScienceDemoS TAR a dd LifeScienceDemoSTAR ay Field v QJ Query results QJ Query results Experiment Name Unmisi 3 FISH FISH Date 29 06 3 Time Lapse fast Gy Time Lapse fast Time 10 10 J Autofluorescence E Autofluorescence Experiment Comment 3 Antibody Labelling Antibody Labelling 3 Unlimited Depth of Foc YFP_only E Unlimited Depth of Foc 3 Histochemistry H Histochemistry 3 Brightfield Microscopy Field y G Brightfield Microscopy 3 HeLa cells with Fura Experi N T H 9 HeLa cells with Fura amme Experiment ame omii H eg Fg Deconvolution Tins 1010 H Ca Deconvolution Experiment Comment 4 LifeScienceDemoSTAR Unmixing J LifeScienceDemoSTAR Unmixing Us 86 Archiving Images Work in the database window otep by step How to work in the database window 1 Open either the example database which you can install together with your im age analysis program or one of your own databases 2 If you need space for images or reports click in the database window button bar Aa on the Narrow view button to reduce the database window gt Your image analysis program removes the form view and reduces the gall
24. e Please note You can save all the database files or only those which have been altered since the last backup If you save all the database files the files with the apl mtb and tnb file name extensions are always written to an individual CD DVD If you only save altered database files clear the include database files check box to abstain from saving the database files Then you should save the database files in another manner 12 Click on the Next gt button gt The system now calculates the number of data media required 13 Before starting the backup carefully check the data in the File Storage dialog box 97 Before starting the backup you will get important information about the backup process Please note that you require at least two data mediums if you want to save all of the database files System Require ments Archiving Images Archive data File Storage x Select Finish to let the storage assistant start the storage process with the Following settings Storage method Backup Database C 4rchiveltestmaizoo4itestmaizo04 apl Destination drive E CD DVD Volume size 703 MB Temporary directory F TmpStorageDir Storage extent Complete backup Mumber of volumes needed 2 lt Back Cancel Help 14 Click the Finish button to begin the backup gt Your image analysis program copies all of the records for the first CD or DVD first to the backup directory and th
25. i Templatel Page Template PageT emplate1 sjej elelee gue lt Record Name gt Height lt Height gt a Position 5 0 10 0 mm Size 160 0 80 0 mm 7 42 Report Generator Report objects oo DE TIEG DURON in the button bar of the report window to Bey 7 Jes Ved Mca ed x xl ele B EIGE insert new database field objects You can select any of the database fields defined in the active image database Report Ob F3 Record Name O Record Version C RecordType C Remote Database ID C Remote Record ID To save an adjusted record object in the report template simply close the edit object mode by clicking on the Finish Object Editing button i Templatel Page Template PageT emplatel ae oo wl a ele 2 fal Channel lt Channel gt Manual lt Manual gt Object templates You can create your own templates for records and field objects These templates determine what your record or field objects look like and their make up Object templates are saved under a separate name within the active document You can then insert them into a report at any time You define object templates within a report template generally so that you can use them with all the reports you create based on this template It is not permissible to import object templates from other report templates Step by step Adding several images from one image database 1 Generate a new report ba
26. 100S ese sauenoes eee aoon paoe 139 17 Button Bars The Stack Navigator button bar En cel E og 2S SS Microscope Microscope Control Opens the microscope control panel For more information on the microscope the microscope control panel For more information on the microscope control click on the help button in the dialog box Acquire Fast Image Se Acquires a fast image sequence as image stack quence The Stack Navigator button bar Multi dimensional images consist of image stacks of different color channels time sequences or Z stacks With the commands in the Stack Navigator button bar you can easily select single images out of an acquired image sequence to be displayed in the viewport Image Stack Navigator Ei ae se hd Wl 1 oD Se Select Color Channel T Only displays the selected color channel see also on page 19 displays the selected color channel see also on page 19 cin 7 Navigate Z Chooses a projection in z dimension for the direction of an image stack Navigate Time Chooses a projection in t dimension for the direction of an image stack Click one of the buttons Select Color Channel Navigate Z or Navigate Time to select the dimension you are interested in A multi dimensional image for example consists of several color channels Click the Select Color Channel button first and the Next button afterwards to step through the individual color channels 7 Gottof Jumps to a certain position
27. 5 Select the Multiple Images tab to define the images position on the page e Define how images are to be positioned when printed out in the Image tiles group Enter the number of images to be printed out across i e horizontally in the Horizontal field and the number of images down i e vertically in the Vertical field e Define the distance between images and the distance to the headers and footers in the Border group Page borders will be defined according to the Single Image tab 6 Click the Print button to open the Print Image dialog box Once your have defined the page layout one time you can simply select the File gt Print command for any future printouts 7 Select Multiple Images in the Page layout list in the Print Image dialog box e This list also includes Single Image to have images printed out one per page 8 Select the All images option in the Print images group to print out all images currently loaded e f your have selected the Range of images option you will need to enter the corresponding image buffer numbers into the field below this option If you enter e g 4 7 3 the images in image buffers 4 5 6 7 3 will be printed out 9 To start printing click on OK gt The number of pages printed will automatically refer to the number of images selected E mailing images Background Information The following is The File gt Send email command is only available if re
28. Arrange Fields command 10 In the View Type list doubleclick on the Insert entry to display all data types for the insertion mask 11 Mark the Image entry gt The Available and Current lists now show all user defined database fields e The Current list already contains the predefined Record Name field and the newly set up Comment field The Record Name field must be filled in for every record which is added to a folder in the database It cannot therefore be removed from the list e The Available list contains available organizational fields The information in the organizational fields relate to all the records in a database folder 12 Mark one of the organizational fields in the Available list for example experi ment description from the step by step instructions and click on the How to define organizational fields auf Seite 66Add gt gt button gt If you now file an image to an experiment the comment can be entered in the insertion form The experiment description will be provided by way of information 13 Use the arrow buttons to change the sequence of the database fields shown in the form view 14 Close the Arrange Fields dialog box by clicking OK 70 Archiving Images Define a database The insertion mask for adding an image contains all of the data base fields that belong to the Add view and the image data type All of the organizational fields e g Experiment Description will ha
29. Both objects now have selection frames around them The selection markers of the object you last selected are gray and the other one s selec tion markers are white 5 Use the first buttons of the Align button bar to align all objects that have white a selection markers in relation to the object that has the gray selection markers Click e g the Center Horizontal button to move the object selected to the left or to the right until all center lines are in alignment i e in the same position gt Objects will only be moved to the edge of the page and no further Image Objects Background Information Image object You cannot insert images into a report directly You have to first create an image object to define the size and position of the image as well as its properties After insertion of the image object the image object remains empty You can identify an empty image object by the fact that the area reserved for an image is hatched Inserting an Click the Image button located in the Report Objects button bar to insert an image image object into a report object Insert Images Use the mouse to drag an image from the image manager or directly from an image database onto an existing image object Keeping the left mouse button depressed move the mouse onto the image object within the report window When you release the left mouse button the image will be assigned to the report document You can insert a number of images simultaneo
30. Channel Ep Document Creation Date Expand pickist on input Document Creation Time Ed Document Files Size Document Insertion Dat Document Insertion Tind Kertica imate Go medis Eni Es E Document Replacement Dat Edit Picklist The client is to be entered each time a new experiment is created Producton OSSO O Production Quality Assurance s j A picklist determines which client can be chosen when a new experiment is created 68 Archiving Images Define a database Define database fields Background Information Open the database Open the database exclusively Data types otep by step Define Fields You can open the database using the Database gt Open menu command or through the file list at the end of the database menu To open a database exclusively use the menu command to open the database and mark the Exclusive check box in the Open Database dialog box If you mark the uppermost entry in the tree structure you will see whether a database is opened exclusively You must open the database exclusively if you want to carry out operations which could alter the structure of a database e g defining or editing fields deleting the database defining a database password changing image and document paths changing the standard image format You can select different data formats for database fields e Text letters and numbers up to a maximum of 2
31. Click the Save button to save the current blue mapping curve in the diagram This mapping function will be saved under the name shown within the Function field The file name extension LUT as in Display1 lut will be suffixed to the actual file name This mapping function is now available in the Function list 203 The Process menu Invert If you wish to generate a new LUT file enter a new name into the Function field Or you can have the current user defined function overwritten by using the same name In this case you will receive a message asking you whether you wish to overwrite the old LUT file Please note that predefined mapping functions cannot be over written Invert Creates a negative of the image If the greatest number for gray value display is Gmax and G stands for a pixel s gray value in the resulting image then this pixel s gray value will be in the resulting image G Gmax Go For 8 bit images Gmax equals 255 For 16 bit images G max equals 65535 In binary images the white areas will become black and vice versa Set Gamma LUT Defines and applies a gamma compensation Gamma is the non linearity function of either the camera or the monitor What are Gamma Many cameras reproduce structures of high or low brightness non linearly such that LUT s for these very bright or very dark image areas are acquired with too little contrast Moni tors can have the same nonlinear problems This prob
32. Field object The template for a field object may include a field object which must however refer to a particular database field e g Calibration unit You can then insert several database fields e g Height and Width which will then always include the record object s unit of calibration 6 text standard text to be printed with every database field 41 Report Generator Record objects Adapting record objects If you are using a database that does not have some of the fields that the record object does then when you insert this kind of a record these field objects will simply be ignored You will not get an error message and the entry in the field object will remain unaffected This deals with placeholders for sheets diagrams or diagrams The placeholder remains empty if no sheet diagram or graph is attached If you wish to use record objects the record objects in the report template will have to be adjusted so that it fits your own database To be able to edit a record object you have to insert an ex isting record object first You can do this on any page you please To alter a record ob ject select it left click Then switch over to the edit ob ject mode by clicking on the Edit Object Template button You can select the field ob jects individually in the edit object mode and then delete them as needed al DataB aseO bjectWithS hadedFields BObject WithN ameAndl mageWidt
33. Image intensity Gray Value Comment af OF Cancel Help Enter a name for the image in the Image name field This name is the one shown in the image manager and the top bar of the image window The image name can be changed it can be up to 31 signs long When you load an image the File name field will contain that image file s complete path An image you have processed in your image analysis software will retain the file name of the original image as long as the processed image has not been saved under its own name When acquiring an image using your image analysis software this field will be blank until you have saved the image File names can be copied into the clipboard Select the complete file name and use the Shift Delete keys Use the Shift Insert keys to get the file name from the clipboard and put it e g into a text document This can be quite useful if you wish to refer to an image within a document When saving an image your image analysis software will automatically suggest the image name for use as the name of the file Note Image name and file name are not the same For example say you name an image yeast 02 25 2003 image 23 its image name you can save this same image under a file name such as 02250323 tif Then when you later reload this image the original image name will appear within the image buffer The Image buffer field displays the number of the image buffer currentl
34. Mean Intensity Projection For each pixel in the XY plane the intensities of the different time frames are analyzed The mean value of all intensities is used for display Minimum Intensity Projection For each pixel in the XY plane the intensities of the different time frames are compared The minimum intensity is used for display Animate Image Stack Clicking the button opens the Animate Image Stack button bar 22 Button Bars The Stack Navigator button bar Reverse Stop Play First Frame Previous Frame Next Frame Last Frame PY In Mark Out aS Options Animate Animate Image Stack Ef Stack gt Here you find the buttons to start Play the animation to stop it and to play it in the reverse mode The slider displayed at the bottom of the Animate Image Stack button bar indicates the position of the currently displayed frame within the sequence Additionally you can define a sub stack within the time lapse image Then only the images belonging to this sub stack will be animated To do so move the slider to the desired starting frame position and click the Mark In button Then move the slider to a subsequent frame click on the Mark Out button to set the end frame for the slide show The selected frame scan will be highlighted in blue in the slider bar Animate Image Stack E 4 0 144 44 o gt voi If you now press the Play button only the range of frames within the blue bar is anim
35. Select the color for displaying graph 2 in the Overlay list 5 Click the Add gt gt button to define graph 2 as on overlay 6 Confirm the choice by OK Horizontal Distance Measures the horizontal distance between two interactively chosen lines Mark the distance to be measured by clicking on both of its ends The ends will be labeled with the letters A and B and a consecutive number The values of the ends and the horizontal distance between them will be written into a new data sheet For each measurement that follows a new line will be added to the data sheet End the measurement sequence by rightclicking Select one column of the data sheet to append further measurements to it Vertical Distance Measures the vertical distance between an interactively chosen line and zero Mark the distance to be measured by clicking on both of its ends The vertical distance between these points is written to a new data sheet For each following measurement a new line is added to the data sheet 319 Graph Arbitrary Distance End the measurement sequence by rightclicking Select one column of the data sheet to append further measurements to it Arbitrary Distance Measures the distance between two interactively chosen points Mark the points the distance between which you want to measure by clicking in the graph The points are labeled A and B each with a running number attached to it The values of the two points and the distance between t
36. Select the fluorochrome from the Fluorochromes list which you want to acquire in the following The list contains only those fluorochromes which you have marked in the Define Fluorochromes dialog box If you cannot find the fluorochrome access the Define Fluorochromes dialog box once again and mark the check box to the left of the required fluorochrome 119 Acquiring images Define Fluorescence Acquisition In the Cube position list you define the position of the filter wheel for the filter allo cated to the fluorochrome An automatic microscope moves the filter wheel into the correct position for the acquisition In the Mirror cube list you select the name of the filter used for this image acquisition The list contains a wide selection of filters made by the Olympus Optical Co e g U MWU In the Objective position list you select the position of the objective used An auto matic microscope adjusts the selected objective automatically In both Objective type lists you define the objective used Select first from the left hand list the magnification of the objective The right hand list then contains the names of different objectives with this enlargement Magnification and the name of the objective are saved with the image information both for the individual image and the composed image In the Description list you define how the images are named by mFIP As a rule you should choose the Fiuorochrome Target entry Then yo
37. Therefore you can be certain that for instance while you are setting up a gray value histogram or reading out pixel values from an image buffer your data will not be falsified by overlay pixels For multi dimensional images there is also only one overlay level The same overlay level will appear irrespective of which frame is on display Images are stored in a bitmap like format that contains information about each indi vidual pixel In contrast to the image the overlay is stored as a vector graphic A line e g is characterized by a starting point an ending point and a color This vector graphic is then converted to a scanned graphic to be displayed with the image on the screen Exceptions are the overlay elements added as bitmaps or icons to the overlay 171 The Image menu Edit Overlay Burning Overlays Burning an overlay writes the overlay information into the image thus overwriting image information The burning of an overlay is not necessary for any application within your image analysis program Burning an overlay is necessary e g when you wish to export an image along with its overlay into another application program such as MS Word This is the only way for the other application program to be able to process the overlay information Please Note Edit Object mode Hi An overlay object must be selected for most overlay operations You can only do this if the mouse pointer is within the image window Click on the wh
38. When transforming pixels can be interpolated Interpolation ensures that transitions between varying colors appear smoother This will give the image a more uniform appearance Select None if you do not want the image interpolated Select Bilinear to smooth gray color transitions The new gray color values will remain within the value range of the transitions within the original image This inter polation is thus a good one for measuring gray color values interactively or automat ically Select Bicubic to obtain an image that has the best visual appearance because its transitional areas are smoothest Overshooting may occur along sharp edges i e gray color values which are outside the value ranges of the transitional areas as in the original image This method is thus not suitable for exact measurements of gray color values Select the Adjust check box to have image size adjusted to fit the new height and width of the resulting image following rotation Image area will be enlarged in such a way that the entire original image remains visible 262 The Process menu RGB Studio What will happen Note Note Clear this check box to retain height and width of the original image as they were Depending on the rotational angle image areas at the corners of the image of varying size will be cut off the original image Use the Margin fields to add a margin of the given pixel width Any additional image areas that occur du
39. When working with a patient database the aliso lalele organizational ID could e atient name Mewe Ausable ea a i J Info Window Lustomer H da Fom View Project name Table View lt Q Query by Example D y ee Remove dd gt gt cord Name fvestigation method Different data types may require different database fields lt HeMmove Arrange Fields View Type Available Current tle FJ Info Window Record Name H Form views Hat name a ustomer HEE Table View Investigation method a Query by Exargpla i 5M Ince The customer is now E Ins indicated when inserting an image Add gt 44 Remove The insertion mask when inserting an image to a project Insert Image Fields 1 4 Record Name IJz02 Project name P 24 6 2003 01 Investigation method ra analysial Customer Production Cancel Help E Skp e t 84 Archiving Images Work in the database window Choose View Background Information Database window Standard full view Tree struc ture Preview window Form View Gallery View Table View O Lon E Button bar Status bar Context menu Several databases can be open simultaneously Each STAR database is repre sented in its own window You can choose between different window views when working with the database Normally the database window is divided into four sections You can arrange the size of the indi
40. accentuated less strongly then select the Laplace filter If you wish to differentiate on the X axis only then select the Differentiate X filter for the Y axis the Differentiate Y filter The Sobel filter can be used for noise reduced edge detection Mean Performs an averaging filter for low pass filtering Filter Matrix 111 111 111 The Mean filter replaces every pixels with the arithmetical mean of that pixel and its eight neighboring pixels This results in noise reduction Abrupt gray value transitions will also be smoothed over however thus appearing blurry after averaging The Mean filter is one of the smoothing group of filters It is a particular NXN filter including a 3x3 neighboring pixel area Other available smoothing filters are the Median Sigma and Rank filters Median Performs a median filter for random noise reduction The Median filter removes isolated erroneous bad pixels Neighboring constant gray value ranges to these pixels as well as edges remain untouched The filtered image thus loses none of its original sharpness The Median filter is a smoothing filter from the rank filter class A pixel s value as well as the values of its eight neighbors will be sorted according to their magnitudes The middle value the 5th one will be assigned to the central pixel Extreme gray values will always be located either at the top or bottom of this listing and will thus never be assigned to any of the pixels The
41. check box has not been selected Select the Use starting point check box to along with the Interactive button deter mine grid frame position in the image If you do not select the check box the grid will always start in the upper left hand corner of the image at the coordinates 0 0 Select the Label fields check box to have grid frames numbered in the overlay rows are designated by the letters A B C AA AB AC WW Columns are designated by the numbers 1 2 3 99 Every cell can be unambiguously identified e g CF12 Grid frames are not numbered if the text does not fit into the frames To have grid frames labeled the grid frames will have to be enlarged If you select the Label fields check box the grid size will not be displayed Select the Automatic gridsize check box to have height and width of grid frames auto matically adjusted to fit the minimum number of grid frames Enter this value in minimum of fields This number is based on the width of the whole image The lowest 294 The Measure menu Intensity Profile value possible is 2 frames the highest 50 With the automatic gridsize grid frames shape will correspond to the X Y proportions of image calibration An X Y proportion of 1 results in square grid frames The current values for height and width are displayed in the Gridsize group Not all adjustments to the minimum number of fields result in a differing frame size because height and width
42. check box selected Click the File button to open the standard dialog box for the opening and loading of files Files will be saved using the LUF format 163 The Image menu Calibrate Image When you click the Linear button all formulas will be reset to n This corresponds to the linear standard LUT coordinates of 0 0 and 255 255 Calibrate Image Interactively defines the ratio pixel real world units The XY Calibration tab is available for any type of image The Image Intensity tab is available for gray value images which are not part of a multi dimensional image The Z Calibration tab is available for image stacks Calibrate Image XY Calibration Defines the absolute size of a pixel Cahbrate Image x XY Calibration 2 Calibration Magnification fio0 Calibrate 2 calibrations 0 067 pm Unit Y calibration 0 067 urn Channel Bey ratio fi I Fixed Calibration f Arbitrary Horizontal Vertical Calibration length fi Lum OF Cancel Help Both input and image must be calibrated in order for scale within the image to be shown correctly via the scale bar To have the scale bar shown simply press Shift F4 Correct calibration is also necessary in order to obtain proper results using the many interactive and automatic measurement functions located e g in the Measure menu Sometimes it is necessary to recalibrate an image e g if you load third party images
43. object 6 Should the desired object not have been found correctly do the following Keep the left mouse button depressed and pull the mouse to the left to decrease the size of the object Keep the left mouse button depressed and pull the mouse to the right to increase the size of the object You can view the result as soon as you release the mouse button 7 Click the right mouse button to end the definition of the first objects gt Anew measurement is created in the measurement display The area and perimeter is given as default measurement values gt The pointer remains in the image You can immediately measure additional objects 8 Click the middle mouse button to end the measurement Background Information Selecting measure ment parameters Especially for two dimensional objects you can measure a lot more than area and perimeter Your image analysis program offers you an entire row of measurement parameters from which you can choose the appropriate one Even after the definition of the measurement object you can always alter the measurement parameters which are to be exported Various measurement parameters are only available for various classes of measure ment objects The measurement of the area of a line is senseless and therefore is not even offered as a function when measuring line objects The selected measurement parameters are shown in the tree view in the Measure ment tab as well as in the measurement
44. 14 point Number of copies f S Displayed mage t Allimages iE Benge oh images f 2 4 Use a comma to delimit single image buffers and a dash to delimit image buffer ranges e g 1 3 7 10 Cancel Layout Hange FLEE E patabase selection Help Define Page Layout Header Footer HeaderFooter Header Footer Single Image Multiple Images Other Documents Image tiles Preview Documents m es Images Graphs a 1 Co Atlas Horizontal po 5 Vertical ja E Border Distance ho ja Header ps E Footer jos B Single image size Width 7 55 cm Height 6 93 cm mef 2 Fe Atlas A 3 Fe Atlas FAF 4 Ag Atlas Gal 5 Al Atlas 1 mepa 6 Al Atlas 2 A 7 As Atlas A 8 Au Atlas m pa 9 B Atlas 1 a 10 E Atlas 2 eA 111A Ahas a OE Cancel Help ed gt Define Page Layout command 311 e The page layout of printed spectra is preset and cannot be influenced by the File Graph Printing graphs Use the Print Graph dialog box to print out single graphs along with graph information TE Hame 20E HP Lazernet 4050 Series FS a Properties Status Default printer Ready Type HP Lazenlet 4050 Series PS Where Sintranet 206 ps Comment Laserdrucker HP 4050 PostScript Print Graph E E Printer Copies Print output Graph only With general info data With all available info data
45. 9 Click the Finish Object Editing button in the report window button bar to exit the edit object mode 10 Now insert the sheet from the database into the adapted record object To do so you can either move the sheet or also the record under which the sheet has been inserted onto the record object gt The sheet and the desired field entries as well will be included in the report Using sheet cells in a record object 1 Use the Record button in the Report Objects button bar to insert an existing record object 2 Click the Edit Object Template in the report window button bar to switch over to the edit object mode 3 Click the Field button in the Report Objects button bar to add a new field object to the record R E 4 In the Select Field dialog box select the Sheet Cell entry and confirm with OK 5 Click the Properties button in the Report Objects button bar 6 In the Record Fields tab enter a description of the measurement value in the Fieldname field i e G Value 7 In the Sheet group enter in the File name field to have the first attached sheet inserted together with the record 53 Report Generator Inserting diagrams 8 Determine the desired sheet cell in the Sheet and Column field Should the inserted sheet not contain the specified sheet cell the field remains empty 9 Click OK to close the dialog box 10 Click the Finish Object Editing button bar located in the report
46. A single color Z stack image consists of monochromatic images which were sequentially acquired at various focal planes H A single color time lapse image consists of single monochromatic images t which were sequentially acquired at different times 4 A single color Z stack in time lapse image consists of single color images d which were sequentially acquired at different focal planes and at different times Mal A multi channel image is made up of several monochrome images Each image stands for one color channel B A multi channel Z stack image consists of multiple color images which were i sequentially acquired at different focal planes First Steps The GUI User Interface mH A multi channel time lapse image consists of multiple color images which H were sequentially acquired at different times H A multi channel Z stack in time lapse image consists of multiple color images which were sequentially acquired at various focal planes and at different times Standard images Your program can naturally also cope with established standard formats Standard images can be depicted as three dimensional data sets the three dimensions being two spatial dimensions x y plus the wavelength color value T A gray value image can be made up of 256 8 bit or 65536 16 bit gray values A binary image is comprised of 2 gray values black and white A false color image is a gray value image whose gray values are sh
47. AOls File Size E E E ution Document Area Menu bar Image buffer box Status Bar gt Am Colorviews I Fw ge 768 576 Many commands are accessible via the relevant menus You can configure the menu bar to suit your requirements Use the Special gt Define Menu Bar command to add alter or remove menus as you wish Each image is allotted its own image buffer within your image analysis program When you start up your image analysis program all available image buffers will be empty While you use the program they will become filled when you load or acquire images and when you perform various image operations that alter an image in such a way that a new image results During any given work session this means that many images are accessible simultaneously Only one image buffer however can be active at any given time ZA Saving GUI configuration 10 First Steps The GUI User Interface Active image buffer Button bars Viewport Viewport manager e The image displayed in the image window will always be the one in the active image buffer irrespective of how many other images are also on display e The active image buffer contains either the live image or an acquired image Any interactive input or measurements are always applied to the active image buffer Commands you use frequently are linked to a button providing you with quick and easy access to these functions Please n
48. As soon as you have defined your own object template for record objects the standard record will no longer be available Therefore if you wish to be able to use the standard record later on then save it Text objects in Object templates cannot include editable text objects If you wish to subsequently object templates insert a record object into a report you can only alter the texts within the text objects in the edit object mode otep by step Creating new field object templates The object template is to include two field objects of varying format One format is to indicate the magnification of each image The other differently formatted field objects are to contain information on the image database image 1 Load the image database which contains the images for your report Ely 2 Select a record object and click the Edit Object Template button in the report z window button bar to switch over to the edit mode for object templates 3 Click the Field button in the Report Objects button bar to insert a standard field object into the record You will be creating a new object template for field objects based on this field object e You cannot insert field objects without an existing database This is be cause without a database no field information will be available You will re ceive an error message if a database is not open gt The Select Field dialog box will be opened This is where you ll find all data base fields tha
49. BESEEEBEEEELE SERESGEESEEELL BEBE EEL BEBE LLL _ EE ee JIS See Help Note This command overwrites the LUT lookup table of the original image i e the orig inal image will be lost This command cannot be undone If you wish to keep the Original image then be sure to copy it into another image buffer before executing the command 265 The Process menu Blend Images The Image gt Image Display gt Edit LUT command provides various other options for editing the colors in false color images What will happen After calling the command a dialog box with a color palette will be opened The palette offers all the image s colors Doubleclick on the field which contains the color you wish to replace The Windows standard dialog box for selecting color will be opened and you can select a new color The new color will replace the previous color in the field of the Edit Palette dialog box The new color will immediately replace the old one in the image Blend Images Superimposes two images by using alpha blending The two images are taken from the source and the source 2 image buffer The Value field sets the percentage each of the two images is to take up in the blended image The lower the percentage the greater the weighting of the source 2 image A higher percentage increases the weighting of the source image 0 means that the second image is visible only 100 means that only the first one is The image wind
50. Database queries Your image analysis program offers three methods of finding images in a database Query by Example You can use this query for a simple search for the entry of a field You can also link several fields using the AND link For a query by example you can use the wild card Query by Example Search targets are Entry all records by Christian Christian all records beginning with letters Gold Gold Database folders created after 16 4 2005 gt 16 04 2003 lt 18 30 all images which were not acquired using the lt gt SlowScan SlowScan logical input channel more all sheets Query by Free Filter You can use this query for a search using several search criteria which you want to connect in a complex way using AND or OR links Query by Free Filter all folders from 2008 Date P 1 1 2008 lt 11 2009 all RGB and gray value im Image Type RGB ae CE ImageType j Monn A all image names which be Image Name gt lt gin with A all images were inserted on Document Inser 20 5 2003 the 20 5 2003 between 15 00 tion Date and 20 00 Document Inser gt 15 00 lt 20 00 tion Time Query by SQL This query uses the standard SQL database administration language to formulate the search criteria Query You can use this button to carry out an initial query by example For subsequent queries you open the dialog box of the pervious query Sa
51. Depress the middle mouse button or the Esc key to return to the Add Shape dialog box Rotated Ellipse This is how you define a shape with the Rotated Elliose method 1 Click the Rotated Ellipse button The mouse pointer moves to the image window 2 Keep the left mouse button depressed while moving the mouse A rectangle is shown in the image The ellipse is within the rectangle 3 Pull the rectangle to a desired size by depressing the left mouse button Then position it by moving the mouse To draw a circle use the shift key 4 Click once using the left mouse button to switch to the rotation mode Rotate the rectangle by moving the mouse If necessary switch to the translation mode to move the object 270 The Process menu RGB Studio Note Magic Wand Delete Copy Invert Shape There are two possible modes with this drawing method the translation mode enables you to change the size and position of the object the rotation mode enables you to rotate the object A click with the left mouse button switches you back and forth between the translation and rotation mode You will recognize which mode you are currently in by the form of the mouse pointer 5 To draw a circle use the shift key 6 Click the right mouse button to end the definition of the ellipse 7 Continue defining shapes by the active drawing method 8 Depress the middle mouse button or the Esc key to return to the Add Shape dialog box Use thi
52. Enter the number of images in your focus series in the Number of images field EFI will then suggest a value This value is determined from the number of successive image buffers starting with the active image buffer containing the same sort of image The same sort of image meaning same image type same image size and the same bit depth Enter the number of the image buffer where you want to have the EFI image inserted in the EF result in buffer field EFI will make a suggestion here as well Select the Create height map check box to be able to view the gray value image put together by the EFI computation Feel free to use it as a height map Enter the number of the image buffer into which you want to have the EFI height map inserted in the Height map in buffer field EFI will make a suggestion here as well Click on OK to begin the EFI image computation How long it takes will depend on your computer and on the settings you ve made in the EFI Options dialog box If your focus series is made up of many images of high pixel count and they all need aligning before the actual EFI computation can begin then the whole process may take 2 3 minutes As long as the hourglass icon is being shown and the blue bar within the status bar keeps expanding you know the computation is still ongoing EFI Acquisition Use this command to start up the accumulative EFI method the EFI image is then computed from a series of live images Dur
53. Even low saturations of a color are detected el When using the color space Hue within the HSI model the tolerance value is 268 The Process menu RGB Studio Fitting Polygon Rotated Ellipse On a color wheel the saturation of a color increases when moving from the middle to the edge of the wheel The intensity is the same for all pixels The color values are arranged along a wheel from H 0 Red to H 360 The color wheel shows the differ ences in use of the different color spaces When using the color space Intensity within the HSI model the tolerance value is added or subtracted from the Intensity value of the starting point Use this color space if the image is a true color image but contains a limited amount of color information The Tools These buttons provide you with tools for drawing shapes Polygon Interpolating polygon Freehand polygon Ellipse Rectangle Rotated Rectangle Magic Wand Undo Polygon This is how you define a shape with the Polygon method 1 Click the Polygon button The mouse pointer moves to the image window 2 Click the left mouse button to set the starting point of the first segment 3 To remove the last point depress the shift key and click the left mouse button 4 Move the mouse to draw the segment and click its endpoint This endpoint will be interpreted as the starting point of the next segment 5 Repeat this for any further segment of your polygon line A polygon sh
54. First Steps The GUI User Interface Within the thumbnail in the viewport manager you can define which image segment is to be displayed within the image window To define the segment adjust the size of the frame and move it to where you want it within the Navigator F Images 1 HER2 173 agr A ala Alle You can hide the viewport manager to create more room for other windows for example To do so use the Alt 1 key stroke Note Move the mouse pointer on the viewport manager and rightclick to open a context menu Image manager The image manager contains numerous tabs Click the different tabs to alter the appearance of the image manager The first two tabs List and Gallery are reserved for the administration of images Note Move the mouse pointer on the image manager and rightclick to open a context menu You can change the appearance of the image manager To do so use the Image Manager Properties command The operands box is for e determining source and destination image buffers used in image processing op erations which alter the original image e g inversion e linking images for certain image processing operations e g addition of two im ages Use the image buffer box e for an overview of the images loaded e for rapid access to image information such as its size and image type e to activate image buffers You can hide the image manager to create more room for other windows for
55. Freehand Polygon method 1 Click the Freehand polygon button located in the Tools group The mouse pointer switches to the image 2 Click the left mouse button to set the starting point of the line Move the mouse by keeping the left mouse button depressed to draw the line The Freehand Polygon line follows the course of the mouse 3 Click the right mouse button to finish the definition of the line If the line you have drawn is not closed your image analysis program will auto matically join the starting point and the endpoint together A closed region has been defined on the image You return to the dialog box The new ROI is imme diately added to the Active ROIs list 182 The Image menu Define ROIs Ellipse The outline of a ROI defined by this drawing method is an ellipse or a circle This is how you define a ROI with the Ellipse method 1 Click the Ellipse button located in the Tools group The mouse pointer switches to the image A circle or an ellipse is displayed in the image overlay 2 Move the mouse to move the circle at the desired position 3 Move the mouse while pressing the left mouse button to distort the circle to an ellipse and or to change its size 4 Click the right mouse button to end the definition of the ellipse A closed region has been defined on the image You return to the dialog box The new ROI is immediately added to the Active ROIs list Rectangle The outline of a ROI defined by this drawing
56. Green n 50 W Limit W Blue In W Limit OF Cancel Help Enter the formula which defines the function of the corresponding LUT in the fields Red Green and Blue The pixel value n is the variable for the X axis If you change a formula the function will be computed with all values between 0 and 255 and the result displayed in the diagram Simultaneously the image is continuously displayed on the monitor using this LUT You can use all expressions allowed in Imaging C Please take note of the following guidelines e Multiplication and division operations will be performed before addition and sub traction Be sure to place brackets around the calculation e All constants should be provided with a point e g 100 to be able to calculate with the precision of floating decimal point operations e n may have a value of 0 You should thus avoid e g using n to divide with Divide by n 1 instead e Take note of error messages that appear in the status bar after you have com pleted inputting your formula Messages appearing while you enter the formula can be ignored If the Red Green or Blue check box is not selected the corresponding LUT will be given a constant value of 0 Select the Limit check box to have your result set to 255 in case of an overflow and to 0 in case of an underflow If Limit is not selected the values over 255 will continue at 0 Values below 0 will continue at 255 We recommend having this
57. However you cannot use the image calculator for multi dimensional images You will receive an error message if you attempt to carry out a calculation for a multi dimen sional image Use the Process gt Arithmetic Operations command instead 247 The Process menu Image Calculator El Image Calculator Destination Frerse Fin Imgl Marmee cantas 7 Intensity scaling Help 8 16 and 24 Bit Gray value images with 8 or 16 bits true color images with 24 bits and numerical Images constants can be used in the operands Using 8 and 16 bits with gray images is allowed and results in an image with the bit depth of the first operand image You can use this to invade the very restrained accuracy of an 8 bit result image by converting the first operand image to 16 bits before calculation e g Image gt Convert Image gt To 16 Bit The calculation is then carried out with higher accuracy and the results are shown within a range of values 256 times higher than before The Display LUT defined in the image manager is taken into account in the display of a 16 bit result image A defined intensity scaling can possibly be taken into consid eration during calculation whereas an allocated LUT is meaningless The calculation of the red green and blue color channels take place separately A mixed usage of true color images and gray value images is not possible Negative Results The pixel values of an image are always pos
58. If the system does not have a communication link to the microscope it is the user s responsibility to adjust the magnification to correspond to the true magnification The term magnification can be defined as the ratio of original specimen size hard copy or original specimen size screen display or some other standard This has to be decided by the user and will reflect the application Using the Calibrate Image dialog box you can calibrate or recalibrate the image of the active image buffer Please proceed as if you were calibrating an input channel The XY Calibration tab is designed like the corresponding tab of the Configure Input dialog box 164 The Image menu Set Magnification If you click the Channe button the calibration of the active input channel is trans ferred to the image This command is useful if the image has been taken with the same values of the active input channel on another microscope where this program has not been installed Calibrate Image Image Intensity Calibrates the gray or intensity values of the active image This tab differs only slightly from the Image Intensity tab in the Configure Input dialog box Instead of the Acquire button you have the Channel button Click the Channel button to apply the calibration values of the active input channel to the image in the active image buffer Calibrate Image Z calibration Defines the Z calibration Cahbrate Image x sty Calibration Z Cali
59. Intensity and Contrast scroll bars Use them to shift the current mapping curve within the diagram around in different directions You ll be able to keep an eye on how your adjustments to intensity and contrast affect the image in the image document Current intensity and contrast percentages are indicated beside the scroll bars The two scroll bars always each start out with a value of 50 0 Both scroll bars can be adjusted to anywhere between 0 0 and 100 0 Leftclick on either the intensity or the contrast scroll bar depending on which one you need The scroll bar you ve selected the active one will blink slowly in two gray tones This makes the active scroll bar easy to see And you ll always know which value you ve just adjusted 202 The Process menu Intensity What does moving the scroll bars accomplish To move a scroll bar Keep the left mouse button depressed and move the mouse to the left or right Percentages can be finely adjusted in this way the finest adjustment possible being 1 0 up or down When your re finished moving the scroll bar s simply let go of the left mouse button You can also click on the directional arrows located to the right and left of the scroll bars This enables you to even more finely tune in increments of circa 0 2 Click right in the scroll bars themselves to make adjustments much greater than just 1 0 per click Here s how the current mapping function G r is changed by moving t
60. OD bars HEE Gl Report LOLS e i aafe g a ghe do alal e e e Fi 0 100 200 a Dei AP TW A lt Report Heading tingle line Martis Tectrobgy Guilty Conds ni a Die adhg or repot ipp wo hess laccri Aniu Chrmoia Jefita Awd hugu Counerk Oce OV aR inanu 2 Bd Iaccri Aniw Chmoiaz a p Aepicwin Aggie amp rege Cone TTET Inenu 3 herci Anw Chroag Appie Agopian EO iil M rage Consert Z Wer lt User gt mgoa Sta Cate 16 12 004 Esed Dy sgan LifeScienceDemoSTAR Brightfielc Page 1 1 Position 0 0 0 0 mm Size 210 0 297 0 mm Ready a F View Il Fw 100 Report button bar The report button bar is a part of the report window Please keep in mind that these buttons functions are not available as menu functions This is why this button bar should remain visible If you like you can use the Special gt Edit Button Bars command to show or hide the button bar or to add other frequently used buttons Turning button bars e Use the Special gt Edit Button Bars command on and off e Rightclick in a report window All button bars linked to report generator are listed beneath the Button Bars command 26 Report Generator Creating reports Paging through a report Go to 7 Add Page Delete Page Report properties File Format First Page Previous Pag
61. Set Split Gain Hinweis oe This command is deactivated for graphs resulting from a Kinetics calculation Set Split Gain Sets a split gain factor Use the Graph gt Set Split Gain command to display a graph with a very large y range in a linear scale For this purpose the graph will be divided into different X areas Increasing the gain factor of the area with high Y values enables the simulta neous display of the all of the Y values Set Split Gain x Split position 127 B Set Gain factor 2 5 Cancel Help Each time you use the command you define one split position You may call the command several times to divide the graph into several areas In this case you should start with the area with the lowest Y value e The split positions and gain factors are integral part of the graph They are saved along with the graph and can only be deleted or modified by using the Modify Split Gain command e You can split the gain factor only for graph in the active graph buffer Overlay graphs are not affected In the Split position field enter the X value by which the graph is to be divided Click the Set button to define the desired energy loss position interactively Use the arrow buttons next to the Gain factor field to set the gain factor for all of the Y values that are greater than the split position All intensity values will be multiplied by the gain factor that has been set The gain factor is indicated in the
62. Split into Standard command The resulting gray scale images can be analyzed in the same way as any other gray scale images Gray scale Images To convert a gray value image into a binary image you must define which pixels of your original image are to be assigned to which gray value i e between 0 or 255 in the binary image This information appears in the form of a gray value range Any 211 Manual The Process menu Gray scale Images pixels whose gray values are within this gray value range are considered set and will appear as the foreground within the binary image All remaining pixels consid ered not set and will appear as the background the background is usually displayed black You do have the option of defining more than one gray value range The Set Threshold dialog box sports two tabs Defines interactively the gray value ranges for your active image You can define several gray value ranges for an image Each of these gray value ranges is called a phase Phases or gray value ranges must be continuous i e there cannot be any gray value gaps within the ranges you define Set Thresholds Manual Auto Settings Preview Phaze L w 3915 FI C None Coo Hight 4222 E Curent Ell Delete PELE Include Pixel Background File Transparent Auto Diagram Smooth Histogram 1 FI _ OF Cancel Help
63. Stage Area Mode When you change from another mode into the Online Mode you will automatically switch to the live image Then when you find interesting areas in the overview image you can zoom in on them in the sample and look at these areas in the live image Offline Modi This mode is only available when you have already created or loaded an overview image In the Single Image Mode you can load images into an image buffer by dou bleclicking the image If you have created the overview image from one single image you will then have loaded the overview image into the image buffer If you have cre ated the overview image from separate images you will then have loaded only the one image you clicked Furthermore it is only in this mode that you can delete images see page 336 The suggested default image name for this area is 1 v lt consec no gt You can change the preference at Special gt Preferences gt Image in the field Prefix for images Change into this mode if you want to load a freely defined area of the overview image into an image buffer You determine the size of this area by keeping the left mouse button pressed while you drag the frame to the size you want Doubleclick the left mouse button to load the image Click the left mouse button once to move the frame to another position The image name suggested for this area is Interest area In the Stage Navigator Properties See page 338 you can also determine another
64. The thresholds you define represent the upper and lower limits of a phase A phase having its thresholds at 0 and 100 is comprised of all gray values G between 0 and 100 this can be expressed in the following way 0 lt G lt 100 Click the New button to define another gray value range After clicking on the button a new entry will be added to the Phase list This new phase entry comprises the stan dard phase name plus its number This new gray value range will now be comprised of all gray values not yet assigned to a specific phase Because gray value ranges have to be continuous the lower gray values will be assigned phases first All gray value ranges you have defined already can be found in the Phase list e Select a phase from this list that you wish to edit e You can alter the standard phase name as you like To do so simply click on the phase s present name and then you can enter a new suitable name for this gray value range The name you give the phase will be used in the phase anal ysis result sheet This phase name can also be used as a particle parameter in an automatic particle detection Enter the upper and lower thresholds for the active phase in their respective fields High and Low The lower threshold represents the lowest gray value belonging to this phase the upper threshold the highest gray value Thresholds can also be set directly by using a histogram Using the mouse simply
65. Word compatible Via the RTF Export function you can have reports exported to MS Word 1 1 This enables you to communicate with fellow colleagues who may not have access to your image analysis program Creating reports Background information Reports and report templates Report window Exporting reports 29 Report templates 54 Ruler 32 Reports are used to document results in standardized form They usually consist of many pages which are similarly structured In order to make report creation easier a report is based on a report template The report template defines all page layouts and object templates that can be used in this kind of report You can never load and edit more than one report or report template at a time The report will be loaded into its own separate window Only one report page can be shown and edited in the report window at a time The report window has its own separate button bar and a status bar There are ruled borders and a grid for use as positioning aids 25 Report Generator Creating reports Reports are opened within a separate win Report Report Objects dow The functionality of Report Window Button Bar Button Bar the report generator is in the button bars The Re pee A O S File Edit Database Acquire P Graph Special Window ala button bars are the two tie cat Uatabase cqure image Frocess asure urap pecial Window lt most important button D 08a 8 amp meam a e Kl 1 OD
66. X position that belongs to the label The dimension of the field depends on the current graph dimension Click the Set button to find the X position interactively A dashed line connects text frame and the current X position Leftclick to confirm the setting and transfer the value into the Position field To move the text label itself click the Set button next to the Label text field or click the Align button Click the Align button to update the position of the text frame The text will be shifted horizontally directly above the current X value in the Position field To move the text frame in the vertical direction click the Set button next to the Label text field Select the Draw line check box to connect text frame and the X position by a line Select the Draw arrow check box to connect text frame and the X position by a line Deletes all labels of the current graph This function as well as the deletion of single labels may also be invoked via the Modify command Copies all labels from source to destination Use Graph gt Markers and Labels gt Copy command to copy all labels of the active graph document into the graph document selected as destination Dest in the Image Manager This tool is very useful to set labels in a series of graphs derived from one experiment at the same position The labels can only be copied if the graph in the dest buffer has the same X axis scaling as the graph of the src buffer 315 Graph
67. all pixels that have more than 5 bright neighbors then you should select the check boxes 6 7 and 8 Bemeeeeee foo I ton n e Po a If Select the Delete option if you want to set all of the pixels at 0 black that have exactly the selected number of neighbors with higher or the same intensity All of the remaining pixels will be set at 255 white The pixels that already had a gray value of O in the original image will under no circumstances be changed The resulting 233 The Process menu Edge Enhance image will therefore be a binary image With this filter thin branches in a binary image can for example be removed if only a 1 has been selected in the Number of neigh bors group In the Number of neighbors group the None 1 and 2 have been selected Every pixel that has fewer than 3 neighboring pixels with greater or the same intensity will be deleted i e set at black In this unfiltered image you see globes On the edges of these globes you see artifacts that the connectivity filter will re move Connectivity Number of Neighbors None 1 2 Delete Select the Extract option to have all of the pixels with the selected number of neigh bors filtered out of the image i e set to the gray value of 255 The filter behaves simi larly to the Delete option except that the gray values 0 and 255 are exchanged Select the Color coding option to have a pixel colored proportionally to the number of brighter or sa
68. alter the properties for all sheet cells Measurement Form A12 680 General Data Specimen Data comuhacted by Mi Specimen EMEE6HS0 conducted om Material emcee 1 tested by Dai Preparations ee tested on Area cosets PhaseAn alysis Phase 1 0 42 Area Phase 2 43 106 Phase 3 107 244 Phase 4 212 255 Pomme s m wT m s m 3 1195 42 16 30 4734 80 23 61 4119 09 56 15 289 16 3 94 1071 42 14 561 1802 80 24 59 3864 44 52 68 595 81 4101 55 45 02 1709 48 23 30 2820 19 1704 25 23 23 083 42 13 45 4543 61 24 14 4158 80 584 19 927 25 12 68 3164 32 29 60 2026 90 1182 45 16 31 1061 74 14 52 1607 87 24 99 3821 73 52 27 819 68 44 24 1 3 912 90 12 49 2017 Ae 27 59 273r OS 37 45 1642 77 227 254 90 12 10 1553 03 21 65 390483 53 44 938 26 12 83 1228 06 16 80 1718 84 23 51 aga 83 AF 58 555 29 12 11 1232 13 16 85 BrO 0G 22 54 551 50 46 99 B27 03 11 31 4192 45 16 31 4726 00 23 62 404 38 56 14 se720 3 93 s5o012 14 63 2400 87 32 96 2568 51 35 12 1482 71 20 28 12 83 4957 80 26 78 2756 25 37 70 4651 48 22 59 p afa AE JE HE Phaze Arslyzix LE _ Phase 1 0 42 me TLUU 300 1 qi J Sunter of Weasiramers Inserting sheets into an empty sheet object 1 Close all loaded sheets Simply use the Window gt Close All command to do so 2 Generate a new report based on the Normal template 3 Select the image object and click the Shee
69. altered You can define a new mapping function within the XY diagram by using your mouse All alterations will be immediately visible within the preview area Continue altering the curve until you have obtained the effect desired Then you can save the new mapping function and when you need it again later reload it for appli cation to another image 210 The Process menu Set Thresholds Channel Reset Select whether you wish to alter the color components Red Green Blue or the intensity within the image in the Channel list The histogram of the channel selected will appear in the XY diagram The mapping function shown will be the curve which was last used for this channel You can define your own mapping functions for each channel The mapping func tions of all four channels are always active The image will be altered in all channels at the same time If e g you have altered the linear mapping of one channel and then switch over to another channel the mapping function of the previous channel is maintained and remains active Click the Reset button to replace the mapping functions with the linear mapping in all channels The preview area will show the original image once again Reset is only relevant for current alterations Any alterations which have already been executed cannot be reversed To define a new mapping function 1 Leftclick on the spot where you wish to edit the curve A little red circle will appear represent
70. analySIS File Edit Database Image Oper Measure Analysis Stage Graph 3D C Module Special Window O e H 2 S4 O0 7GH in eo XY M a HI e FA images 1 T 7 47 Z X Se Es Stage Navigator Unnamed 7 fe 222 na Su Se 50 2s Daeke awaah mf SANE SS Here is an onscreen layout that is typical for what you will see when working with the Stage Navigator An overview image has been acquired and is now displayed in the Stage Navigator The user employs it as an orientation guide that shows him which area of the sample is currently being displayed in the live image The overview image also enables him to move quickly to another area in the sample The sample analysis takes place in the live image The view port and image manager have been hidden to provide a larger image window You can memorize interesting areas on the overview image by saving their posi tions and giving them an individual name You can then return to these positions later to analyze the areas in more detail This provides you with a good possibility to make a comparison of two areas in a sample The Stage Navigator is a modeless dialog box i e you can leave the dialog box open and still be able to use a lot of important functions You can e g select another image buffer or activate the image window without having to close the Stage Navi gator 328 The Stage Navigator Preconditions for using the Stage Navigator Preconditions for using t
71. are set at standard sizes in the unit of image calibration such as 1 2 5 10 20 and so on Determine the color of the grid and the overlay text in the Colors group The Grid and Text palettes contain a selection of eight colors each The Grid field is for determining the color of the grid The Text field is for determining the color of cell numeration and cell size display A gray frame around a color indicates it has been selected Click on the Draw button to have the grid at its current settings drawn into the overlay In addition cell size or alternatively numeration of the grid frames will be written into the overlay depending whether the Label fields check box has been selected or not The dialog box will remain open so that various settings can be tried and immediately checked on screen Each new setting will delete the previous grid and its settings Depending on the position of the dialog box it may have to be moved per mouse to be able to view results of setting changes in the image window Click on the Interactive button to be able to determine grid frame size and position interactively within the image window A red rectangle will appear in the overlay displaying the dialog box settings If a grid has been written into the overlay it will be deleted when you click Interactive Keeping the left mouse button depressed move the mouse to enlarge or diminish the rectangle to set the size of the grid frames If the Use starting point
72. arrai e a a a a e a aia eS 255 ROB SO eea a N A E A use dadueenas Gastetoae 258 Spectral UNM X N assnssnnnnnsnnnnnnnnnnnnnrnnnnnnnnnnnnnrnnnnnnnnnnnnnennnnnnnnnnnnnnn nnmnnn 275 Interactive Image Measurement cccscceeeceeeeseeeeeeeeeseneeeeseeneeeesenees 280 Save Load and Edit Measurement Results 2 0 0 0 eecccccceeeceeeeeeeeseeeseeeeeeeeaeeees 282 Create measurement sheets ccccecccecccsceceeeceeeceecceeeceeecaeeceeeeeesaeeeseeeseeessenes 284 Using Stats ics FUNCIONS mirositor ena E a accu A Aei aa e TEER 286 Measuring Arbitrary Structures cccccccccccseecceeeceeeeeeeeeeeeseeeseeeseeeeseeesaeeseeeeaeeeaes 288 THE WEASUIE MENU crunn a a 292 PKO WV AUS aaien E 1Saasebaandatusmeebuaieeueanracencn 292 LIES Cele 1 72 ea cee nen serra Cerne a a a E eee Oe eee ee eet ee 292 POMAD e Sits lt er alata cone ts ade ells anit E tenatiommetommaun 292 Gda ices tanh screech a cle fesse olecs vasedulmode cast 294 ensiy Prole 22 205 222 eacechenssacsaaneasen tenet E n E are AT E A RE A 295 E A E S E E Gok ot ae cs eerane aes 297 KNE HIG reati a a a a a a AE S 298 Phase COlOn COGING reeni i e a a a a A iA 300 Prase ANAIVSIS one eia a S 301 Define Classifications sesser ieena A e aeaa A eona eNi 302 B EEE E E E A EA E E E ye va E E N E EA E E tetas 304 Contents AS GS faa eras sat catat scatter aaigae coe ine nat sae eee ataatccee eoaaeee tale ce eee asians aes nee 305 Graph Sesietcseteaieesscarsctiassenccic
73. bar click the Graph button 7 Define a rectangular graph object on the report page The size of the graph object determines the sizes of the displayed graph You may change the size and position of the graph any time gt If more than one graph has been loaded the Available Graphs dialog box will list all of them Select the desired graph from the list and confirm with OK 8 Use the File gt Report gt Save As command to save the report Use the File gt Print command to print out the report Graph Information Use this dialog box to obtain or enter detailed information on the active graph The Graph Information dialog box contains additional information that is saved along with the graph The information includes calibration data the name and your comments on the graph 312 Graph Markers and Labels Doubleclick on any graph buffer within the Graph Manager to view information on that graph you can also alter this information Graph Information x General Title Line profile 1 of Histochemistry Graph data Date 02 05 04 Graph start 0 0 SON Graph end 706 56 Ume 16 30 Channel width 0 345 User Graph range 06 56 Components 1 Comment Channels 2045 Data type Version 4 13 1616 m gRs Y anis Title x Coord Title Gray Value Unit fum Unit Abbrechen Hilfe In the Title field a name for the currently active graph buffer is proposed If you enter a new title into t
74. be represented in the gallery by a schematic folder icon 5 Click on the newly set up project in the tree structure gt In the database window you will find a form view on lower right You will now find information regarding the new project here The form shows all database fields which you have defined in the Arrange Fields dialog box for Form View and the Project data type 73 How to create a new database folder Projects2004_apl Archiving Images Create a new database folder Jol ES el lela EE Bala 7 wales alele Projects2004 No records available File Name Projects2004 Path F 4Projects20044 Projects2004 apl Created 07 12 2003 14 38 44 Last access 01 12 2003 Modified 07 12 2003 1 4 38 52 Onanad auchisively yer na Set Beraut User Feld elves er ANSTS BKK Goto Record Images DA Projects2004 Load Diocunient mages _ 0 0 kE Z lined with Documents mage Kie Arrange Fields aata Conn Table Daeuner its a Wacanen Re Settings VEn Ebon Ba Define Context Menu Organizational ID for describing the project Insertion mask when creating a new project folder Fields 1 2 The project name can be created automatically by the system Projects2004 apl 3 project 1 Emal ele aea Projects2004 yect e O04 ap Field value File Name Projects2004 Path F 4Projects2004 Projects2004 apl Created 01 12 2003 14 46 50 Last access
75. before it is saved as an image and you can also trigger other devices here For example a noisy image source can be aver aged to reduce the noise The same logical channel could trigger the selection of a filter wheel or select a microscope magnification 111 Acquiring images Set Input An input channel may contain macro commands These commands can for example define different procedures before and after the acquisition For example it is possible to select a microscope magnification or a special filter automatically before an exposure Your program supports up to 256 logical input channels Input channels belonging to cameras that are currently not available are not visible However they are not deleted Therefore that you may not be able to add a new input channel although the list contains less than 256 entries ali Set Input aj Colorview II FW BW Colorview Il FW BW lt Y Call Each entry in the Set Input dialog box contains three information elements e The camera icon e The logical channel name e The description To change the order of channels in the Set Input dialog box first select the channel entry you wish to reposition within the list Then position the mouse cursor do not leftclick yet precisely between the two entries where you wish to have the selected entry moved to When the mouse cursor changes shape then you can leftclick The selected channel entry will be inserted there Click the New Channel
76. blank current page number total number of exis tent report pages Header The header of the report window shows the The header of the template window shows the name of the report If the report has not name of the report template along with the been saved yet it will have a standard name name of the active page template which is Report consecutive number yee Report container src Template container stc Report srd Report template srt Report template The Report Template button bar is a part of the report window It differentiates itself button bar in some functions from the button bar used for creating reports Please keep in mind that these buttons functions are not available as menu functions This is why this button bar should remain visible If you like you can use the Special gt Edit Button Bars command to show or hide the button bar or to add other frequently used buttons 55 Report Generator Creating saving new templates ajal 75 m h o L Finish Object Editing Edit Object Template Leave Page Templage Save Page Template Select Page Template New Page Template Save Report Template Set Zoom Redo Undo Background objects Page templates can include background objects Background objects that have been defined for a page template will appear on each report page created based on that page template Background objects cannot be edited on a report page This is why you can
77. by step PhaseAnalysis E E 23 E 24 BPE 1802 1708 48 2a aE 21 Insert and edit open sheets directly into a report 1 Load the sheet you want to insert into a report For example use the Measure gt Histogram command to create a sheet 2 Now maximize the sheet window To do so click the middle button in the upper right corner of the sheet in the document header This will enable you to have the greatest number of sheet cells inserted into the report 3 Activate the report window Select e g the report name located at the bottom of the Window menu in the list of files 4 Select the image object and click the Sheet button located in the Report Objects button bar and pull open the sheet object gt Should you have loaded numerous sheets the Available Sheets dialog box opens Select the desired sheet and confirm via OK gt Your image analysis program will insert all lines and columns of the measurement sheet into the report that are visible within the sheet window Cells that are without content will be left out 5 Click the Ungroup button in the Order and Group button bar to be able to edit the sheet within the report gt What to do if the button bar is not available Use the Special gt Edit Button Bars command To have a button bar displayed select the check box next to the name of that button bar gt Asheet that is inserted into a report is comprised of a group of text objec
78. compress the X scaling You may also change the graph s scaling by using the buttons on the Graphs button bar 307 Graph The Graph Window i Graphs 3 Line profile 1 of GFP TFP CRECER ts AIP os ffrectontme none ros Fe Line profile 1 of GFP YFP Gray Valle 6 0 Distance Scroll bars You may use the scroll bars located at the right side and at the bottom of the diagram to shift the graph along the axes without a change in the axis scale Graphs Button Bar The button bar at the top of the window is used to change the displayed X and Y range and to edit graph overlays X Scale The different buttons are combined into functional groups The first sets the scale for the X axis of the graph Zoom In Click this button to decrease the displayed X range i e to stretch the graph in the X direction X direction To scale the Y range up or down keep the corresponding button pressed The command will be repeated automatically at short intervals While working with these two scaling operations the initial X value remains unchanged Scale X Click this button to enlarge any X segment of the displayed graph to full axis size In the desired graph click on the borders of the desired X value segment X range The first clicking produces a blue line cursor the second a green one Both diagram cursors can be moved with the depressed mouse button The current X position is continuously shown on the status bar Rightclick
79. dialog box 5 Generate a new report based on the Normal template 6 Insert at least one image object To do so click the Image button located in the Report Objects button bar 7 Pull an image via drag amp drop right out of the image manager onto the image object within the report gt The image will be inserted into the report and shown in the report window Image Manager Fal Report OF x Ey ey MB te cep A cee memm a ee Sic fe Dest Se A ae ners BL Beez x X Be ia Ca i i 2re 300 x2 5 M sox 1024124 Jetl SS 6 E 500x600x8 D Fem mia yu commen on ha rege hee 7 E shia BOO x g 0 M ones 2048 xe 9 scree o E 37 er0x24 Catalyst 11 OW pean inzaxz4 ee List lal G 4 G M Position 0 0 0 0 mm Size 21 4 36 Report Generator Report objects Note o Changing the print size of an image 1 In order to display an image at a different magnification level select the image object by leftclicking on it 2 Click the Properties button in the Report Objects button bar to alter any of several image object properties especially print size 3 Click the Image Properties tab This is where properties specifically for image objects are made available You can alter the image properties that have been proposed within the report template for individual images within a report at any time e The Fit image into the frame option located in the Magnification gr
80. display mode is part of the image information If you save the image its current display mode is saved as well LUT Name of the LUT Uses a color LUT for the single color channel display Use the Image gt Adjust Color gt Load LUT command to select the LUT to be used for display Fluorescence Color Uses wavelength LUT for the single color channel display Use the Edit Fluorescence Color command to modify the color information to be used for the display To open this dialog box load a multi channel image To open the image information simply doubleclick the image in the image buffer On the Image Information gt Dimen sions tab doubleclick the color field next to a fluorescence 19 Note Button Bars The Stack Navigator button bar Load LUT Use the Load LUT command to set the LUT to be used for display in the False Color mode The normal entry dialog box will open In it you can select one of the numerous predefined LUTs Ge RGB Lookup Tables of Color Chann fel Fa Cyan T a Cold armH ot Help E Click the Load button in this dialog box to load the selected LUT The application automatically switches to the False Color display mode Overlay Transmission Activates and deactivates the overlay mode The selective fluorescence labeling of sub cellular structures often creates an image in which a particular cell is no longer visible To enable you to see where the fluores cent structures
81. example with four differently focused single frames The four object planes are each represented by a continuous line All parts of the spec imen are sharply focused in at least one of the single frames depths of focus of the 4 images comprising the focus series depth of focus of the computed composite image The 3 methods for calculating an EFI image Depending on the software version you use there are three possible ways of calcu lating an EFI image 1 Calculation from an image series EF Calculate command use the standard method when you already have a focus series of images These images must have been saved in successive image buffers so that EFI can recognize them as a successional image series 2 The accumulative method EF Acquisition command use the accumulative method when you want to have the EFI image calculated from a sequence of live images The EFI image will be continually newly 129 Acquiring images Extended Focal Imaging acquired while you change your microscope s focus setting by hand With this method EFI will have the current live image appear simultaneously with the preliminary EFI image on your monitor If the live image contains areas that are in better focus than in the preliminary EFI image these will be adopted by the latter 3 The fully automated method EF Focus control command use the fully automated method when EFI is to acquire the focus series accordi
82. fined fluorochrome into the Dye list If this is not done your entries will not be adopted by the program Click the Delete button in order to delete the selected fluorochrome from the Dye list Enter the names of the fluorochromes into the Dye field A Dye 1 Dye 2 term is preset when you define a new fluorochrome Enter the excitation wavelength of the fluorochrome in nm into the Excitation maximum field Enter the emission wavelength of the fluorochrome in nm in the Emission maximum field The Color field shows the defined color of a fluorochrome The color of a fluoro chrome is the color in which it is represented in the composed image You can alter this color in two different ways Click on a color field to select one of the seven colors on option or on the More Colors button This opens the Choose Color dialog box in which you can define any color for the fluorochrome When composing the fluorescence images you can also change the color represen tation of the individual fluorochromes Note You may not allocate a color to a contrast image fluorochrome lt Brightfield gt lt DIC gt or lt Phase Contrast gt so that you can use it as an overlay image 118 Acquiring images Multiple Fluorescences Define Fluorescences Defines the acquisition parameters objective and filter together with the target for a fluorochrome Define Fluorescences Me Taget Fluorochro Cu Mirror cube
83. from the Labels picklist 314 Graph Markers and Labels Delete All Copy Hinweis Click on the Delete button to delete the selected label Click on the Delete al button to delete all labels defined in the active graph In the Label text field enter the text for the annotations The amount of text is limited to 116 signs Click the Set button to position the text frame in the graph document Note that the text frame can only positioned in the visible part of the graph You cannot scroll or zoom the graph while you set the labels Select the Anchor text check box to position the text frame at a fixed position of the graph document The position of the text frame does not depend on the currently displayed part or zoom factor of the graph Use this option for general descriptive text referring to the whole graph Clear the Anchor text check box to position the text frame at a special position of the graph When you shift the graph or change the zoom factor the position of the text frame will change accordingly Use the Centered text check box to format the text in the text frame When the dialog box is opened the text frame is indicated in the graph document by a dashed rect angle Clear the Centered text check box to left align the text in the text frame Select the check box to center the text Use the features in the Relative position group to add a label to a special X position of the graph In the Position field enter the
84. green blue in the Gamma group The new color values for each pixel will be calculated using the 3 gamma corrections The resulting image will also be a true color image and will be written into the destination image buffer Enter the gamma values into the Red Green and Blue fields for the respective color components Values from 0 01 to 10 are available Note To correct image contrast first select the same gamma value for each of the 3 color components This way you can emphasize e g the contrast of a dark background without changing the whole image s coloring If you use varying gamma values you will be shifting the image s coloring in the resulting image This can of course be used to correct color tinge s Click the File button to open the standard dialog box for the opening and loading of files Files are saved using the GA3 format Select the Preview check box to be able to view any changes to the original image resulting from alterations in the gamma values A red preview window will appear within the image window showing you any results from gamma corrections Click the Window button to define the preview window The window can be moved around enlarged or decreased using the left mouse button just like a frame White Balance Balances the true color image s colors to white Many cameras tend to adulterate falsify image colors at acquisition This kind of color displacement can be corrected retroact
85. gt is preset If you select this option those colors from the defined colors of the respective palette are used that come closest to the colors of the original image Dithering involves taking the pixels of the defined palette colors and placing them alongside one another so that to the eye of the observer a mixed color or color blend is the result This means that in many cases a more realistic representation of color transitions becomes viable The Colors in input image field indicates the number of colors contained in the orig inal image When working with a true color image this involves all occurring colors of all pixels When dealing with a false color image these are all colors being used by the current LUT Click OK to have the image currently being shown in the preview window written into the destination image buffer The dialog box will be closed The preview window will also be closed The image document window will be re maximized and will show the resulting image Transform Image Applies various affine transformations on the image From the Mode list choose the transformation desired The parameters in the group will change their function correspondingly The following transformations are avail able Shear Rotation Decompression Size and Translation You can apply several transformations successively without having to close the dialog box Adjust the relevant parameter s and the transformation will then be executed
86. have the height information at its disposal due to the uncontrolled manual focus changes that are made 130 Acquiring images Extended Focal Imaging 3D presentation You can have the height map presented perspectively You can create an especially realistic topographic impression of the sample if you use the EFI image that has been calculated as texture for the 3D model A three dimensional reconstruction that you have created in this manner can be rotated and viewed from every side To do this use the Process gt 3D Surface gt Create command EFI Options Defines settings relevant to computations you will be making with EFI later EFI Options Stack General OF IY Align Neighborhood LK Cancel WE Help W Accelerated The Stack group is only relevant to EFI computation methods used on focus series of images that have been saved as a stack This is true for both the standard and the fully automated methods Before EFI image computation select the Align check box if you wish to have indi vidual images of a focus series brought into alignment with one another This is needed for example when acquiring images via stereo microscope and lateral displacement has to be compensated for Another situation requiring compensation is when a specimen begins to drift noticeably during image acquisition If you ve selected the Align check box the Accelerated check box will become avail able Select
87. have the optimal Z Spacing calculated The value of the stop position may then be slightly reduced This assures the acquisition of a Z stack without any redundant data concerning the Z position Please note that you can only use image stacks with an optimal Z Spacing if you want to make a 3D recon struction e g with the VoxelViewer add in or if you want to employ deconvolution 2nd Howto The Z range will be determined by a Reference Position 1 If necessary open the Acquire Z Stack dialog box The live mode will be acti vated 2 Clear the Use absolute range check box 3 To define the start position focus the specimen and click the Reference button to adopt the current stage position 4 Enter the distance to be traveled in each direction from the reference position in um 126 Acquiring images Acquire Z stack Alternatively change the focus position on the microscope by turning the wheel in one direction until you reach the position at which you want to make the first acquisition then click the button When you have done that change the focus position in the opposite direction and click the button gt After every change the Z Distance field will show the complete range that the stage will move through 5 In the Z Resolution group define the number of images to be acquired in the Frames field 6 Click the Acquire button to start the acquisition This is how you acquire a multi channel Z stack
88. image to a 16 bit image and multiplies the gray values with 256 An 8 bit gray value image is converted into a 16 bit gray value image Gray values remain unchanged They will not be spread to the 16 bit value range An 8 bit false color image is converted into a 16 bit gray value image To begin with the false color image will be converted into an 8 bit gray value image via applying a linear standard lookup table LUT The current LUT of this false color image will be ignored and the image s actual gray values will be displayed This operation corre sponds to the Image gt Image Display gt Set Gray LUT command From here the image will be converted into a 16 bit image its gray values remain untouched A binary image is converted into a 16 bit gray value image Gray values remain unchanged i e black image areas retain a gray value of 0 in the 16 bit image and white ones the gray value 255 Converts an 8 bit gray value or false color image or a binary image into a 24 bit true color image An 8 bit gray value image is converted into a 24 bit true color image The three color components R G and B will be assigned to corresponding gray values i e color components are equal to one another R G B and also equal to gray values An 8 bit false color image is converted into a 24 bit true color image The three color components are given the values in the LUT lookup table of the image being converted The resulting true co
89. intensity values of a true color image to a gray value image The values of the intensity component I 0 255 will be assigned to the gray values 0 255 Extracts a subset of data out of a multi dimensional data set This command opens the Extract dialog box Use the Extract dialog box to define the color channels and the range from to of Z Layers and or time points t Frames Only the images defined here are extracted By setting a step size higher than 1 you can opt to extract only every 2nd 3rd and so forth layer or frame Dimensions ROIs Color Channels From EE to Em step Hm All TittG Frames Fron oF co DHE step i 6 All copy non processed data Cancel Apply Help Combining fluorescences Creates a multi dimensional data set from several single images Your image analysis software allows you to combine individual data sets within the color time and Z dimensions to generate larger data sets Prerequisite for such an operation is that the data sets have the same size in the two other dimensions and in x and y width and height For example it is possible to combine two full frame time sequences of 50 images each into one sequence of 100 images as long as both contain the same number of color channels Another example would be two mono chrome time lapse images that are each made up of 50 frames Every time lapse image was acquired with another color channel In this case you can create a
90. left to shorten it The exposure times for live acquisitions 113 Acquiring images Multiple Fluorescences and snapshots are equally altered during correction as long as the exposure time for snapshots is lower than 125 ms The exposure time for the live acquisition remains unchanged for exposure times exceeding 125 ms Doubleclick on 0 below the Expo sure time correction slide control in order to use the automatically calculated expo sure time values once again Note Note Moving the control to the left only leads to a hardly visible change in bright ness in the live image This is due to the automatic gain display function which is always enabled when using intelligent Exposure Exposure Lock Click the Exposure Lock button to lock the current exposure time for intelligent Expo sure As of now no more alterations will be made to the exposure time Click the button anew to return to the dynamic adjustment of the exposure time Preview Click the Preview button to use the setting for snapshots with live images as well Now the resolution and exposure time for snapshots will be used Click the button anew to once again use the settings for the live acquisition White Balance Click the White Balance button to carry out a special white balance when using intel ligent Exposure In the image document move the ROI s red rectangle to a white or uniformly gray position on the specimen Change the size of the ROI by keeping the right mo
91. list contains predefined classification schemes such as Sample Area or Sample Form Factor It also contains your user defined schemes All classification schemes can be edited as needed or simply consulted as sample schemes The Unit field displays the unit of the current classification scheme The unit is set in the Set Unit dialog box The Classification parameters group contains information on the current classifica tion scheme The Minimum and Maximum fields display the upper and lower limits of the class distribution The Bins field indicates the number of classes The Mode field indicates which function has been used for determining the range of values for the different classes The two possible entries are either Linear or Logarithmic 302 The Measure menu Define Classification This group is only available when you have had the classification scheme computed and the sheets in the dialog box have not been edited by you at a later point The New classification field is for the name of the new classification scheme you wish to define The New button will not be available until you have entered a new name Select the Show sample objects check box to have standard lengths and circular areas of varying sizes displayed in the overlay of the image in the active image buffer These standard sizes will be displayed in the unit of image calibration Class divi sions i e classifications can be more easily estimated using these st
92. loading of files Here s where you can save and or load color threshold settings The Undo and Redo buttons can be used to reproduce all previously set threshold settings as long as this dialog box is open These buttons refer to the settings located in the RGB and HSI tabs Click the Undo button to undo a previously set threshold setting one step at a time The Redo button will only become available once you ve undone a threshold setting Click this button to reproduce a threshold setting you had undone HSI Sets thresholds based on the HSI model Set Color Thresholds x RGE HSI Preview B Phase Hue 258 8 254 5 a One Color Jak oR 255i Curent New Include Exclude Ok Cancel The RGB and HSI tabs both have the same structure Below is a description of only those controls that have a different function than in the RGB tab Every color is made up of the color value itself saturation and intensity To define a phase you have to enter a range between 0 and 255 for both saturation and inten sity For Hue you can enter values between 0 and 360 steps of 0 5 are accept able The Hue Sat and Int fields indicate the upper and lower limits for the respec 218 The Process menu Binarize Binarize Multidimensional images Binarize Why binarize images tive hue saturation and intensity channels The easiest way to define color
93. menu RGB Studio Fill Border Generates a new image by filling the image areas which surround the shapes with a particular color If you wish to reposition the particles before you fill the surrounding area then press Shift when you select this command The Select Fill Color dialog box will be opened This is where you select the color or gray value for the image background This dialog box varies depending on the image type Select the desired color or gray value Click on OK A new image will be created in the destination image buffer All pixels that are not part of the shapes will be assigned the color selected The shapes will reappear in the overlay Create Mask Creates a binary image from the shape or its background SHIFT key The binary image will be created in the destination image buffer Display Shape Draws the active shapes into the overlay 273 Spectral Unmixing Spectral Unmixing Spectral Unmixing General Information Absorption and emis sion spectra of eGFP and eYFP Now that the genetic make up of more and more species has been deciphered modern biology is now confronted by the next challenge the exploration of the func tional significance of protein and its role in physiology and what influence it has on diseases The development of computerized microscopes capable of acquiring spectral infor mation together with the development of new life cell fluorescence dyes and new color variants of f
94. method is a rectangle This is how you define a ROI with the Rectangle method 1 Click the Rectangle button located in the Tools group The mouse pointer switches to the image A rectangle is displayed in the over lay 2 Move the mouse to move the rectangle at the desired position 3 Move the mouse while pressing the left mouse button to change the rectangle s size 4 Click the right mouse button to end the definition of the rectangle A closed region has been defined on the image You return to the dialog box The new ROI is immediately added to the Active ROIs list Rotated rectangle A ROI defined by this drawing method is a rectangle of arbitrary orientation This is how you define a ROI with the Rotated rectangle method 1 Click the Rotated rectangle button located in the Tools group The mouse pointer switches to the image A rectangle is displayed in the over lay 2 Click the Rotated rectangle button 3 Pull the rectangle to the desired size by depressing the left mouse button Then position it by moving the mouse 4 Click once using the left mouse button to switch to the rotation mode Rotate the rectangle by moving the mouse If necessary switch to the translation mode to move the object 5 Click the right mouse button to end the definition of the rectangle A closed region has been defined on the image You return to the dialog box The new ROI is immediately added to the Active ROIs list Virtual Combines two RO
95. move the red and blue lines representing the higher and lower thresholds to where you want them on the histogram The values in the High and Low fields will reflect the current position of these lines even as you move the lines 212 The Process menu Set Thresholds Click the Include Pixel button to expand the active phase s gray value range Once you have clicked on this button you will be able to use the mouse cursor to adjust the phase thresholds onscreen When you then move the mouse cursor to a position where the gray value is beyond one of the active phase s thresholds High or Low the relevant threshold will be adjusted to that at the mouse cursor s position The preview will continually be updated to display your adjustments The zoom function i e the buttons showing a lens with a and a sign respectively is for spreading a histogram on the x axis or reversing this spreading Zooming is essential when working with 16 bit images because any gray value range of interest is often quite small in comparison to the total number of possible gray values Possible spread factors for 8 bit images are 2x and 4x 16 bit images can in addition be spread to 8x 16x 32x 64x 128x and 256x The current spread factor is displayed on the left of the dialog box next to the scroll bar Use the X scroll bar to spread a histogram on the x axis The Diagram list has 4 different options you can choose from Histogram Smoothing 1s
96. multi channel time lapse image 187 The Image menu Combining fluorescences Before you use this command activate one of the images that you wish to combine into a multi channel image The appearance and functionality of the dialog box depend on what type the image is Multi dimensional images such as time lapse images or Z stacks will open a different dialog box to 8 bit or 16 bit standard gray value images You can combine two or more multi dimensional images into one image Combine Mode Feasible image objects Color Channels el f Time Sort according to M real time 2 position Use the Combine Mode group to determine here in which dimension the combination is to be performed Feasible image objects field All loaded data sets with the same frame size as the active data set and the same size in the two dimension that are not chosen in the Combine Mode group are listed here For example sequences with different number of images cannot be combined into a series of Z stacks By default no data set is selected The data sets to be combined into a multi dimen sional image with the active data set have to be selected by mouse click To select an arbitrary number of options simultaneously keep the Ctrl key depressed To select options that are lined up one after the other keep the Shift key depressed In the Feasible image objects list the images are listed in the order that they were lo
97. no image information or image distortion in the image border Normally the logical input channel is used to cut off these irrelevant image borders during the acquisition Use this command to correct the artifacts after acqui sition Clipping B order Pixel 150 Acquiring images Multiple Image Alignment Note For an automatic alignment it is necessary to clip off artifacts This is because image artifacts caused by the camera are fixed at a certain image position Pattern recog nition cannot distinguish between artifacts and real image structures and will possibly assemble the images in such a way that the artifacts overlap instead of equivalent image patterns In case the imaging system in use causes systematic image artifacts you should use the Acquire gt Camera Configuration Configure Input gt Format tab to correct the arti facts during image acquisition A clipping frame will appear within the image in the active image buffer It will be auto matically computed in such a way that all of the undesired single color edges remain outside of the frame The only lines or columns along the borders of the image which will be located are those in which the entire line or column has the same gray or color value The dialog box will show the excluded lines and columns accordingly In the Clipping Border Pixel group you enter the number of rows Top Bottom and columns Left Right that are to be cut The clipping frame with
98. no preceding or overlapping structures are located within the fitting area Furthermore switch to a linear graph presentation the background subtraction func tion does not work for a logarithmic graph presentation As soon as the command has been confirmed the graph will be fitted and extrapo lated to the left hand side of the specific structure The X range of the graph that has been used to determine the background will be defined by an upper and a lower reference The proposed lower reference is the lowest X value that is displayed in the graph document Background Subtraction x Assumed function Power ad Y Z26007 sx 221E 00 Reference Lower f r Set Lower Upper E r Set Upper The calculated background curve and the lower and upper references will be shown in the graph document Use the commands in the dialog box to adjust the references and to set the back ground fitting method Choose a suitable fitting method from the Assumed function list After a new fitting has been chosen the background will be automatically recalculated from the graph values and displayed in the graph document The dialog box shows the fitting form and the computed fitting parameters for the current graph 322 Graph Filter Invert Absolute FFT Filter Envelope The following table supplies you with an overview of the available fitting methods Ip stands for the background intensity here Fit
99. of any shape To do this you mark a typical point within the object Your image analysis program then automatically searches for points in the area which have similar gray or color values An object which can be collected with a magic wand must be able to differentiate itself from the background based on its color or its gray value Magic wand Use the magic wand located in the measurement button bar to quickly and comfort w Examples of objects which can be easily 4 located with the magic wand KD 5 step by step This is how you define an object with the magic wand 1 Load the image you want to measure into the active image buffer 2 Select a zoom factor of 100 This is the best setting for the magic wand 3 Use the Alt 4 key stroke to switch to the measurements environment 4 Click the Magic Wand button located in the measurement button bar gt The pointer moves to the image window 5 Click on a typical color value in the object you would like to measure gt The point selected in the object is marked by a blue point in the overlay Your image analysis program immediately looks for the object and high lights it with a red lining in the overlay 288 Interactive Image Measurement Measuring Arbitrary Structures The pointer in the left image is located on the object s selected point Only a part of the object is found Keep the left mouse button depressed and pull the mouse to the right to select the entire
100. of pixels whose RGB or HSI values define a phase Areas of gray value ranges not assigned a phase will not be calcu lated Phase analysis will only be displayed on the image area within a frame if a frame has been set Phase analysis will only be applied to the image areas which lie under the white areas of a mask if a mask has been set A measurement sheet will be generated by this command which contains the abso lute areas of the gray value phases as well as the area of each phase relative distrib uted by percentage to either the total image area or the area within the active frame Sheet column headers contain phase name and lower and upper thresholds The column headers color corresponds to its respective phase Phase analysis of 301 The Measure menu Define Classification various gray value ranges will enable you to e g determine the surface area distrib uted by percentage of a particular material on a background Surface area can be calculated in true color images using selected color ranges Phase analysis can be applied to other images and or other areas within the same image using the same thresholds Measurements taken will be appended to the measurement sheet This insertion will take place automatically as long as you do not open the Set Thresholds dialog box between measurements As soon as you adjust the thresholds your image analysis program will generate a new sheet Conduct the measurement as done with th
101. on the header of the first column and use the Graph gt Measure gt Integration command afterwards The results are then appended to the active measurement sheet Enclosed Angle Measures the angle between two interactively chosen points Gradient Measures the gradient and offset between two interactively chosen points 320 Graph Calculation Point Value Measures the point value of an interactively chosen point The cursor jumps into the graph with a vertical line attached to it Move the cursor into the desired position and click once to evaluate it A label will be set to mark the position The name of the label is X1 X2 X3 Additionally a data sheet is opened for the point values In case of a multi graph one column is used for each single graph therein For each following measurement a new line is added to the data sheet To end the process of measuring point values rightclick once Select one column of the data sheet and use the Point Value command again to append data to the same sheet You may change the name of the labels using the Graph gt Markers and Labels gt Modify command You may change the headers of the sheet using the Edit gt Sheet gt Edit Column Header command Calculation 1 Derivation Calculates the first derivation Tst Difference Smoothing width El g0 Absolute derivation Cancel Help Enter the desired width of the range you want to use for calcula
102. polygonal figures desired within the image Rightclick to return to the Edit Lines dialog box Sharpen Performs a gradient filter for edge enhancement Filter Matrix 0 1 0 1 5 1 0 1 0 The Sharpen filter emphasizes image detail The image will thus seem more in focus The Sharpen filter does however enhance noise just as it does genuine image information The Sharpen filter differentiates the image with a particular emphasis on the central pixel on the X and Y axes The Sharpen filter is one of the derivative class of filters The central pixel emphasis is limited If you need stronger emphasis and thus stronger focussing then select the Sharpen II filter Sharpen Il Performs a gradient filter for pronounced edge enhancement Filter Matrix 1 1 1 1 9 1 1 1 1 The Sharpen II filter emphasizes image detail The image will thus seem more in focus The Sharpen II filter does however enhance noise just as it does genuine image information The Sharpen II filter differentiates the image with a particular emphasis on the central pixel on the X and Y axes and in the diagonals The Sharpen II filter is one of the derivative class of filters It emphasizes the central pixel very strongly resulting in a sharp focussing effect If you need merely a limited emphasis of the central pixel you d do better selecting the Sharpen filter Differentiate X Performs a gradient filter for horizontal edge enhancement Filte
103. polygonally 161 The Image menu Edit LUT sheet Polygon Formula Linear Polygon points Count b Define Color C Red Green Blue T Monochrome Within the diagram lines or polygons in the respective color represent the LUT s three primary colors The horizontal axis represents gray values from 0 255 The vertical axis represents LUT entries i e color intensities from O 255 A LUT polygon is defined by its points represented by small white squares These points are joined by lines There will always be at least two points at the left or right edge of the diagram that cannot be shifted horizontally To add a new point to the polygon simply click on any arbitrary point within the diagram If you move the mouse cursor over any point of a polygon the cursor will turn into a hand Then pixel value and corresponding intensity of this point will be displayed at the right edge of the diagram If you keep the left mouse key depressed you can alter the position of this polygon point All changes made in the diagram will be displayed on the monitor immediately A polygon point can be shifted between the two neighboring points to the right and left of the point in question This makes it easy to define a rectangular or saw tooth shaped polygon To delete any point of a polygon rightclick on it How to define a point of a polygon with greater precision keep the Shift key depressed while
104. priori N Variance check box to calculate N variance The Variance parameter will then be calculated for a previously known a priori mean not calculated from the values measured In this case the divisor N 1 in the formula is substituted by N Select the Color rows of result sheet check box to have statistical functions displayed in color in the result sheet A statistic sheet is generated by the Statistics command The Description field contains a brief description of the statistical parameter selected The Edit button is only available if you have selected one of the user defined statis tical functions of an Imaging C module from one of the two lists Available Current Click the Edit button to load the SFM source file of the relevant Imaging C module into the text editor Click OK to close the Define Statistics dialog box Now you can edit the source file Then select the Special gt C Module gt Module Manager command Select the module desired from either the Loaded or the Other lists Click on the Build button to compile the newly edited source file and to generate the newly executable SXU file Click on the Install button to open the Install Modules with Statistic Parameters dialog box and to register a module containing a user defined statistical parameter Creates a sheet with statistics about the currently selected sheet Use this command to have statistical parameters computed from measurements in one sheet col
105. remains depressed the line cursors will remain visible and can be moved again by using the mouse cursor Click this button to stretch the Y range automatically from the minimum to the maximum Y value of the displayed graph segment Click this button to automatically rescale the displayed Y range of the graph to the maximum Y value The scaling is automatically updated when moving the graph in the X direction by using the scroll bars of the graph window Click this button to zoom up a rectangular area of the current graph window Clicking the button opens the Define Display Area dialog box Click the Set button to draw a rectangle into the graph Use the mouse to resize and move the rectangle to the inter esting part of the graph Click the right mouse button to return to the Define Display Area dialog box Alternatively you may enter the absolute X and Y limits of the graph area in question into the fields of the dialog box Click the OK button to zoom the selected area of the graph to the whole graph window Click this button to rescale the graph and show the whole graph that the current graph buffer contains Click this button to change the X axis scale type from linear to logarithmic scale Click this button to change the Y axis scale from a linear to a logarithmic one Click this button to delete all text labels in the current graph When this button is clicked all graph processing operations that produce a new graph will affect only t
106. report pages numbered Your image analysis program will automatically fill in the correct page number when you e g delete a page located before the current page AutoText properties can be altered just like the properties of any other report object in the Report Object Properties dialog box In addition to the properties to which you can assign text objects you will find the Auto I ext tab where you can decide the type of AutoText and the way AutoTexts are updated Text variables In order to change a text object to a text variable you have to alter its name in the General tab This tab is located in the text object properties The name of a text vari able has to start with the symbol Anytime you alter the contents of a text variable your image analysis program will automatically update all other text variables in the report of the same name You can change the contents of a text variable as often as you like in order to e g correct spelling errors Text variables are usually defined in a report template so you can structure headers and footers This means you only have to enter a heading that is to appear on every report page once You can define as many text variables as you like in your report templates e g for headings author project title department You can however also define text variables on a report page within a report When you change the contents of a text variable within a report this chan
107. report template that reflects its contents into the File name field 6 Select the report template directory you determined in the Preferences dialog box in the Save in list Warning Your image analysis program will propose the current directory in the Save in list Be lt P sure that you save your report template in the correct directory If the report template is saved in another directory it will not be offered in the New Report dialog box 7 Click the Save button to save the report template Save button to have the report template in its previous form overwritten by the 8 If you wish to save the report template after having made changes click the most current version Object templates Background Information Object templates You can create your own templates for records and field objects These templates determine what your record or field objects look like and their make up Object templates are saved under a separate name within the active document You can then insert them into a report at any time You define object templates within a report template generally so that you can use them with all the reports you create based on this template It is not permissible to import object templates from other report templates Edit object Mode The edit object mode is for selecting and editing the separate elements of a record object you have selected All other report page elements are not available for editing and are only shown o
108. reserve space for a sheet is always empty Properties of sheet Selecting sheet cells your image analysis program will insert precisely those objects sheet cells of the measurement sheet that are visible within the sheet window This means you can alter the size of the window to clip lines and or columns Empty sheet cells are ignored at insertion You can make use of the autofilter functions located in the Edit menu in order to have specific kinds of data not shown Any filter you have set will be considered upon insertion into the report When moving a sheet from a database to a sheet object only the sheet cells are inserted which were displayed during the insertion of the sheet If you ex plicitly want to determine exactly the sheet cells which are to be inserted into a sheet define a sheet object within the record object e Formatting sheets a template cannot be defined for a sheet Each sheet you insert has to be formatted individually e Sheet object structure your image analysis program converts measurement sheets before insertion into a report into a complex object made up of numer ous separate text objects Every single sheet cell is in fact a separate text ob ject You can completely disassemble a sheet into its component parts in a re port for editing purposes e Editing sheets to be able to edit each sheet cell separately you have to break up the grouping ungroup Regroup each sheet after you have finished editi
109. results particularly if you often work with various image sizes This is how you set a frame 1 Select the Image gt Set Frame command A red rectangle will denote the current frame in the image overlay The mouse cursor is attached to the lower right hand corner of the frame 2 Press the left mouse button and pull the lower right hand corner of the frame until you reach the desired frame size 3 Move the frame to the desired position in the image 4 Click the right mouse button to set the frame You may change the color the frame is displayed during frame definition Use the Special gt Preferences gt View tab The frame color is determined in the Mouse cursor gt Primary color palette Red is the default An activated frame will always be displayed with a red and white outline Enable Frame Enables or disables the frame for image processing functions Note For image editing functions that effect multi dimensional images a frame is of no relevance The frame has to be defined using the Image gt Set Frame command Convert To 8 Bit Converts 16 bit image to an 8 bit image The conversion of a 16 bit gray value image into an 8 bit gray value image is done in the same way as it s displayed on the monitor 184 The Image menu Convert To 16 Bit 8 bit gray value images 8 bit false color images Binary images To 24 Bit 8 bit gray value images 8 bit false color images Binary images Converts 8 bit
110. selected during the transformation an interpolation of the desti nation point from the neighboring pixels in the environment of the source point will take place Interpolating really only makes sense and is in fact only possible for gray value and true color images it is not available for binary or false color images RGB Studio Apply Filters Should the current configuration of your image analysis program not offer you the Process gt RGB Studio menu you will need to take the following steps to activate this add in 1 Select the Special gt Add In Manager command 2 Inthe Available add ins list select the check box next to the RGB Studio option 3 Click the Close button 4 Exit the program then restart it gt After you have installed this add in the Process menu will additionally con tain the RGB Studio sub menu Use this command for processing true color images You can define a series of filters and then have them applied to the respective image Apply Filters x Filter List Cancel Hue Saturation Intensity Contrast Histo Contrast Sharpen Remove Noise The Filter List contains a series of various filter functions Select the filter desired The following three filters Histo Equalize Stretch Intensity and Invert are predefined For all other filters there are one or two filter parameters which will appear beneath the list in the Parameter group after you have selected a filter The
111. small height range The rest of the image is blurred The following is generally true The better the lateral resolution is the smaller your depth of focus will be That means we are confronted with a physical problem that cannot be overcome by making any adjustment to the optical system 128 Acquiring images Extended Focal Imaging What is the Depth of focus Points on a specimen located precisely at the microscope s object plane will appear sharply focused Points on a specimen located between two planes one slightly above the other slightly below the object plane will appear fairly sharply focused The distance between these two planes is the depth of focus depth of focus fairly focussed out of focus in focus N immage plane object plane 3 dimensional specimen Digital image processing offers us a way out of this problem The basic idea is quite simple One acquires a whole series of images one after the other of the same sample respectively sample segment While doing this one turns the microscope s focusing wheel a little further for each acquisition so that in every image another level of the sample is sharply focused Subsequently one only then needs to cut out the sharply focused parts of each single frame and then put these together just like a puzzle to form a composite image which is sharply focused all over In this way one can simulate an unlimited depth of focus An
112. successive image buffers Please note that the images have to be arranged in the order of the color channels of the color model desired Activate the image buffer which contains the first image Using the Separate Colors command you can have the color components comprising a true color image separated into color levels The individual gray value images can be edited and processed and then using the Merge Colors command recombined into a true color image A shape is an arbitrarily shaped area within an image Each image may contain several shapes Shapes can be copied from the image and inserted into another image Or you can clip out shapes and use them to generate a new image In addi tion you can fill these image areas or the remaining areas of the image with color Adds shapes to the image Add Shape Magic Wand Options Tolerance E 5 Fill holes Smoothing jo W Outline Color pace 2 i i s Cancel Help 267 The Process menu Shape Click the Undo button to delete the shape that was defined last Continue clicking on the button to successively delete shapes in the reverse order that you defined them This button is only available for shapes that you have currently defined via the dialog box Any shapes that you have defined and then closed the dialog box are not affected by the button Click OK to confirm the settings The defined shapes are indicated in the image overlay The comma
113. sure to use a zoom factor of 100 Click on OK to confirm the current overlay and to execute separation using the settings in the Define Separator gt Result group Click the Cancel button to return to the Define Separator dialog box Any alterations you ve made in the edit mode to the computed lines of separation will be lost when you click on Cancel Click the Erase All button to have all lines of separation erased from the overlay Click on the Set All button to reconstruct all lines of separation that you d erased since opening the Edit Lines dialog box All lines either computed by the separator and or drawn in by yourself interactively will reappear in the overlay Click the Erase button to erase individual lines of separation from the overlay You can define a circular image area interactively All lines of separation within this circle will then be deleted as well as all lines that intersect with the circle A line segment is defined as a line between two points of intersection Until you confirm deletion by rightclicking all deletion of outlines can be reversed one step at a time To do so press Shift and rightclick simultaneously 241 Filter The Process menu Filter Click the Trace button to select specific outlines Before using this function we would recommend you click the Erase All button Click on the Polygon button to be able to add missing outlines freehand or free mouse Draw the arbitrary
114. the Acquire gt Acquire Fluorescence Images button in the Acquisition button bar The messages will conduct you through the acquisition In the preview mode you have the possibility to focus the images for each fluorescence individually by using your microscope s Z drive Only with the motorized microscopes of the BX51 61 type or the IX71 81 type will the complete acquisition run automatically 4 Use the File gt Save command to save the fluorescence image You should save the fluorescence image in the TIF image format Define Fluorochromes Defines fluorochromes for the acquisition and editing of fluorescence images and to set their subsequent color representation Define Fluorochromes Mil OL Thiadicarbocyanine 651 67 OC Thiazole Orange 510 530 Cancel OL T0 FRO 514 533 OL T0 FRO 3 642 660 select all O T0 PRO 5 748 768 OO TOTO 509 533 _ Deselect al O toT0 3 B42 GED Default OU ted 490 695 O14 wild Type 396 508 396 508 Help O14 wild Type 475 503 475 503 DO ww 721 605 639 Fluorochrome Dye TRTE Search Excitation masiman 555 rrr New Emission masimum 5850 B nm Apply Color a Delete The term fluorochrome describes a certain colorant used for fluorescence micros copy A fluorochrome must be defined or selected in the first step so as to be avail able for future work In the Dye list are the names of all predefined and user defined fluorochromes with the appropriate excitation Ex
115. the Channel first option to go to a defined Z position bring the first mirror cube into the light path and acquire an image of this fluorescence e g DAPI Subse quently the second mirror cube will be activated and the corresponding fluorescence image e g FITC acquired Only after images of all fluorescences have been acquired at a defined Z position will the stage move to the next Z position Here the first mirror cube will be brought into the light path again and an image of the fluorescence will be taken Every mirror cube will be brought into the light path several times with this option For this reason the complete acquisition will take longer than when you use the Z first option You should use this option when your sample bleaches quickly Extended Focal Imaging What is EFI With the commands in the Acquire gt Extended Focal Imaging EFI group you can acquire images with your microscope that have a virtually unlimited depth of focus To make this possible EFI calculates a composite image out of differently focused single frames Focus series that is sharply focused all over Too little depth of focus in microscope acquisitions is a well known problem Espe cially light microscope users often work with samples that cannot be reproduced as images that are sharply focused all over because their surfaces are too uneven The microscope s depth of focus is not sufficient to make it possible to sharply focus on more than a
116. the color channels before you can adjust any thresholds Leftclick on one of the phases in the color bar Keeping the left mouse button depressed you can move both thresholds simultaneously Use the buttons located in the Include pixel group to define color ranges within the image interactively Select a color range within the image in the color of your choice Your program will then take the pixels belonging to that part of the image and deter mine minimum and maximum color values Use the options located in the Preview group to see whether all the image structures you re interested in have been included If need be simply correct the thresholds by adjusting them directly in the histogram When you click the New button the color thresholds that had been set for the active phase will become obsolete New color thresholds will be defined within the image Use the mouse to define circular image area s containing the colors within the active phase All pixels located within the circular areas so defined will be the basis for color threshold calculation e Leftclick to have a circle appear around the mouse cursor e Keeping the left mouse button depressed you can alter the radius of this circle e Confirm the size and position of this circle by rightclicking e You can select several image areas in this way To return to the dialog box sim ply doubleclick using the right mouse button Click the Include button to add color values to the curr
117. the image background to match the image The background is the area of the image that contains no image information This background is usually displayed in white After you have clicked the button a red rectangle will be attached to the mouse cursor Move the rectangle to the area of the image with the gray value you want to use for coloring the back ground Rightclick to have the background colored in the rectangle s mean gray value Click on the Equalize button to adjust the gray values of the individual images to match one another This feature is especially useful if the images were acquired with different illuminations and as a consequence with a different mean gray value For equalizing the gray values the histogram of the different images is calculated for the overlap region The histogram of the second image is adjusted to the histogram of the first image i e the first image of the series determines the mean gray value of the composite image Any further change of a parameter in this dialog box e g the image positions or the color of the background is related to the original gray values so that the matching has to be performed once again The Equalize button is not available for true color images You should match the gray values after positioning the individual images because the matching takes a relatively long time if a lot of images are concerned Click on the OK button to confirm the current composite image The composit
118. the images is ignored and the images are justified side by side You can position the images manually after wards 0 85 has proved to be reasonable for light microscopic images If the images could not be aligned automatically for a certain prescribed quality you should vary the correlation to greater as well as to lower values Thumbnails of the image series are shown in the image area in the arrangement selected Use this preview to check the chosen arrangement method The numbers below the thumbnails refer to the image sequence in the image manager You can also use the image area to change the image arrangement by drag ging amp dropping Move the mouse onto an image When the mouse cursor becomes a hand symbol leftclick to pick up the image and drag it to the new image position The number below the image is connected to the image buffer not to the image posi tion Color images are displayed as gray value images Click the OK button to start the automatic image alignment The individual steps of the search are displayed in the status line If the quality value entered is not achieved a message will inform you of this 145 Acquiring images Arrange The Align Multiple After the automatic image alignment has been completed the Align Multiple Images Images dialog box dialog box will be opened in it you can check the resulting composite image and if necessary reposition any images manually Overlap Area Side by side
119. the images to be analyzed During the application you can acquire the images evaluate an existing series of images or read the images out of the image buffer block 251 The Process menu Define Processing Define Processing Image Input Processing Measurement Source of images File Ur DETARE Image manager DB Query DB Selection Test PKR Mas Mo of Iterations Test image E OF Cancel Help In the Image Source group you select the image source for the automatic process The image sources Snapshot Live acquisition Stage Image file series Image manager DB Query and DB Selection are available The appearance of this tab depends on the image source selected Select the Image manager image source to read in the images from the image buffer block After each image the system advances one image buffer If the next image buffer is empty the application is aborted even if the maximum number of images entered has still not been reached In the Max No of iterations field you enter the maximum number of images to be acquired processed and evaluated during the application The system will continue reading out new images from the selected image source until the maximum number of images has been reached In the Test image field you enter the number of the image for a test image Click the Test button to acquire a single image from the selected image source Select the St
120. the mathematical magnitude used to judge the similarity of two image patterns It is a positive real value in the range 0 to 1 The nearer the correlation is to 1 the better the congruence between both patterns From the results of the correla tion calculation the approximate position of the reference pattern in the search image can be found In the immediate neighborhood of the pattern that has been found a pattern recognition routine will be employed to pinpoint the exact position for the image alignment Should the reference pattern not be found in the search image with the required correlation the reference pattern will first be increased in size to 256x256 pixels then to 512x512 pixels The search area in the search image s border will also be corre spondingly enlarged Should the required correlation not be reached you will have to mount the images manually 139 Acquiring images Acquire Acquire Defines the parameter for image acquisition Note The acquisition functions require additional software that is not included in the stan dard packages Use this command to acquire an image series for an automatic image alignment After completing the acquisition you can compose an overview image by aligning the individual images automatically First of all your stage must be initialized Special gt Preferences gt Stage tab Calibrate the position of the camera with regard to the axes of the microscope stage using the
121. the ordered sequence is to replace the central pixel The pixel environment in the following example consists of 5 pixels arranged in the shape of a cross The rank filter then generates an ordered sequence and assigns the mean value of that sequence to each central pixel Gray values of Sorted sequence Gray values of the original image MaX q the filtered image 84 85 99 x gt 80 85 104 109 120 gt 80 109 120 72 104 155 Application of various smoothing filters Original NxN image Iterations 2 150 150 o Size 10 g 3 S T 100 100 q O O 50 50 0 0 Mean Median T gt 4 T gt 100 100 5 50 50 0 0 Rank Sigma Size 10 Size 10 Rank Order 50 o 150 150 o Sigma 10 5 3 S S T 100 100 q O 50 50 0 gt l 0 X Position X Position 227 The Process menu Define Filter The illustration shows a comparison of the effects of various filters used for smoothing purposes The original image consists of a simple rectangle of light shading upon a dark background The image has strong noise amplitude interfer ence In addition the image contains so called shot noise i e individual pixels have an intensity deviating strongly above and below the image s average gray value Each image has its intensity profile displayed superimposed on the rectangle within the image The Mean filter and the NXN filter broaden the structure of the rectangle The more powerful the smoothing effec
122. this latter check box if you want EFI to only take up 25 of the image area and not all of it As long as your images are of fairly clear structure you ll be getting the same top quality EFI results in a fraction of the computation time In the General group enter a value greater than 1 up to maximum 5 in the Neigh borhood field Then each time before the most sharply focused single frame is calcu lated the image noise will be suppressed This kind of averaging is convenient if you have an image containing extreme and random gray value variation giving the application the impression the image is in excellent focus when it really is not The greater the value you enter in this field the larger the neighborhood of surrounding pixels used in the EFI computation will be Select the Create height map check box when you wish to also have the height map issued at the same time as the EFI acquisition is made EFI Calculate When you ve already got a focus series prepared use this command to start up the standard method of EFI image computation Number of images EFl result in Buffer 20 EB Cancel IY Create height map Height map in butter jig is Help First individual images of a focus series must be stored in successive image buffers The first of these image buffers must be activated 131 Acquiring images EFI Acquisition After initiating the command the Compute image dialog box will appear
123. to be drawn Cyclically varied colors are used by the program However you can select a color from the Color list Click the button with the arrow besides the field to display a list with all available colors The Active ROIs list shows all of the ROIs that have currently been defined in the active image Number label if applicable and frame color are displayed for each ROI Use this list to select the ROI you want to work with Select the check box next to a ROI to have that ROI displayed in the image overlay 181 The Image menu Define ROIs dialog box Click the Draw Particles button to have all of the selected particles drawn into the image overlay Click the Delete Al button to have all of the ROI that are currently defined deleted Click the Load button to load ROIs that have been saved out of a file Click the Save button to save your defined ROls for later use with similar images Tools These buttons provide you with tools for drawing and managing move combine and specifically delete ROIs The following drawing methods are available Polygon Interpolating polygon Freehand polygon Ellipse Rectangle and Rotated rectangle Polygon The outline of a ROI defined by the Polygon drawing method is a polyline made of successive segments This is how you define a ROI with the Polygon method 1 Click the Polygon button located in the Tools group The mouse pointer switches to the image 2 Click the left mouse button
124. to be detected at high acquisition rates or if spatial resolution is of minor importance The Binning group is only available for cameras with a binning function Enter the number of adjacent pixels to be integrated into the Horizontal and Vertical fields Enter a value of 1 to not use the binning option If you want 2 adjacent image lines or columns integrated then enter a value of 2 Some cameras will not allow you to adjust the Horizontal and Vertical values inde pendently If this is the case when you alter one of the two values the other will be adjusted accordingly Enter the line and column numbers to be clipped and the pages they re located on into the Clipping border Pixel group The corresponding format will appear in the Image format field A graphic of this format will be displayed in the Active area field The result will then be added to the Image format list Enter the line and column numbers to be clipped into the Left Right Top and Bottom fields Click the Interactive button to be able to define borders within the image directly This button is only available if the active image buffer contains an image with the same resolution as the logical input A red frame will then appear Frame size can be enlarged or reduced by depressing the left mouse button Position the frame by moving the mouse around Confirm frame size and position by rightclicking The image area remaining outside the frame will then be clipped
125. to destination image buffer 256 The Process menu Image Geometry Crop Properties Dimensions i Vertical FFH Horizontal pas Diagonal Diagonal o OF Cancel Help The Mirror group is where you determine which axis is to be used for mirroring the original image Click the Mirror button to initiate the mirroring process Crops an image by selecting a ROI The cursor jumps into the image with a rectangle attached to it Resize the rectangle by moving the mouse with the button depressed Move the rectangle by moving the mouse Rightclick once to fix the rectangle and cut out the parts of the image which lie outside of the frame Correct Aspect Fits the image aspect to that of the image display This command is for displaying images that have non square pixels via the correct aspect ratio Some detectors provide you with images that have a pixel aspect ratio X Y ratio that is not equal to 1 When loading these images they may appear distorted Using the Correct Aspect command the program computes a new image that has an X Y ratio of 1 The number of pixels in the X and Y directions will have changed Therefore the size of the image will have changed as well 257 Align The Process menu Align Aligns source 1 with source 2 and writes the results in the destination image buffer When the Special gt Preferences gt Image gt Apply interpolation after Align function check box has been
126. txt SKB Sheet1 sfs SKB Command txt SKB Text1 txt SKB Spheres tif 260KB 16 Button Bars Button Bars Button Bars Some functions are only available via the standard button bars This chapter describes the most important button bars The Standard button bar GHA Hal S 3 3 Sel Creates a new text document B Loads a file from disk Di Saves the active document to disk Print Directly Prints the active document with the current print settings Image Information Displays an overview of all image data see also on page 194 Protect Image Toggles the read only mode for the active image buffer on off O i ae xo lt lt D mmama Copies the active image or selection to the clipboard Paste Pastes the contents of the clipboard ion button bar TR A 4 a a SS ire Starts continuous acquisition using active input device Stops continuous acquisition or acquires single image Camera Control j Adjusts the camera parameters For more information on the camera parame ters click on the help button in the dialog box gt D gt O Q O mri Define Fluorescence Defines the acquisition of fluorescence images see also on page 120 Acquisition Intelligent Exposure Starts up the automatic mode of the camera see also on page 113 Acquire Z stack 7 Acquires an image stack see also on page 125 Acquire Image Acquires image sequences see also on page 134 o ieunes E
127. unit adjustment does not convert into the following units cm and ngstr m Scale Bar Properties Size Defines the font size of the scale bar label for the different purposes Scale Bar Properties x Display Format Size Clipboard Burn image W Adjust to printer frame width e0 E om Height Eo is cm Font size le fF paint Overlay Print Font size fe B5 point Cancel Apply Help Select the size of scale bar lettering in the Clipboard Burn image group for the following application areas clipboard copying and burning Select the Adjust to printer frame check box to select a constant lettering size for both clipboard copying for printing and for burning into the overlay Lettering size is computed such that it corresponds to the set image size Use this function when you wish to export images along with their scale bars into another application program via clipboard such as MS Word Scale bar sizes will then be adjusted to actual print size This assumes of course that the print size is entered correctly Enter print size of the image into the Width or the Height field These fields are only available when the Adjust to printer frame check box has been selected Enter the desired size of scale bar lettering i e font size into the Font size field this applies to the lettering of all scale bars Your image analysis program uses the font size selected when printing images and
128. view Use the Superordi nate record button in this view to move within the database You will find the buttons in the database window button bar 2 Select one of the views offered and confirm by clicking OK How to configure and open the info window 1 Select the Database gt View gt Arrange Fields command 2 Unfold the Info Window branch in the tree structure 3 Mark the Image data type 4 In the Available list mark the user fields that you want to display in the info window and click on the Add gt gt button e Remember that you can only display a maximum of seven database fields o in the info window e Remember that only one line will be displayed per field Database fields of the Memo type are therefore of limited suitability for the info window 5 Close the Arrange Fields dialog box by clicking OK e 6 In the gallery view click on the info symbol on the lower right in the thumbnail gt The info window opens It shows your selection of seven fields 7 In the info window click on the Keep visible button with a free notice board pin I ial to fix the window on the database document gt The button now shows a sunken notice board pin e Now you can click on any thumbnails in succession in order to look at the selected fields in the info window 8 Click on the symbol with the cross on the upper right to close the info window 89 Archiving Images Find data Find data Background Information
129. when images are copied into the clip board The font size selected is irrelevant to monitor display The font selected will only be considered if the Adjust to printer frame check box has been selected Otherwise the scale bar will be burnt into the overlay the same size as it would have when shown within a viewport at 100 zoom Font size is an absolute value i e scale bar lettering will always be the same abso lute size independent of the actual print size of the image This ensures that scale bar lettering always remains legible even when printing out several images at one 169 The Image menu Show in Viewport time or one extraordinarily large image To have the scale bar printed out along with an image the Printer check box in the Scale Bar Properties gt Display tab must be selected Show in Viewport Shows or hides the selected scale bars in the viewports A short cut for doing this is by pressing the Shift F4 keys this allows you to switch back and forth with without scale bar Select the Display tab in the Scale Bar Properties dialog box Select the Show scale bar for Viewport check box and confirm with OK Draw into Overlay Draws the currently selected scale bars into overlay This command is not available for multi channel data sets If you transfer the scale bar to the overlay it will be linked to the image and saved along with the image when you use the TIF file format In contrast to when you burn the s
130. will be z 21 displayed The image buffer box is displayed 5c Je Dest Sez Mask when an image window is active If a graph window is active the spectrum buffer box is shown Click on the graph window to change from image processing operations to graph processing oper ations The elements Src Source Dest Destination and Src2 Source 2 of the operands box now refer to the corresponding graph buffer The destination graph buffer is used for all graph operating functions creating a new graph The Source2 graph buffer is needed for graph opera tions with two source graphs e g for the addition of two graphs Image Manager The Graph Window To measure individual points To change the XY scaling in the diagram lt _ gt 4 To display a graph activate a graph window Select the graph buffer containing the desired graph The whole graph in the active graph buffer is displayed as a diagram in the window Use the mouse to determine the exact coordinates of a plotted data point Bring the cursor to the diagram s area A black line cursor which can be moved by moving the mouse sideways will appear The exact X and Y values will be shown in the graph window s status bar Click with your left mouse button in the graph window The mouse cursor changes its shape to a vertical double headed arrow Move the cursor up and down to stretch or compress the Y scaling Move the cursor to the right or to the left to stretch or
131. window of the project 4 In the case of several images the Insert Images dialog box will open gt The check boxes in front of the selected images are marked You can still change your selection at this point e f you only insert one single image into the database the insertion mask will open directly 5 In the Prompt for data input group select the Always option 6 Confirm the image selection by clicking OK 7 The Insert Image dialog box will open for each image which is to be inserted gt The name of the image buffer is given as a suggestion in the Record Name field unless you have defined another in the Database gt Adminis tration gt Database Seitings dialog box e g automatic record names gt The insertion mask contains all user defined database fields which you have selected using the Arrange Fields command for the Insert view and the Image data type 8 Fill out the database fields and click on the Insert button to open the insertion mask for the next image gt The inserted images are now displayed in the gallery view of the database window 9 Click on the plus character in front of the selected database folder in the tree structure gt In the tree structure the images are arranged as an image folder under neath the selected order You can now insert new images or other docu ments one organizational level below the images 10 Click on an image folder in the tree structure 76 Archiving Image
132. work with an automated microscope the whole image acquisition will run automatically When your microscope cannot be steered by the image analysis program a wizard will guide you through the acquisition process step by step Acquire Z stack Acquires an image stack which shows the same specimen area at different focal planes BS Acquire Stack Ea Acquire Range urn _ ae Reference 5922 78 a 2 Distance 4 00000 Use absolute range Help Resalution Frames 2 S Color Channel Properties Spacing 4 E Hm lt Options Multi Channel Stack 2 First i Channel first 125 Acquiring images Acquire Z stack This is how you acquire a Z stack There are two possible ways of setting the parameter in the Z Range um group by defining the start and the stop position as described below step by step in the first set of directions or by defining a reference position and the distances to travel in each direction as described in the second set 1st Howto The Z range will be determined by the Start and Stop Positions 1 If necessary open the Acquire Z Stack dialog box The live mode will be acti vated 2 Select the Use absolute range check box 3 Focus the specimen manually at the microscope Then turn the focus wheel in one direction until you reach the position at which you would like the first image to be acquired or do this with the help of th
133. 01 12 2003 Modified 01 12 2003 14 49 04 Opened exclusively Wes Anew project folder is created Read only ro Database identifier 4N5TSay2 Converted from mdb ro Database version 1 02 101 Poeci Z 74 Archiving Images Insert data How to change the standard name for a new database folder 1 Open the database exclusively e Select the Database gt Open command In the Open Database dialog box select the apl file and mark the Exclusive check box 2 Select the Database gt Administration gt Settings command 3 Replace the organizational ID e g Project in the upper Format field with any other text You can use the wildcards lt Date gt and lt Cons No gt 4 Click OK to close the dialog box 5 Set up a new database folder Insert Images gt The new standard name will now be offered in the insertion mask Background Information Insert Images There are several options for inserting images into a database folder Menu commands mark the database folder in the tree structure of the data base window Select the Database gt Insert gt Images command Context menu mark a folder in the tree structure of the database window and click on the right mouse button to open a context menu Select the Insert gt Im ages command Drag amp Drop in the image manager drag the images from the image buffer down onto the database symbol in the image manager The images will then be inserted into t
134. 5 Thus it is possible to define a maximum value beyond the maximum intensity value of the image Use this possi bility to compare the intensities of two images on the monitor Intensity Use the Intensity slider to change the brightness of the display Increasing the Inten sity will shift the mapping range to lower intensities thus increasing the brightness of the pixels shown Contrast Use the Contrast control to change the contrast of the display Increasing the Contrast will shrink the mapped range thus increasing the contrast in the mapped range Gamma Use the gamma slider to change the mapping function from the default linear one Using a gamma value greater than 1 the image as a whole appears brighter The contrast of dark image regions is increased Use a gamma value of less than 1 and the image as a whole appears darker The contrast of bright image regions is increased 154 The Image menu Image Display Use the Detail Mapping s tab to define a display Adjust Display L O function of your choice Adjust Display Detail Mapping for the way the image is _ E GFF displayed 176 3156 Close Reset Linear Help The buttons displayed in the dialog box above the histogram stand for the different color channels Click on one of the colored buttons to switch the appropriate channel on or off You can recognize which color channels are currently on display by the gray bar below the colored buttons The name
135. 5 ms In doing so a quick live image is guaranteed which simplifies the microscope s settings when the camera is running It is possible that the live image is very dark should the changes you made to the setting result in very long exposure times for snapshots Please alter the microscope settings accordingly in such cases Click the Acquire button to start the live acquisition using intelligent Exposure The live image is displayed in the image window You can make the necessary settings focus stage settings with the help of this live image The speed of the live image has absolute priority when using intelligent Exposure For this reason the image resolution is automatically reduced to increase the live image s frame rate These changes however do not affect the snapshots which were made with intelligent Exposure Click the Snapshot button to initiate the acquisition of a snapshot intelligent Expo sure acquires a single frame and saves it in the active image buffer A hint box informing you about the acquisition process appears if the exposure of a snapshot exceeds one second The Exposure time correction slide control enables you to manually influence the exposure time for snapshots calculated by intelligent Exposure Position 0 on the slide control does not influence the exposure time which was automatically calcu lated by intelligent Exposure Move the slide control to the right in order to increase the exposure time or to the
136. 55 characters e Long whole numbers e g 10 O or 500 e Decimal number whole numbers and fractions e g 1 2 whether a comma or a period is used for decimal fractions depends on the local settings of your op erating system e Date Time The permitted date and time formats depend on the local settings of your operating system e Memo any length texts e Yes No For fields of this data type there is either the status Yes or No A check box appears during insertion and editing of the record How to define the database fields for the characterization of individual records A database should contain the field Comment For every record that is added to an experiment in the database the comment should be included For images existing organizational fields are also to be shown in the insertion mask for information purposes 1 Open the database exclusively e Select the Database gt Open command In the Open Database dialog box select the database file and mark the Exclusive check box You can recognize the database file because of the APL file name extension 2 Select the Database gt Administration gt Define Fields command 3 Click the Add New Field button 4 Change the standard name of the new user field in the Field Name field Enter for example Comment as the new user field 5 The Data type list offers various types of data Select the Memo option when you want to enter a lot of text in the comment fie
137. Adjustment Preview IY Preview Window aA Execute Gamma Brightness 1 jo 0 Contrazt File E El pes E Eee eesti E EEN ee Gamma Structures with either high or low brightness are often non linearly imaged by many cameras This means that very bright or very dark image areas are acquired with too little contrast Monitors may also display this kind of nonlinear behavior Using the gamma parameter you are able to correct these non linearities and thus generate more realistic images or image displays Move the slider to the left The image will become darker as a whole Contrast will be enhanced in bright image areas Move the slider to the right The image will become brighter as a whole Contrast will be enhanced in dark image areas Brightness The parameter adjusts the intensity of the HSI color space i e the mean value of the three color components Red Green Blue A constant offset is added to the intensity values of the pixels Values vary between 1000 and 1000 Hue remains unchanged Contrast Use the parameter to improve the image quality of images which have too little contrast In addition high image contrast makes it easier to determine thresholds to separate different phases e g for phase analysis or particle detection The parameter alters the intensity histogram The intensity values are multiplied by a constant factor and thus spread or compressed What m
138. Animate Opens the Animate Image Stack dialog box see also on page 22 JE 18 Button Bars The Stack Navigator button bar Select Color Channel Use the Select Color Channel menu to set the display mode The menu offers the following commands Image Stack Navigator E fd All Color Channels 1 GFP wv 2 YFF Gray Scale F alse Color Fluorescence Color Load LUT Overlay Transmission Select Transmission All Color Channels A multi channel image is made up of several monochrome images Each image stands for one color channel For example A sample is labeled with three different fluorochromes The resulting multi channel image acquired with three different excitation filters is displayed as an overlay of the three different color bands To view the individual color bands open the Select Channe menu All color channels of the active image are listed Select the desired color from the list The currently displayed color channels are marked in the shortlist The selected color bands will be displayed exclusively in the viewport the other channels will be hidden You may select only one or more than one channel To select more than one channel keep the Shift key depressed when you select the channels Gray Scale Uses a gray LUT for the single color channel display This does only change the display not the image itself Thus you can simply switch to another display mode Note that the
139. B Size 5 Cancel NxN Execute Preview i Windows Help Enter how often the filter is to be applied to the image in the Iterations field Select an entry between 1 and 25 The preset value is 3 The greater the number of iterations the greater the extent of the averaging The number of iterations and matrix size also determine the time required to complete the filter operation Enter filter matrix size into the Size field The preset matrix size is 51 If you choose to make your matrix very large the filter will affect large image structures 230 The Process menu Define Filter Pseudo Reimer Why the Reimer Filter Defines the accentuation for the topographical effects of the Pseudo filter This filter is for making objects look three dimensional After the filtering process has been completed the object will look as if it is being illuminated by a virtual light source Dark objects will appear as hollows and light objects as ridges The resulting image will seem to have a topographical appearance This pseudo three dimen sional display of image structure is why the filter is called what it is Filter Matrix O 10 1 2 9 128 0 0 0 The Pseudo filter registers transitions from light to dark pixels Transitions going from light to dark are considered positive and transitions going from dark to light negative The image will be normalized such that the ze
140. Border To Single Image A P Header area Header ultiple images Header Footer Multiple images Footer Border Bottom Non printable paper area Singen image A Field codes for Use predefined field codes in headers and footers to have certain document proper headers footers ties or information automatically included in your documents Field codes are always introduced by the following symbol They are placed in curved brackets To have an image s name printed out along with the image you would enter the following Name Name Comment Buf Page PrintMag Date Time Now Context sensitive The File gt Print command is context sensitive and thus dependent on what kind of print settings document is active If the active document is an image the Print Image dialog box will be opened Different document types open respectively different dialog boxes Print Directly S Click the Print Directly button in the Standard button bar to print out the active docu ment without having to go through a dialog box The active page layout will be used when you print directly Draft mode Use the draft mode for trial printouts In the position where images are located gray rectangles will be printed Headers and footers will also be printed as rectangles The actual images will not be printed out as image prints can be time consuming Draft mode printing is a fast and easy way to check out what your layout looks like e g
141. Colors button the Color dialog box will be opened You can select a color from either User defined Colors or from Basic Colors Colors are defined via the Red Green and Blue components they are comprised of or by their Hue Saturation and Intensity values Append Colors transfers the defined color into the selected box of User defined Colors Copy Image Note Duplicates the contents of the source image buffer into the destination image buffer You can most easily copy an image by dragging amp dropping the image from the Image Buffer Box into the image buffer desired No warning message will appear if you overwrite an image in the destination image buffer To copy an image segment into another image buffer you first select the desired image segment using the Image gt Set Frame command Then use the Edit gt Copy command To copy an image segment into another image buffer you first select the desired image segment using the Image gt Set Frame command Then use the Edit gt Copy command or simply press Ctrl C to copy the image segment into the clipboard Then activate the destination image buffer and insert the image segment using the Edit gt Paste command or by pressing Ctrl V To copy an image or an image segment into the clipboard you can use the Edit gt Copy command or simply press Ctrl C Delete Image Deletes the image in the active image buffer Use this command to remove images no longer require
142. Correction Select the Polynomial fit option to have the correction image calculated based on a two dimensional fit of the original image least square fit If this option has been selected the Polynomial fit group will appear in the lower left corner of the dialog box This is where you determine whether the two dimensional fit is Linear Square or Cubic Select the Save shading image check box to save the image used for shading correc tion The program then saves the shading image internally When you apply a shading correction to an image using the Process gt Shading Correction command this is the shading image which will be used This means that the shading image will not be recalculated This speeds up shading correction This approach only makes sense if e g you are applying shading correction to series of images which all have the same shading Do not select this check box when working with images with varying shadings Click the Execute button to apply shading correction to the image in the source image buffer The resulting image will be written into the destination image buffer You may also use the Process gt Shading correction command When you select the Preview check box a window within the original image will display the results of shading correction immediately after alteration of any param eter You can decide how large you would like this window to be by clicking the Window button and using the mouse The Execu
143. EA hen e e l an aiiogmnslaumade auttibe tel Mares tobmtehudacties 188 Combining IUOFESCONCCS isc sirrinin ne wales Wid ddewes tied a aE a aaaea aa aal 188 OIG MINI ACG a ra aaa a N 190 CODY MAGS serorea eeni Banda Ue utes in vida awe ssid a cia duet 192 DEICIE NAGS en N e tea E dh lawen E E ieemiananevaunwe tema 192 POLE CU IIMAGS naei a a a E e tidal 193 Deine lmage THISIONY sex narena e a e e a a 194 mage TATONIMAUOM s 2 cr a a E age eat mae 194 THe PROCESS MENU monica n 198 NLSN UY oe aaa 198 PXGIUIST COONS reizenn E A E A a AE E AE A 208 DOL INNS SMOIGS 2 soi cakeottcosacslarticcecac dant hacshattannss sat asaacsenlecdacmeed a lenmssankalesne ms sauhewees 211 EVAN IZ Oe mn ra E e E E O mmsantees 219 Define Shading Correction ccccccccsccceccceecceeecceeccaeecseeceeeeceeecseeseeesseeeseeeseeesaees 220 SMaCING COMeClON eaea A N A O aA 222 BackOround S btractioN secc enoar a ere eee aa OaE aa Raia 223 General Information on Filter Operations c ccc cceccseeeceeeceeeeeeeeaeeeseeeeeeseeeseeees 224 Denne FIOT csc ae seuss nes tensnch ies eel eca nau cea ccns mad suet scam taate wits Wioaa ss emtatcecaiee vay se maleuanceas 228 BMG Ft as rte ta ett ta aie IE A ANER AV AIN MEAT SI A eset ar E Nantes 242 Mage Calculator sor na e A aa e e a a aa a a 247 Arthmetle Operations ardei a E ain techies esta EE A aaa aiia 250 Delne PFOCCSSING tee arannana a a E 251 EXECUIC PIOCESSING wosan aa e EEE N a SEE A ATL 254 Mage GEOMElY
144. In the lower right hand area of the dialog box an overview of the acquisition process will be shown The grid that is shown there corresponds to what you have set in the parameter group Acquisitions that have already been made will be displayed reduced in size in this grid The position of the next acquisition is indicated by a red cross In the Parameter group you set the size of the composite image how the process is to run and the settings for tools In these two fields you enter the number of frames in the horizontal and in the vertical direction you want to have combined into the composite image From this list you select whether you want to have the acquisitions made in rows Horizontal or in columns Vertical In the Overlap field you set which percentage of an image shall overlap onto its neighboring images The greater the overlap is the easier it is for you to align the images However you should also take into consideration that the larger the overlap is the more images you will need to acquire to portray a given area In the Transparency field you set to which degree the orientation strip shall cover up the new image The higher the value of the transparency the less clearly will the ori entation strip be displayed Click the Close button to stop the acquisition and to close the Manual Acquisition dia log box Click the Acquire button to start the acquisition process or to resume it The live image will be adopte
145. Interest Area again click the Edit AOI Edit Area of Interest button Change the frame size in the Stage Navigator window and click the right mouse button again gt The entries in the Count Y and Count X fields will be updated since a change in the size of the Interest Area influences the number of images that will be acquired 7 Click the Scan button to start the acquisition gt The program will switch to the live image and start the acquisition A window will inform you of the probable duration of the acquisition You can cancel the acquisition at any time by pressing the ESC key 334 The Stage Navigator Save overview image gt After the last acquisition in your display the live image will be switched off and the overview image will be shown gt The Stage Navigator will switch to the Online Mode and only the area of the overview image will be displayed Save overview image When you have acquired an overview image by means of one of the methods described above it will be subsequently displayed in the Stage Navigator E Stage Navigator Testi Duan Dweaaearte 2 position 3815 00 The overview image will to begin with be located in a temporary folder with the path lt Installation Folder gt StageNavigator To save it permanently proceed as follows 1 Click the Save Folder button 2 In the Create an image series folder dialog box go to the location where you want to save it 3 Ifthe fo
146. Into Overlay 171 Shape 268 Driver not initialized 329 Driver not loaded 343 Edge Enhance 234 242 246 Edit Image 190 LUT 160 Overlay 173 Palette 266 Edit AOI 334 Edit record 80 EFI 3 Methods 129 Acquisition 129 Focus control 130 How it works 130 Emboss Filter 259 Enclosed Angle 320 Enhanced Focal Imaging see EFI Index Envelope 323 Equalize Contrast 198 Export images 92 Exposure time 113 Express database 64 Extended Focal Imaging EFI 128 F False color image 7 Fast Image Sequence FIS 134 file name 195 Fill Border 274 Image 265 Shape 273 Filter 224 228 242 259 323 Connectivity 232 General 224 Matrix 224 Fine adjust stage alignment 329 Fire Wire Cameras 329 Floyd Steinberg 263 Fluorescence 119 159 Fluorochromes 117 Focus 124 Focus Control EFI 133 Form View 82 Fourier image 7 Frame 185 frame 330 fully automated method EFI 130 G Gallery View 85 Gamma Correction 204 Get separate images of high quality 333 Goto record 92 Gradient 320 Graph Information 325 Graph information 312 Graphs print 310 Gray value image 7 Modify 200 Grid 294 Grid in the grid 341 GUI 2 Height map EFI 130 Histo Equalize Filter 262 Histogram 292 Horizontal edge enhancement 242 how to print images and graphs simultaneously 310 HSI Adjustment 209 i Image Acquisition 102 Calculator 247 Display 152 Geometry 255 Intensity 108 Scale Bar Scale bar 167 Image Analysis button bar 24 I
147. Manager command or use the Alt 3 key stroke gt You will find all of the open document windows listed in the document manager The document type and the title of the document window are given for each document E 2 Select the image window gt There is always just one image window 3 Click the Activate button located in the document manager gt The image window will then be in the foreground Loading images into specific image buffers 1 Click the Gallery tab in the image manager 2 Activate the image window by simply leftclicking within the window e Ifthe Images window s header is colored this means it is active 3 Select the Standard button bar gt Open command 4 Leftclick on the image file you wish to load 5 Drag the file directly onto any one of the image buffers while keeping your left mouse button pressed drag amp drop 8 First Steps The GUI User Interface gt The image buffer will show a preview of the image you have loaded 6 Repeat the last step as often as needed 7 To quit loading just click the Close button Use the mouse to drag amp drop images into UESLE E al Open Image the image buffer a images desired Ef D m E fz Look in Sy Images MS Explorer a file Sic e Dest Sic2 Ma Auclabel HE manager can also be ES used for drag amp drop erat loading a Za a E Steel tif E z Wood tif esktop My Computer aol hid PS hy Network F aD
148. Multiple Image Alignment gt Calibrate command If your stage is not initialized correctly you will have the opportunity to define the stage areas after selecting the Calibrate command If the stage is not calibrated the Multiple Image Alignment gt Calibrate dialog box will be opened After calling the command the Acquire Multiple Images dialog box will be opened Here you specify the number and the size of the individual images and the complete image area recorded at acquisition Acquire Multiple Images Close Acquire Yj UY ee GO 115 mm 90 mm Image Tiles Preview Horizontal 2 B Vertical j2 B Fisel Overlap Horizontal a56 B Vertical 256 B Final Image Size width 115 2 gt mm Height 89 6 gt mn Shift type f Specimen shift Focus Use EFI Options Logical Camera Preprocessing Only first image Fostprocessing Only last image Driving i Meander C Comb mage shitt In the Image Tiles group you specify the number of images you want to acquire for the overview image The images are arranged in an image matrix shown in the Preview group The preview is automatically updated when the image matrix is changed Changing the number of images increases the final image size but does not effect the size of an individual image and the width of the overlapping region The values shown in the Final Image Size group ar
149. Note The Cut Objects function do not make use of the usual Windows clipboard It has its own memory for data you copy You can not use the Crtl X keys to cut objects Copy Objects Copies the selected objects from the overlay into the overlay clipboard Click the Copy Objects button to transfer a copy of the selected objects to the overlay clipboard Use the Paste Objects button to transfer the objects into the overlay of the active image Note The Copy Objects function do not make use of the usual Windows clipboard It has its own memory for data you copy Please keep in mind that you cannot copy overlay objects via Ctrl C To copy an entire overlay 1 If the data layer is currently protected cancel its protection You do this by click ing on the Layer button and selecting the Protect Data Layer command 2 You select all the objects of an image by clicking the Select All button 3 You click the Copy Objects button to copy the entire overlay into the overlay clip board 175 The Image menu Paste Objects 4 Activate the destination image in the image manager 5 Click the Paste Objects button to insert the overlay If the overlay is larger than the destination image only as much of it as fits within the image starting at the upper left corner will be shown Paste Objects Pastes the objects from the overlay clipboard in the overlay of the active image Click the Paste Objects button to transfer the objects from
150. Number of copies Print to file Cancel Help Print output Choose the Graph only option to print only the graph in the active graph buffer Choose the With general info data option to print the information from the General tab of the Graph Information dialog box below the graph The general info data comprises the graph range the initial and final values the number of channels the data type and a user defined comment Choose the With all available info data option to print the information of additional tabs of the Graph Information dialog box onto a second page Using the Report For printing out graphs you may just as well use the report generator integrated into Generator your image analysis program The report generator is for creating professional multi page reports The layout possibilities it offers are of the highest quality 1 Load the desired graphs into the graph buffer box 2 Change the display of the graph so that the interesting structure is clearly visible For example you may switch from a linear to a logarithmic scaling 3 Select the File gt Report gt New command gt The New Report gt General tab provides you with several predefined page templates 4 Select the Normal template 5 Click OK to create a new blank report gt The report will be opened in its own document window Additional button bars will be added on the right hand side of the graphical user interface 6 In the Report Objects button
151. Obj p Magn Objective Aktine FITC A U MNU 5 60 00 oe I Cancel A Erightfield lt Brightfieds E U MNE 5 UPLFL Ol A ONA DAFI D Aucun 5 60 00 UPLFLOI A Myosine TRITC C U MNG 5 60 00 UPLFLOI zeal Deselect all Help UL Fluorescence Target Aktine Search Fluorachrame FITC T Mew Cube position Miror cube Apply A U MNU Delete Objective position Objective type 5 eooo uPLFL oi Description Fluorochrome T arget ir In the Target Fluorochrome list you can see an overview of the fluorescences defined by mFIP at the last accessing together with information regarding filter block and objective If the fluorescence defined here is to be adopted the check box on the left in front of the name of the target must be marked When the dialog box is opened for the first time or if all fluorescences have been previously deleted the picklist is empty In this case click the New button in the Fluo rescence group in order to define a new fluorescence In the Fluorescence group you create and edit existing fluorescences In the Target input field you name the structure which is made visible with the selected fluorochrome You can select any name The name is saved with the image information on the mFIP tab Furthermore it is used to name the image which is created if you have selected the Target Fluorochromes Target or Target Fluorochromes entry under Descrip tion
152. Port dialog box If a remote control is available you may enable or disable this feature with the On check box in the Remote group Use the Read button to transfer the current magni fication via remote control Click the Set Unit button to open a dialog box In this dialog box you define a pixel s unit The unit selected is displayed above the button If you activate the Automatic check box the values of the resulting calibration will be displayed automatically using the correct scale In the Resulting Calibration group you see the values for pixel width and height corre sponding to the magnification and the current X Y ratio 106 Acquiring images Camera Configuration With a fully automatic microscope the objects will be adopted from the Control Panel In this case the Add button Note will be inactive Translation This group will only be available when your microscope is provided with a motorized stage that can be steered in all three axes X Y and Z by your image analysis program When you change an objective it is quite possible that the image segment on display will have been moved The reason for this is that the objective s optical axes are not positioned exactly the same This fault will be corrected by the XY shift When you change an objective it is quite possible that the image will be a little blurred The reason for this is that objectives are set at different heights in the objec tive tur
153. Q480 40x HQ500 20x Q515LP and HQ535m Reference images of an eGFP sample Reference images of an e YFP sample Note YFP exciter dichroic emitter observed emission 450 500 550 nm YFP exciter Measured is the relationship Ratio of the mean intensities of the selected structures ROIs The intensities will be determined after a background correction of the two excitation wavelengths The relationship results in a constant that is characteristic for every fluorochrome under the given experimental conditions These two ratios are the prerequisite for the Spectral Unmixing of the images of double labeled samples The reference images have to be taken only once for each fluorochrome in a given experimental set up e g filter set labeling method cell type 277 Spectral Unmixing Using Spectral Unmixing Using Spectral Unmixing Spectral unmixing is composed of two steps Calibration and Unmixing It is currently possible to separate two or three different color channels Calibration The first step is to calibrate the contribution of the fluorochromes to the different color channels Depending on the number of different fluorochromes to be analyzed different reference samples stained with only one fluorochrome have to be prepared 1 Acquire multi channel reference images of the reference samples under the same imaging conditions as used later for the experiments with your multi la beled sampl
154. R illustration system and were made available by kind permission of Dr Paulo Magalhaes and Prof Dr Tullio Pozzan from the University of Padua Italy 279 Interactive Image Measurement Interactive Image Measurement Interactive Image Measurement Your image analysis program offers a wide range of measurement functions They enable you to quickly count objects and measure segments areas and angles All the results are saved together with the images and can also be exported to a sheet All the measurement on the measurement display button bar button bar Simply begin a measurement by clicking onthe appro priate button EBS x EA eTe File Edit Database Acquire Image Proce Measure Graph Special Window 7 4 4 Gait Ta ua Aa A H Images 6 MIA Clematis 36 ob Haa a mm The measurement display which contains 3 6 MIA Clematis a all of the measured ES sei ail A 7 i es mM m s values is activated in BZ 2D Object i ANA Sea 235 42 pr Perimeter 60 10 prm Fi 2D Object Ares 243 45 une Perimeter 64 50 pm Elf 2D Object Area 125 42 pr the image manager Perimeter 46 45 prm BS 20 Object Area 132 22 pnt Perimeter 45 43 pm 2D Object Area 105 06 ur Perimeter 52 38 pm Statistics for Area Name Value Count 5H Mean 168 31 purr Minimu 105 06 pir F Maximum 243 45 urr Standard Deviation 65 75 w
155. Record command 80 Archiving Images Insert data How to insert docu ments Pale Es Be ExampleD atabase apl c5 C4 E Ble There are other documents inserted below this record The folder Field name Field value contains es ample Grain size analysis odule Grains 1 image record foe bila Planimetr a FES Planimetyy 2 documents Ca oe ee as H Cy Filtering Insert Documents The list contains all loaded sheets diagrams To be inserted one sheet and Sheet Histogram one diag ram Sheet Graphite4nalyse E Diagram histogram O Diagram histogram O Diagram Graphite nalyse Select All Unselect All Insert Documents I Prompt for file names Current document histogram File name Type Eaei Curent document histogram i File name ristogram sft Type Diagram Selection index iia Siler Cancel Help amp ExampleD atabase apl Diagram and sheet is inserted Grain size Sheet sfs Example Grain size analysis Module Grains E Sek 1 kB 4 xampleD atabase Grain size analysis 81 Archiving Images Work in the database window Work in the database window Arrange Fields Background Information Image data type All images which have been loaded into the image manager represent a data type of their own This m
156. Record Objects 38 Resize 31 Select 31 Sheets 50 Text Objects 46 Text Variables 47 To adapt record objects to your database 45 To change the print size of an image 37 To create a new report template 57 To insert a sheet 51 To insert images from image database 43 To insert images from the image manager 36 To insert text objects 49 To use sheet cells in a record object 53 To use sheets in a record object 53 Report Templates 54 Object Templates 59 Page Template 56 To create new filed object templates 60 To plan report templates 61 To save report templates 58 Resize image 255 RGB Adjustment 209 RGB Studio 258 Rich Text Format 29 Roberts Filter 245 ROI 181 215 257 297 Area 297 Average Gray Value 298 Kinetic 297 Perimeter 297 Rotate image 256 RTF 29 S Save Objects Overlay 176 Savitzky Golay 324 Scan button 333 Scan Interest Area 334 Select Color Channel 19 Selecting Measurements 289 Send email 13 Separate Color channels Z layer time 187 Colors 268 Image 239 Separator Filter 239 247 Sequence when switching on the devices 329 Session password 100 Index Set Frame 185 Input 111 Magnification 167 Split Gain 316 Threshold 211 Shape 268 Copy 272 Crop 273 Delete 272 Display 274 Fill 273 Invert 272 Sharpen 242 Sharpen II 242 Show in Viewport 171 Show Markers 181 Sierra 263 Sigma Filter 236 247 Single color time lapse image 6 Single Image Mode 337 Single Z stack image 6 Smoothing 323 smoothing fi
157. Scale Bar Note Select the Palette bar check box to have the current lookup table LUT of the image displayed in addition to the vertical scale bar This function can be used for 8 bit and 16 bit gray value images and false color images The palette shows a gray value wedge from 0 255 for a gray value image and all colors displayed onscreen for a false color image As soon as the viewport becomes smaller than a certain size the palette will disap pear The height of the palette will be adjusted to fit viewport size There are 2 different heights This applies to printing situations as well i e if several images are being printed out the space for an individual image may be insufficient for the palette to be printed out as well Select the false color palettes only check box to have palettes displayed for false color images only This check box is only available if the Palette check box has been selected Select the application area for the scale bar in the Show scale bar for group You can have the scale bar shown in the viewport have it printed out with the image or copy it into the clipboard All three application areas can be selected independently of one another Select the Viewport check box to have those scale bars shown in the viewports that you ve selected in the Scale bar selection group This setting is a property of the viewport and not of the image i e you can only have the scale bars shown either in all viewport
158. Soft Imaging System An Olympus Company User Manual Imaging Software cell Olympus cell Family We at Soft Imaging System GmbH have tried to make the information contained in this manual as accurate and reliable as possible Nevertheless Soft Imaging System GmbH disclaims any warranty of any kind whether expressed or implied as to any matter whatsoever relating to this manual including without limitation the merchantability or fitness for any particular purpose Soft Imaging System GmbH will from time to time revise the software described in this manual and reserves the right to make such changes without obligation to notify the purchaser In no event shall Soft Imaging System GmbH be liable for any indirect special incidental or consequential damages arising out of purchase or use of this manual or the information contained herein No part of this document may be reproduced or transmitted in any form or by any means electronic or mechanical for any purpose without the prior written permission of Soft Imaging System GmbH Windows Word Excel and Access are trademarks of Microsoft Corporation which can be registered in various countries Adobe and Acrobat are trademarks of Adobe Sytems Incorporated which can be registered in various countries Soft Imaging System GmbH All rights reserved Soft Imaging System GmbH Johann Krane Weg 39 D 48149 Munster Tel 49 251 79800 0 Fax 49 251 79800 99 E CELL 0206 Conten
159. Stage Navigator or load another overview image the saved posi tions will be lost Trouble Shooting Problem Cause 1 Correction 1 Cause 2 Correction 2 Problem Cause Correction The Stage Navigator button bar is not shown The button bar has been faded out 1 Make it visible by moving the mouse pointer over any button bar and clicking the right mouse button 2 Select the Stage Navigator option in the context menu The Stage Navigator add In has not yet been activated 1 Activate the Stage Navigator add in 2 In the menu access first Special then Add In Manager and select the check box for the Stage Navigator option 3 Restart the image analysis program to make these changes effective The stage cannot be moved In the Special gt Preferences gt Stage menu the entry Driver not loaded appears behind the stage name The stage controller was not switched on when the image analysis program was started 1 Select the stage you want to use in the Special gt Preferences gt Stage menu and click the Connect button 2 In the Preferences gt Stage dialog box select the Virtual stage option in the Selection list again and close the dialog box Problem Cause Correction Problem Cause Correction Problem Cause 1 Correction 1 Cause 2 Correction 2 The Stage Navigator Trouble Shooting The stage cannot be moved In the Special gt Preferences gt Stage menu the entry Stage not initialized a
160. The active database s name can be seen beneath the database icon located in the lower part of the Image Manager Define Image Sequence Monitors an image sequence The Image Sequence Processing command enables you to acquire a series of single images stacks or mFIP images in predefined intervals If you are appropriately equipped it is possible to automatically operate the entire procedure When selecting this command the Define Image Sequence dialog box will be opened with a previously defined image sequence processing loaded Define Image Sequence General Buttons The buttons described here are common to all tabs Click on the OK button to close the dialog box If you have entered a name into the Process field located on the Info tab the current parameters will be saved and auto matically reloaded the next time the Define Image Sequence command is used Click the Start button to begin the process with all the current settings Click the File button to save a set of parameters The ISP save input output dialog box is opened A saved file receives the file name extension STD and is stored in the ISP subdirectory within your image analysis application s root directory It contains all of the settings of a process Click on the Details button to show hide the Sequence group The Sequence group for the definition and organization of the individual sequences of a process is defined here 134 Acquiring images Define I
161. The Process menu General Information on Filter Operations What will happen recalculated is situated at the center of the filter matrix Most filters employ a 3x3 matrix In this case the intensity of a pixel will be influenced by 8 of its neighboring pixels Every pixel within a 3x3 matrix will be multiplied by the matrix element above it by the so called weight factor For the filtering of the whole image the filter matrix will be shifted across the original image and the gray value of each pixel will be recal culated W Wg are the weight factors of a 3x3 filter matrix l4 lg are the original intensities of an image area comprising 3x3 pixels The intensity of the central pixel I5 after filtering is calculated as follows Bild Filtermatrix Normalization factor olee Filtering l Normalization In order to prevent the filter operation from altering the mean image brightness the result can be divided by a normalization factor Normalization usually consists of the division of the sum of all weight factors W W Wol If the result is negative it will be set to a gray value of 0 i e black Negative weight factors in the filter matrix will yield negative intermediate results When an offset is added negative values will be displayed in the resulting image If the result turns out to be greater than 255 it will be set to 255 white The application of a filter for a numerical example is
162. The other two modi are designed for you to use when you are loading images into an image buffer You can then use all of the functions of your image analysis program on these images e g filter operations or particle detection Furthermore you can insert the images in a database For these two modi it is irrelevant whether you have a microscope and a camera connected or not One could therefore also call them Offline Modi This enables you for instance to make all the images you need at a 336 The Stage Navigator The Modi of the Stage Navigator Stage Area Mode Online Mode Single Image Mode Interest Area Mode workplace equipped with a microscope then later start the Stage Navigator at another workplace without a microscope and edit and analyze these images there in these modi Modi for image acquisition This mode is activated when you start the Stage Navigator You can see the com plete stage area defined by the X Y boundaries as a gray area You acquire the overview image in this mode When you have acquired the overview image the change into this mode will occur automatically As opposed to the Stage Area Mode in the Online Mode only the area of the overview image will be displayed all other stage positions will be faded out This makes it possible to display the overview image in a much larger size Should you wish to acquire further overview images of other areas of the sample you will have to revert to the
163. The procedure described above is slightly different when acquiring a Z stack of multi channel fluorescent images To acquire a Z stack of multi channel fluorescent images please proceed as follows 1 The Acquire gt Acquire Z stack command presupposes that you have already defined a fluorescence acquisition Use the Acquire gt mFIP gt Define Fluores cence Acquisition command to do so This fluorescence acquisition should contain all of the fluorescences that you want to use for the acquisition 2 Open the Acquire Z Stack dialog box 3 Define the settings in the Z Range um group 4 Click the Options gt gt button for additional settings 5 In the Multi Channel Stack group select the Acquire check box 6 Click the Color Channel Properties button Color Channel Properties Ea Color Channel DAPI bd Properties Numerical Aperture 0 3 Refractive Index Air 1 0 Emissive Wavelength 455 cme tb gt Inthe Color Channel list you will then find all of the fluorescences that you have defined with the Acquire gt Define Fluorescences command 7 Enter the numerical aperture of the objective you are using if you use the IX or BX software steering this value will be automatically written in Select the immersion medium in the Refractive Index picklist The wavelength for fluores cences will be automatically adopted from the Define Fluorochrome dialog box For brightfield acquisitions a
164. These lines are the thinnest ones possible They serve to outline particles for particle analysis however only if you ve selected the Adjacent borders 4 option in the Connectivity group in the Define Detection dialog box Otherwise particles will not be separated since some pixels will already belong to another particle even if they only touch diagonally In the Zoom field define the zoom factor of the image area displayed Select the zoom factor or enter it via keyboard Maximum brush size will depend on the number of pixels comprising the ROI 190 The Image menu Copy Image Note Decide what gray or color value you want to alter your image with in the Color selec tion group Select a gray value or simply enter it via keyboard into the Pixel field You can also select a gray value from the grid in the lower right of the dialog box using the mouse A bar in the gray or color value you select will be displayed in the upper part of the field By rightclicking on the zoomed image area you select the gray or color value of the pixel you clicked on and adopt it in the Color Selection group This gray or color value can then be used to edit other pixels If a true color image is edited the Color Selection group will provide you with the Colors button Instead of defining pixel value you define the red green and blue color components from anywhere between 0 and 255 RGB for the colors to be added When you click on the
165. aded into the image buffer Select the Sort according to real time z position check box to sort the single images in the new multi dimensional image Should this check box not have been selected the active image will be the first single image in the new multi dimensional image The rest of the images will then be combined in the order of their data slots You can combine two or f Combine Fluorescences x more standard images into one image Feasible image objects Available Fluorescences Image Fluorescence 4 3 Images FITC FITC 1 Imaget TRITC DAPI 2 Image DAFI TE OK Cancel Help Use the Combine Fluorescences command to have images converted into a multi dimensional image format During the conversion each single standard image will be treated as a one channel image For this reason you should not use this command to convert colored images that contain more than one color channel The Combine Fluorescences command is only available when a standard image has been selected in the image manager 188 The Image menu Edit Image Note The Feasible image objects list in the Combine Fluorescences dialog box contains all of the loaded standard images with the same resolution in X and Y direction and that are of the same image type The Available Fluorescences list contains all of the available fluorescences together with the color that belong to each of these fluores cences Select an
166. age 6 Now it s time to alter the size and position of the image segment e Select the red frame delineating the image segment You can alter its size and position within the image any way you like gt After every alteration to the image segment the detail image will be changed accordingly The position of the detail image is not affected by this A zoomed image detail FAE Eee E called Detail Zoom is shanu g comprised of the image haic 7 PETIA CT TTR 18 segment the image de tail and two auxiliary lines Image segment Auxiliary lines 7 Now you format the zoomed detail e Select the detail image and click the Properties button in the Report Ob jects button bar You ll find all properties for image objects for the detail im age in the Properties tab All Zoom levels and automatic labeling are avail able e Select the image segment and click the Properties button You can set col or line type and width in the Line tab e Select one of the auxiliary lines and click the Properties button You can set color line type and width in the Line tab Record objects Background Information Image database An image database enables a structured storing of all your images It enables you to access a great number of images fast and easy Database fields Database fields determine the structure of your database Database fields define the criteria relevant to all informat
167. age This is determined by the size of the image matrix i e the number of images arranged hori zontally next to one another and vertically below one another The size of the single frames will be determined by the XY calibration of the active input channel The values of the Image Tiles and Pixel Overlap groups and the preview are auto matically updated when the final image size is changed In the Width field you enter the desired width of the overview image The width is restricted by the fixed width of the individual images and the maximum possible overlap region MIA computes the possible width that comes closest to the desired value Use the arrow buttons to add or remove an image without changing the pixel overlap Assuming a 3x4 image matrix the final image width is obtained via Width 3 width of the individual images 2 width of the overlap region In the Height field you enter the height you want for the overview image The width is restricted by the fixed width of the individual images and the maximum possible overlap region MIA computes the possible width that comes closest to the desired value Use the arrow buttons to add or remove an image without changing the pixel overlap Assuming a 3x4 image matrix the final image height is obtained via Width 3 width of the individual images 2 width of the overlap region In the Preview group you can check the position of the individual images and the size of the overlap r
168. age dots Whilst the selection speed increases as a result the spatial resolution of the images falls For this reason you will as a rule choose a high binning value for the preview in order to obtain a live image that reacts quickly Where the single image acquisition is concerned you should as a rule choose binning 1 in order to acquire images with a high resolution Select the Link exposure times check box to have the exposure time for the acquisi tion optimally matched to the exposure time for the live image If the Binning setting is identical for single image and preview the exposure time for the single image will be set to the value which has been defined for the exposure time for the preview Every further alteration of the exposure time for single image or preview is adopted automatically for the other mode If the Binning for a mode is now altered the exposure time is adjusted for the other mode A doubling of the binning value results in an exposure time that is 4x as long in the other mode This adjustment of the exposure time following an alteration of the binning takes place so as to harmonize exposure conditions for preview and single image When you clear the Link exposure times check box you can enter any exposure time you want for the single image and a completely independent exposure time for the preview In the Integration field you enter how many images are to be integrated into the single resulting image The ma
169. age image source to use the previously defined cluster for the image acquisition The stage moves consecutively to all stage positions within the cluster and takes an image The camera settings and the image calibration are determined by the active logical input channel 252 The Process menu Define Processing The Processing Tab Save Image Series Binarize Shading Correction Auto Thresholds Rank Filter NxN Use this tab to specify processing steps for each individual image within the applica tion Define Processing Ed Image Input Processing Measurement Available functions Current functions Eila Auto Thresholds detault r Binarize Run Test Eeli Wp awr Color Separation Blue Color Separation Green Color Separation Hue Color Separation Intensity Color Separation Red Color Separation aturatior Color Thresholds Convert image to 16 bit Convert image to 8 bit DCE Filter Differentiate T 2 Hemove I M Visualize OF Cancel Help All processing steps contained in the Current functions list are applied to each processed image The order of the processing steps in the list corresponds to the order for the application The Available functions list contains all functions that can be used for the automatic process Some of the available functions Use this command to specify an image series in which the images of the current process are to be stored You c
170. age is marked Here you decide how the measurement results of several images are to be imparted All in one table or in separate tables Interactive Image Measurement Using Statistics Functions Create Measurement Sheet Select the images whose measurements will be included in the sheet ao i Imagel lz Imagez TE Images Cancel Select All Image Imagez Images Images Unselect All I Show image name in the First column Generate statistics of the sheets One sheet per image PELE Help 9 Inthe Create Measurement Sheet dialog box click the Select All button to export the measurement results for all images Select the Show image name in the first column check box to additionally acquire the names of the images in addition to the values into the sheet You can thus match the individual measurement values in the export sheet clearly to an image when using numerous images Clear the One sheet per image check box to write all the measured values to one sheet Confirm by clicking OK gt The measurement sheet is created and displayed In order to make the measurement sheet visible and to avoid having it covered by the image your image analysis program automatically returns the image window to its original size 10 Use the Alt 4 key stroke again to hide the Measurements button bar Using Statistics Functions Background Information Statistic display Create sheet 287 Your image a
171. ailable in every case 120 Acquiring images Multiple Fluorescences Define Fluorescence Acquisition Cancel Fluorescence Exposure time TRAITE Myosine KOD m FITC Aktine 200 me DAFI DNA 50 ms ACME Brightfield gt Brighttield 2 m e Pixel Shift r lkt RE Help Acquisition options Delay 0 is Manual I with preview Camera settings Exposure time Binning Snapshot 500 ms i Preview s0 ms i W Link exposure times Integration if Frames ls Remote Olpmpus Dummy Bs 2 W Ho Messages C Automatic 2 Corection 0 B pm ZPosition 5000 Eum Epeus Bead Beset Use pixel shift In the Fluorescence list you can see the list of the fluorescences which have already been selected for this series of image acquisitions together with the individually defined exposure time The names of the fluorescences are represented in the form defined in the Define fluorescences dialog box To change it access the Define fluorescences dialog box once again Using these four buttons you organize the fluorescences and the order of the image acquisitions Add fluorescence Remove fluorescence Move fluorescence up a Move fluorescence down a P Click the Add fluorescence button to open a dialog box with all the fluorescences which were defined and selected in the Define fluorescence dialog box Select a fluo rescence
172. ainly happens is that the saturation of the HSI color space is altered Values vary between 1000 and 1000 Hue remains unchanged 208 The Process menu Adjust Colors Define HSI Adjustment Intensity Hue Saturation Sets hue saturation and intensity Use the Execute HS Adjustment command to apply the current settings in the Define HS Adjustment dialog box to the image in the source image buffer Define HSI Adjustment ren vate IM Previews Windows Cancel Intensity j poo B io S o Reset 0 B Eea Saturation Values vary between 1000 and 1000 Hue remains unchanged Move the slider to the left The pixel values will be shifted downwards along the inten sity axis of the HSI color space The image will become darker At the minimum of 1000 the image will be black The absolute value of all pixel intensities is O Move the slider to the right The pixel values will be shifted upwards along the inten sity axis The image will become brighter At the maximum of 1000 the image will be white The absolute value of all pixel intensities is 255 The values vary from 360 to 360 Intensity and saturation remain unchanged Move the slider to the left right The circle of colors will be traversed in a clockwise counterclockwise direction The original image will reappear at 360 Values vary between 1000 and 1000 Intensity and hue remain unchanged Move the slider to the left Pixel va
173. alues can be displayed in radically different colors This will give the resulting image a coloring distinctly different from that of the original image You can decide whether you wish to permit this function to be applied to false color images or not in the Special gt Preferences gt Image tab To permit it select the Allow operations on false color images check box here The original setting will leave this check box clear thus some commands will at first not be available for false color images Moreover there are a number of commands that in any case can be applied to false color images In these instances you are also only working with the image s gray values The LUT will not be taken into consideration remaining unchanged An Overview of Tabs The Edit LUT command provides you with three possibilities for altering the LUT e You can input all LUT values directly into a sheet e You can generate the LUT interactively by defining polygons e You can use formulae to have the LUT computed In effect you re editing a separate LUT in each of the three tabs i e each time you go to another tab you ll not only be switching to another tab and different editing method but also to an altogether different LUT of the image itself In general it is not feasible to automatically convert between the three LUT display methods In fact there are only a few exceptions where this would make any sense There is however one important excepti
174. alysis program offers you a great number of acquisition commands with which you can not only acquire single images but also a complex time lapse experiment You will find the commands for acquiring images in the Acquire menu The Acquire button bar offers you a separate button for every acquisition process en cel E og a SS Starts continuous acquisition using active input device The live image is displayed in the active viewport The camera icon in the status bar will blink and the name of the camera will appear in red lettering As long as the live image is being shown in the active viewport the program will only make commands available to you that you can use in this mode Stops continuous acquisition or acquires single image Camera Control Adjusts the camera parameters Use the Camera Control dialog box to set and modify camera parameters Camera Control allows you to control the camera during live acquisition Elements functions and layout of the dialog box depend on the camera in use Click on the help button question mark in the dialog box to call up camera specific infor mation Camera Configuration Note Configures the active signal source The command opens the Configure Input dialog box This dialog box provides you with several tabs for determining all input settings All settings are linked to the active input To access settings of another input you will have to activate that input first Then you
175. an actually write a clear request within the text object Enter image comment here Configuring the Graphical User Interface GUI Take the time to configure the Graphical User Interface optimally to your needs when working with report generator define window width of the report window and of the database window such that both windows may appear at the same time Position both 61 ke Report Generator Planning report templates windows so they do not overlap Place the button bars which belong to the report generator to the right or the left within the user interface not below the standard button bar at the top If the button bars are positioned below the standard button bar the display of the whole Graphical User Interface will move up each time you switch documents If you are inserting images from only one database then you can increase the amount of space for report generator significantly by minimizing both the image manager and the Viewport manager while you edit a report To show or hide the windows use the following keyboard shortcuts Alt 1 and Alt 2 Reference Page Create reference pages for all elements that you use frequently in your report template This could e g be a group comprised of an image object text object and lines Text objects which can be edited cannot be defined within a record object This is why you define a record object along with a text object on the reference page Define all the elemen
176. an also specify or change the basic settings for an image series with this command With the Edit button you open the Define Image Series dialog box Use this command to define the threshold for a binarization and then to binarize the image With the Edit button you open the Set Threshold dialog box Use this command to subtract the background from the current image Here the background is either a saved reference image or is calculated from the current image With the Edit button you open the Define Background Correction dialog box Use this command to binarize the current image The thresholds for the binarization are calculated from the image histogram With the Edit button you open the Set Thresholds gt Auto Settings dialog box Use this filter to remove spot noise from the original image The rank filter adjusts the pixel values in the center of the filter matrix to the gray values of the immediate surrounding area With the Edit button you open the Define Rank Filter dialog box Use this command to apply an averaging filter to the image You can freely define the size of the neighboring environment which is considered in the median filtering With the Edit button you open the Define Filter NxN dialog box 253 The Measurement Tab The Process menu Execute Processing Phase Analysis Use this tab to define measuring operations for each individual image which are executed while running an automatic process Defi
177. an be enclosed but they cannot be filled A polygon s position can be subsequently altered however you like The object s size or as the case may be the course of a line cannot be altered This is how you draw a polygon 1 Click the Polygon button The mouse pointer jumps into the image window 2 Click the left mouse button to define the polygon s starting point Set additional points 179 The Image menu Highlighter 3 This is how you delete points Keep the Shift key depressed and with the left mouse button click to delete the last segment step by step 4 Keep the left mouse button depressed to change to a free hand drawing mode 5 Click with the right mouse button to close the contour Highlighter Creates a rectangular highlight object in the overlay The segment highlighted will look as if someone has placed a colored transparency over it Bit depth should be raised to 24 bits before burning an overlay containing high lighting onto an 8 bit image The highlighted area would look patchy in an 8 bit image Highlighted objects have their own separate Object Properties dialog box You have 6 highlighting colors to choose from in the Fill palette Besides using the transparent color effect to highlight something you can also have the image area selected displayed inversely Simply select the Invert entry in the palette Highlighting objects can be superimposed upon one another resulting in interesting and useful eff
178. and emission wavelengths Em Select the check box in front of a fluorochrome so as to make it available for the other mFIP commands If the check box is not activated although the settings are saved they are not available in other mFIP commands Fluorochromes which you have defined yourself are flagged in the list with a special f symbol 117 Acquiring images Define Fluorochromes Click the Select all button to select all fluorochromes Click the Deselect al button to deselect all of the fluorochromes Click the Default button to reverse all the parameter changes in the predefined fluo rochromes This command causes the original fluorochrome list to be reloaded In this way fluo rochromes which might have been deleted by accident can be restored The user defined fluorochromes are not affected by this command In the Fluorochromes group you can alter the data for individual fluorochromes and define your own Click the New button to define a new fluorochrome In the Dye field there are preset entries Dye1 Dye2 etc The values Excitation maximum and Emission maximum as well as the Dye will be adopted from the last fluorochrome that was selected Click the Search button to find the first entry in the list the name of which begins with the entry in the Dye field In this way you can quickly find fluorochromes which are known by name Click the Apply button to adopt changes made to a fluorochrome or to adopt a rede
179. andard sizes and you can check image calibration Any overlay present will be faded out while you define classes If the active image buffer is empty the standard sizes will not be displayed Select a color for the active class from the Set Color list All particles belonging to this class will be displayed in this color in the overlay during successive processing in sheets and in histograms Determine the color you wish to assign to a particular class by clicking the sheet row of that class Doubleclick on the class number and class ID in the first sheet column The values belonging to that class in the sheet row will also be displayed in the color selected The sheet located in the lower dialog box contains the class divisions i e classifica tions of the current classification scheme The left column indicating the running numbers of the classes indicates class ID The From column contains the lower limit of each class the To columns the upper limit The Name column is for entering a name for each class You can have classifications computed i e the upper and lower limits of each class in the Compute Classification dialog box If necessary from to values in the sheet can be corrected To do so doubleclick on the field where you wish to alter a value The value in the To field must always be greater than the corresponding value in the From field Values here are calculated to two digits after the decimal point The value of t
180. ape needs at least three points 6 Click the right mouse button when the definition of the polygon line has been finished Your image analysis program will close it automatically 7 Continue defining shapes by the active drawing method 8 Depress the middle mouse button or the Esc key to return to the Add Shape dialog box Interpolating A shape defined by the Interpolating Polygon drawing method is defined by a line Polygon fitted to the points you set 1 Click the Interpolating Polygon button The mouse pointer moves to the image window 2 Click the left mouse button to set points You need at least four points to define an interpolating polyline 3 Click the right mouse button when the definition of the polygon line has been finished Your image analysis program calculates and draws the best fit line through the points you have set 269 The Process menu Shape 4 Continue defining shapes by the active drawing method 5 Depress the middle mouse button or the Esc key to return to the Add Shape dialog box Freehand polygon This is how you define a shape with the Freehand Polygon method 1 Click the Interpolating Polygon button The mouse pointer moves to the image window 2 Click the left mouse button to set the starting point of the line Move the mouse by keeping the left mouse button depressed to draw the line The Freehand Polygon line follows the course of the mouse 3 To remove the last point depress th
181. area according to the upper right pixel position Origin and the ROPs size in units of pixels Size The position of the ROI in the image can be changed by clicking on the Set ROI button A red rectangle will appear within the image It can be positioned using the mouse Press the left mouse button to confirm this position Note To be able to see and edit the largest possible image area at one time click on the window icon i e the maximize button located in the upper right corner of the Image Edit dialog box Define the size of the paint brush in the Brush field Maximum brush size will depend on the number of pixels comprising the ROI Select the Grid check box to have a black grid placed over the relevant image area Each unit of the grid represents a single pixel This provides a convenient visual separation of the pixels It also means that you can distinguish pixels from one another that are the same color as long as they aren t totally black the same color as the grid The grid will also be placed over the paint brush sector The color of the grid over this paint brush sector will depend on the color setting of the sector itself When working with a gray value image the gray value of the grid lines will be the inverse of this sector s gray value meaning if this sector is black P 0 the lines will be white P 255 for an 8 bit image When working with a true color image the color of the grid will be t
182. ase Click the Delete button to delete the current classification scheme from the Classifi cation list Click the Unit button to open the Set Unit dialog box and set the unit of measure for the current classification scheme Click the Compute button to open the Compute Classification dialog box to set the number of classes and their respective value ranges Clicking OK will save the new classification scheme and any other alterations you have made The CLASSES CIF file will contain these Define Statistics Selects the statistics to perform for a sheet Define Statistics E Available Current Average Deviation Mean Minimum a Masimum Standard Deviation Skewness Sum Vanance hoe Add gt gt Install The total number of all values Description Help AE FLEE Mean is known a prion NW arance W Color rows of result sheet Statistic functions calculate statistical parameters such as Count Minimum Maximum Mean Standard Deviation and others from the values of any particular sheet column First select statistical parameters with the Define Statistics command Then select the relevant sheet column and have the statistical functions calculated with the Statistics command The following statistical functions may be selected e Average Deviation e Kurtosis e Count e Standard Deviation e Maximum e Sum e Mean e Variance 304 The Measure menu Statistics Statistics e M
183. asuaciaunetenecsisevesageauassaaawamenesiteaaeouneaemuasbeaeteas 307 Markers ANG ADCS skimere die ts th duce a a E iuansele 313 SOL SOT Gall z sasn hee Neves ng E AA TEA 316 WIOOITY SDNt Galles cess Jcsteencdseasaseencnsac a A E a EA 316 ROLE CE MAD My ero a a a E a N naveocar 317 PEE O E A ree eran eee eee oe 317 11161 10 6 eerie innr etree ea ese tae co nt nt NT reo een ny CTT ee eee RE re 317 Overlay SCIOCIO Nis arrasi irene a acetal tence ly aA EEA 319 WICA SUNG aie E ar aencamn had sesashag acnetanan quads O E lt aGiGeshassie mane ipa A nauesaneS 319 CAICUIATIO MN sees 054 aie Sad eters a Avante eee E AAE 321 RIES DETAI E AEN E tated A A A E A ETA A A E A E EA T 323 ANAME UC eeni a a a a E Med recentaloe he acer 324 Grabmal O M e ceras o a a E a does eee as 325 Define Graph HISTORY emesis e e e r e dance eE E aE AEAN 326 CONVER O renina aa aa a A T E G 327 The Stage Navigator assasssnnnnnnnnnnnnnnnnnnnnnrnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nnmnnn 328 Summary Of Feat reS city siicts a titcadenath nii a i a a S 328 Preconditions for using the Stage Navigator ccccccccececeeeseeeeaeeeeeesaeeeaeeeaees 329 Start Stage Navigator exc steers Sas cant ie See end Sew banana ects cece acs Sac naneeaeeaee aa 330 How to acquire AN overview image ccccceecceccceeceseceeeceesaseceeceesaeeseeseseseesaeees 331 Save OVEPVIEW IMAGE secsrirerecan Aan E EA O eee 335 Loading an overview image in the Stage Navigator
184. at acquisition Click the No Clipping button to have line and column numbers reset at 0 No borders will be clipped The Image format field will display the maximum image format possible according to the resolution of the logical input The Active area field displays a preview of the current image format this enables you to estimate how much of the borders you want to clip A blue frame indicates the maximum image format possible according to the resolution of the logical input The crosshatched area represents the image area White areas indicate borders to be clipped Selects the active signal source The image acquisition functions within the program are based on the concept of logical input channels Logical input channels are built around the physical input devices that allow you to acquire an input signal As a rule the device used for image acquisition is a CCD camera However it can also be a video camera a Slow Scan Interfaces or a scanner If you need more than one acquisition method just select the suitable input from a list of several predefined input channels The acquisition conditions are specified by the settings of the input channel used You configure the hardware that has been connected in the logical input channel Give the input channel a meaningful name and add a short description You can here choose between several methods for the editing of digital data You can determine how the camera signal is to be pre edited
185. ate 95 Protect with a password ccccccceccsecceeeseeeceecueeseccueceeesaeccueeueesueseuseueeseeseeeseeeseees 99 Acguning Image S eresse E 102 ACOUNG cacai a a a a a a 102 Snap ane ne een eee a a e n a a eee a er en 102 Calera COMMON ennaa i a e A E ced das nice un a 102 Camera Configuratio s sedina e E a 102 Set anette te oe e RT Cre een ne nan E enn een ner 111 HENGEN EXOOSUIG syiti nissantancuiaandt st eyanand teed a A ieee 113 Multiple Fluorescences ccccccecceecceeeceeeceeceeecaeeaeeceeceeeceesaeeseeeasesseseeesaesaeesans 114 PACOUMC Z SlAC Kies sean na N nu Maasai e cadet Nadaiduuben e 125 Extended Focal Imaging 2 csc seraceeate Sh aumncan lamaastiens iareiant Scataaenute a oameeatous castes taaetme 128 Define Image SEQUENCE eve sereante e e a oa EEN 134 Multiple Image Alignment oanoanannannannanannnnnnnnannnnnnnsrnnnnnnnnnenrnrnnrsnenernrrnrsnesnenrenne 139 tne image Men s a 152 image DISDIAY airera a E 152 CANIDFATC IMAG Cx iee a aa E EE E EE E ioe 165 OL IVIACMMICAUOM ees eira E ot E E TAA 167 DEEE gt EA A E E E A E E E E 167 Overlay Bar secs cid ecc eesti ens aa ade oe E EE 172 Delete Overlay ssnsti deities luak aed slab E Eai 181 SOW M S i a a N 181 Contents DENE HES log tee teee ttt to oe aera Gos th oot eee ett N sa tratsae 181 SO ee UM ie ae rte a a torstene case Ges tee ape see 185 FMD Geir Lane aias R NE aly 185 CONVO caren n Ea a AS rE a N R 185 SEPE an aan eria a E A 187
186. ated Click the Options button to define the parameters for the animated slide show Frame rate fi B Framers Loops Play f B time s Auto Repeat cae In the Frame rate field you can adjust the number of frames displayed per second 23 Button Bars The Image Analysis button bar In the Loops group you can choose how often the image stack is to be played Select the Play option to have the animation repeated n times Enter the number of times you want to have the animation repeated in the field Select the Auto Repeat option to repeat the image stack animation continuously until the Stop button is clicked In the Direction group you can select the direction of the animation Choose here whether the stack is animated unidirectionally or meandering back and forth The Image Analysis button bar Image Analysis x Sakae ooo O e Perene neos nen ene 0 e E a a Separates and resorts the contribution of different fluorochromes to the total signal in each color channel see also on page 275 Adjust Display a Adjusts the display of the image see also on page 152 Auto Adjust Display JRA Adjusts automatically the display of the image see also on page 158 Load LUT Load display LUT for a single color channel see also on page 160 Combines different images in one see also on page 188 Extracts the selection see also on page 187 24 Report Generator Report Generator Report Generator What exactly
187. ator Rank Sigma DCE Separator Performs a rank filter to suppress noise Use this filter to remove spot noise from the original image The rank filter adjusts the pixel values in the center of the filter matrix to the gray values of the immediate surrounding area To filter statistical noise out of the image use the Sigma filter Parameters of this filter can be defined using the Define Filter gt Rank command see page 235 Performs a sigma filter to suppress noise Parameters of this filter can be defined using the Define Filter gt Sigma command see page 236 Performs DCE Filter The DCE filter Differential Contrast Enhancement enhances weak contrast differ ences only This means that image structures that are barely distinguishable from one another in the original image will become visible in the resulting image Resulting images become more detailed and appear more focussed The parameters of this filter can be defined using the Define Filter gt DCE command see page 238 Separates parts of an image Use this filter to compute the outlines of connected gray value areas and to separate particles in contact with one another Parameters of this filter can be defined using the Define Filter gt Separator command see page 239 Image Calculator Calculating images means applying a formula to the pixel values of one or more images The result of a calculation is once again an image
188. ay value modu lations are to be enhanced 238 The Process menu Define Filter Note Separator The result of the DCE filter depends on the size of the image area in which you apply it This means that the image area in the smaller preview window may look different than the completely filtered resulting image This difference will be even greater if the image areas not shown in the preview window have very light or very dark pixels Enter a whole number between 0 and 100 into the Bandwidth field or do the same using the scroll bar The bandwidth parameter defines the range of gray values inten sity the lesser gray value modulations are comprised of Enter a whole number between 0 and 100 into the Enhancement field or do the same using the scroll bar The Enhancement parameter determines the factor by which lesser gray value intensity modulations are to be enhanced Which value is the most suitable for differential contrast will depend on the bandwidth selected Employing a narrow bandwidth in conjunction with strong enhancement will yield the greatest differential contrast Image detail will be accentuated whereas larger image struc tures will take a step back into the background Select the Quality check box to have image artifacts of the DCE filter reduced This will lengthen the time required to apply the filter The DCE filter can produce image artifacts that appear to be scratches These are only found within large
189. b documents It provides file formats for all docu ment types Cancel Help Diagram Formats Module Formats 4 Click the Up One Level button to move up a level in your computer s directory structure gt In the field below the button bar you will find a list of all sub folders and documents of the file types selected 5 Doubleclick on one of the folders listed to get a listing of its contents i e all subdirectories and files the folder contains e Your program s root directory contains the Images subdirectory In it you will find a selection of TIF images 6 Click the Preview button to view thumbnails of image files Select the image files one ata time 7 Select the images you wish to load 8 Click the Open button to load the images selected gt The Open Image dialog box will be closed gt The images will be loaded into successive image buffers The first image will be loaded into the active image buffer e g 5 The next images you load will then be inserted into image buffers 6 9 if you have loaded a total of 5 images simultaneously Activate image window Sometimes the image window will be hidden behind another window This is the case if a document window has been maximized or if numerous other documents have been opened The following step by step instructions show you only one of the ways of bringing the image window back to the foreground 1 Select the Window gt Document
190. b is not automati cally updated by changing the adjustment of the device 103 Acquiring images Configure Input XY Calibration Configure Input XY Calibration Calibrates pixel size Configure Input x Info Calibration Magnification Macro Input Display Format Magnificatior fi Calibrate amp Calibration 0 00345 mm F Unit Y calibration 0 00345 mm F Eepe part patio fi I Fixed Acquire Snapshot Calibration iiel f Arbitrary Horizontal c Vertical Calibration length fi mm OF Cancel Help The Magnification field shows the magnification taken from the Image gt Set Magni fication command as the default value If necessary the magnification can be modified here The X calibration Y calibration X Y ratio fields display the values of the last calibra tion When changing one of these parameters the two other values are adapted auto matically When the Fixed check box is selected the X Y ratio remains the same when cali brating at different magnifications In this case it is only necessary to calibrate one direction The other direction will be adapted automatically If the check box is switched off you have to calibrate in both directions When calibrating the system for the first time switch off this check box and calibrate both directions Otherwise the results may be incorrect for later measurements In the Calibration group you determine the direction o
191. background is the entire area defined by the text object e Use the Line tab to set the frame s properties its width and color Warning You always determine the properties for the whole text within a text object This is lt gt why individual words cannot be put in bold or italics If this is what you want then you ll have to compose the expressions using separate text objects 46 Report Generator Report objects Grouping text objects Aligning ct texts Insert Auto texts Different page templates Formatting text variables Select and group several text objects so that you can alter font properties type size and color background color and text frame for all the text objects simultaneously When you wish to align text objects using the functions of the Align button bar the vertical alignment of all the text objects being aligned should be the same The reason for this is that all the functions are for the text objects not the texts them selves If you want to align text within the text frame then go to the Text tab in the text properties a Autotexts Autotexts are a specific kind of text objects whose contents are independently deter mined You can use autotexts for reports and report templates Click this button in the Report Objects button bar to insert an AutoText The Select AutoT ext dialog box offers you all of the available AutoTexts For example Insert the autotext called Page Number to have
192. bjects select and Dell 8 Click the Field button in the Report Objects button bar to add a new field object to the record gt The Select Field dialog box will be opened This is where you ll find all data base fields that are defined in the active image database 9 Select the check box in front of the desired database fields and confirm with OK gt The mouse cursor will change its shape Click on the spot approximately where you wish to have the field objects placed Once the field objects have been inserted you can position them more precisely gt The Select Object Template dialog box will be opened This is where you select a template for a field object Should your report template contain a single format for field objects or not the field object will be automatically inserted in a standard format gt All selected fields are inserted in the record object underneath one another 10 Click the Finish Object Editing button in the report window button bar to save the altered record object 11 Enter a name for the altered record object into the Finish Object Editing dialog box and click the Save Object Template button to terminate adaptation of the record object to your database 12 Use the File gt Report gt New command to save the report templates under another name 45 Report Generator Text objects 13 Use the File gt Report gt New command to produce reports based on the newly adapted report
193. bration Calibration of the plane Scale s 0 067 pm Seale r 0 067 pm Calibration of the range Frames Offset 2 5 pacing Enter the offset in Z direction in the Z Offset field The offset determines the position of the first image of the Z stack relative to frame Z 0 In most cases a Z Offset is not needed Enter the distance between two frames in the Z Spacing field Click the Unit button to select the unit for the Z calibration The Set Unit dialog box will be opened Set Magnification Sets the magnification of the current input channel or the currently active image This command opens the Magnification tab of the Configure Input dialog box see also on page 106 The new magnification will remain valid for all subsequently acquired images of the selected input channel 165 The Image menu Scale Bar Note Be sure to change the magnification on the Magnification tab if you change the magnification of your microscope You might want to reconfigure the system to always prompt this dialog box when issuing the command Acquire or Snapshot To do so use the Macro tab located in the Configure Input dialog box For correct cali bration make sure that you have calibrated the active input previously If you have not established a calibration of your input device the Set Magnification command has no meaning since the image will remain uncalibrated You may also use this command to set the magnificatio
194. bring the fluorescences into the order you want In the Acquisition options group select the Manual option Select the with preview check box Select the no message check box Only with the motorized microscopes of the BX51 61 type or the IX71 81 type do you select the Automatic option in the Acquisition options group instead In the Fluorescence list select the first fluorescence In the Camera settings group select the Link exposure times check box not available for all camera types Leave the binning of single frames at 1 Click the Preview button in the dialog box Set the exposure time in the Preview field in such a way that the fluorescent structures in the live image are optimally visible and the image noise is minimal Only with motorized microscopes of the BX51 61 type or the IX71 81 type do you set the absolute Z position for the first fluorescence by focusing and for the subsequent fluorescence the relative Z position by reading instead Click the Snapshot button in the dialog box Then define the exposure times for all of the other fluorescences in the same way Click the File button and save the acquisition sequence Click OK to close the dialog box 116 Acquiring images Multiple Fluorescences Acquiring a multi channel image 1 Delete all of the images in the image buffer box 2 Use the Image gt Set magnification command and enter the objective s magni fication Then click on OK 3 Click
195. button to define a new input channel The Select Device dialog box will be opened This is where you select your acquisition device or signal type from the list of Available devices Click the Duplicate Channel button to copy a selected channel along with all its settings and thus to create a new channel with these settings The name of the copied channel will have a 1 appended to it Click the Configure Input button to open the Configure Input dialog box There you can define the parameter for the new input channel This button is not available if the active channel is write protected You may also open this dialog box simply by doubleclicking on the input channel Click the Delete Channel button to delete a selected channel This button is not avail able if the active channel is write protected Click the Device Properties button to define general settings for the GrabBit PCI These settings are global i e they apply to all input channels which use the GrabBit PCI This button it opens the GrabBit Device Properties dialog box is not available for all frame grabber boards Click the Close button to exit the dialog box The selected input will become active i e the next image acquisition will be via the settings defined for this input 112 Acquiring images intelligent Exposure intelligent Exposure Calibration Exposure times Acquisition Snapshot Exposure time correction Starts up the automatic acquisition
196. calculated as follows Gray values of the original image Gray values of Matrix the filtered image 20 8 29 10 53 32 0 27 80 Filter classes The various types of filters can be divided into various classes This division has more to do with the application of the filters than their mathematical real ization Negative weight factors are admitted in the filter matrix of derivative filters If the sum of the weight factors equals zero the derivative filter will yield a value of 0 for homo geneous gray value areas These filters provide a scale for the intensity of transi tional gray values Derivative filters accentuate gray value edges i e the edges of objects within an image These filters can aid you as far as edge extraction is concerned The occurrence of differential terms will however increase image noise 225 The Process menu General Information on Filter Operations Application of various derivative filters N on on Gray value Differentiate X Laplace Sharpen Il Pseudo X Position The illustration shows a comparison of the effect of various derivative filters The orig inal image is comprised of clearly defined light as opposed to dark structures which have relatively clear edges To the right are the intensity profiles before and after filtering via two conductors After application of the Differentiate Y Laplace and Sobel filters the only image structures sti
197. cale bar the image information where the scale bar is will not be lost Note Please keep in mind that other application programs cannot read nor display an image overlay This means you will need to burn the image overlay including the overlay scale bar via the Burn button in the Overlay button bar before exporting it Burn into Image Merges scale bar permanently into image Use this command to have the current scale bar selection irreversibly drawn into an image All image information where the scale bar is burnt will be lost Note Please keep in mind that this command cannot be reversed because it in fact results in a new image being generated The original image will be overwritten when you apply the burn command A scale bar that has been burned into an image can of course no longer automatically be adjusted to fit image size because it has become a part of that image One of the main reasons for burning a scale bar into an image is when you are preparing to export an image into another application program e g MS Word which cannot read the image s calibration data Even though you have burnt the scale bar into the image the image calibration remains accessible In this case you should be sure to burn the scale bar when copying the image into the clipboard To do this select the Clipboard check box in the Show scale bar for group in the Scale Bar Properties gt Display tab This allows you to retain the unchanged image for
198. call up this tab histogram calculation will be based on the whole Decide whether image structures are to be light or dark in the Background group ground Histogram limita E Overflow U l EA image Select the Low option if image structures are light and the background dark The Select the High option if image structures are dark and the background light 214 The Process menu Set Thresholds Note Select the None option if you wish the whole gray value range to be divided up into n phases n the number of phases desired Select the Preview check box to have the phases displayed in color The phases will be displayed in the standard colors as shown in the color bar beneath the diagram Defining the portion of the image to be used in histogram calculation is done in the Histogram limits group You can exclude gray values from either the upper or lower end of the histogram Select the Dynamic option to have the relevant gray value range calculated for each image individually The values in Underflow and Overflow are for you to define what percentage of an image s pixels at the upper and lower end of the image s histo gram are to be excluded from the threshold setting procedure Select the Fixed option to have your thresholds set within a fixed gray value range The values you enter into Lower limit and Upper limit define this fixed gray value range For the calculation of your thresholds enter the lower gray valu
199. ce is to be carried out and how much time is to be left between two cycle starts Enter how often the defined cycle is to be carried out into the Number box Enter how much time there is to be between two cycle starts into the Repeat every box The value will generally be limited by the length of the acquisition processes which are carried out within the cycle If your defined cycle requires more time than allotted by the entry in the Repeat every box your image analysis program carries out the acquisitions as fast as possible The Acquisition group enables you to determine which acquisition method is to be used in the process The methods at your disposal depend on the add ins of your system and hardware attached to your system Click the Define button after having selected the desired mode from the picklist Select the desired acquisition method from the Mode picklist 136 Acquiring images Define Image Sequence Enter whether or not you want to save the images acquired directly as files on the hard disk or if they should be saved to a database in the Save image group Define Image Sequence Settings Defines how any connected hardware components are to be used for the sequences MS Define Image Sequence Processing Sequence m sari 7 ca gl EIR Stage Camera uer EEF Sequence Frame 1 setting C Info Flow settings Macro Stage Fath E Default New position 3350925 pm Y pos
200. ch new ROI The color makes it easier to iden tify the ROIs on the image 4 Exit the Define ROIs dialog box via the Close button You can measure the area of any defined ROI To do so use the Measure gt RO gt Area command You can measure the perimeter of any defined ROI To do so use the Measure gt ROI gt Perimeter command You can measure the pixel heights within a defined ROI To do so use the Measure gt ROI gt Average Intensity command You can measure the average intensity within a defined ROI To do so use the Measure gt ROI gt Average Gray Value command Define ROIs dialog box Define ROIs Ea Ej Draw Parties Color Delete All Active AOls Tools Load The label you enter in this field will be assigned to the next ROI to be drawn The label is displayed on the image in the overlay and appears in the Active ROIs list If a name has already been given to a ROI the program adds a number to it when it is assigned to a new ROI Use the button with the arrow pointing to the right besides the Label field to open a menu containing three commands e The Delete Selected Label command clears the entry in the Label field e Change Selected ROI Label allows you to edit the entry displayed in the Label field e The Change Selected ROI Color command assigns the currently active color to the selected ROI The Color field shows the active color It defines the color of the next ROI frame
201. check box has been selected the starting grid frame i e the red rectangle can be positioned as desired within the image The grid will then start not in the upper left corner rather where the red rectangle has been positioned Rightclick to have the grid drawn as defined and to return to the dialog box The current values for height and width are displayed in the Gridsize group Intensity Profile Intensity Profile gt Horizontal Creates a graph of the gray values along a horizontal line How to measure a horizontal intensity profile 1 Select the Measure gt Intensity Profile gt Horizontal command A horizontal red line will appear in the overlay The corresponding curve is instantly displayed in the graph window 2 Position the red line where you wish to measure gray value intensity and then leftclick The line will be drawn into the overlay 3 If you drag the mouse another red line will appear and can be placed as wanted 4 Terminate the measurement with a rightclick The line profiles of all color channels will be drawn into a separate graph for each of the lines drawn into the image overlay 295 The Measure menu Intensity Profile gt Vertical Intensity Profile gt Vertical Creates a graph of the gray values along a vertical line How to measure a vertical intensity profile 1 Select the Measure gt Intensity Profile gt Vertical command A vertical red line will appear in the overlay The corre
202. check box to rotate the image counterclockwise to the left Click the Interactive button to define the rotation angle interactively on the image The illustration shows the order in which you make the necessary clicks 3 1 263 Note Fill Image The Process menu Fill Image Image calibration remains unchanged following rotation Decompression Use this transformation to blow up or shrink an image in a user defined direction The decompression is a combination of a shearing and resize function The image is first sheared in the given Direction and then resized by the given Facior The illustration shows three sample decompressions The left image is the original image the right image shows the resulting image The direction is indicated in red the used in blue Size Use this transformation to resize an image Enter a factor greater than 1 in the Size field and the image will become larger a factor of less than 1 makes the image smaller If you enlarge an image to a zoom factor of 2 this means that for every pixel in the original image 4 pixels of the same color will be used in the resulting image When you use odd zoom factors such as 1 5 not all pixels will be doubled This may result in e g beam or bar structures that were symmetrical in the original image being uneven in width in the resulting image Much of this can be balanced out if you use interpolation as you zoom When you reduce an image i
203. circle the three color components will be distinctly different than 1 You can then use white balance to bring many of the image s pixels to saturation This means that many pixels color components will be given a value of either 0 or 255 such that any shadings of colors within the image will disappear This result is something that can be used as a kind of comparative criterion for determining whether white balance has in fact corrected an actual color tinge If any details within the image in question are no longer visible you know that you ve altered the actual existent colors and that you ve corrected either too much or in the wrong way Leftclick within the image A red circle will appear in the overlay Position this circle with the mouse To enlarge or decrease the circle keep the left mouse button depressed as you move the mouse To confirm the circle and apply the white balance rightclick If you decide to cancel the white balance simply middleclick you will thus not alter the image and will return to the Graphical User Interface GUI To Gray Scale Image Converts a false color image to a gray value image 207 The Process menu Adjust Colors Adjust Colors Define Intensity Adjustment Sets the gamma brightness and contrast value Use the Execute Intensity Adjustment command to apply the current settings in the Define Intensity Adjustment dialog box to the image in the source image buffer Define Intensity
204. ck the Top button to set the maximum lift your microscope stage is to have A message will appear instructing you to move the stage via joystick to the topmost position desired and then to click on OK You ll know you ve reached the relevant topmost position when the lowest regions of a specimen appear in focus Click the Bottom button to set the minimum lift your microscope stage is to have A message will appear instructing you to use the joystick to move the stage to the lowest position desired and then to confirm this You ll know you ve reached the rele vant lowest position when the highest points of a specimen appear in focus These stage positioning limits called end positions could also be set the other way around EFI makes no distinction between where top and or bottom are The Z Distance field displays the distance between the two end positions in micrometers Your microscope stage must be height calibrated Click on the Execute button to begin automatic image acquisition of your focus series according to your settings The Auto Archive button is available if you ve opened one or more databases Click this button to have the focus series you ve just acquired directly inserted into the active database The focus series will then be stored in the database including the 133 Acquiring images Define Image Sequence height map and the EFI image as a single data record plus appended images
205. closed The image document window will be re maximized and will show the resulting image Click the File button to open the standard dialog box for the opening and loading of files Save the filter sequence you are currently applying to an image by clicking the Save button To reload filter sequences you ve saved click on the Load button Files will be saved in the FLT format When you click the Load button in the Save Load Filter Sequence dialog box the filter sequence you have selected will be applied directly to the original image That is to say the filter sequence is applied to the unfiltered image before executing the Apply Filter command not to the image as it may currently be displayed A filter sequence you have loaded can be incrementally reversed by clicking the Back button in the Apply Filter dialog box As you reverse the filter sequence the dialog box will show the respective filter function and its parameters as you reverse them This is how you can have a look at what the component filters of a sequence are any time You can also replace one filter with another or set a different parameter value You then execute the subsequent filters and then save the altered sequence One fairly typical application for filter sequences is saving various standard filter sequences and then applying them to an image and trying out which one obtains the best results 259 The Process menu Apply Filters Filter List Available fi
206. command is only available for 8 or 16 bit gray value images and for binary images not for 24 bit true color images or multi dimensional images Modify Gray Values Function Change Save Brightness ct l AIE Cancel Contrast a sao Help ddd 200 The Process menu Intensity Keeping track of gray value changes in the image document Active Frame The Modify Gray Values command allows you to define a mapping function for adjusting each gray value of your original image assigning new gray values for the resulting image This is a way for you to define the intensity and contrast of the image as you see fit such that image structures of similar gray values can either be accen tuated or suppressed This command is global and affects all gray values and pixels of an image To edit the gray values of individual pixels use the Image gt Edit Image command The XY diagram of this dialog box displays the two curves gray value distribution histogram of the original image in red and the mapping function in blue You can define a new mapping function by either drawing a new curve directly within the diagram or by using the two scroll bars beneath the XY diagram If you wish you can save the new mapping function for later use and application on any other image s Any gray value changes effected by the current mapping function will immediately appear in the image Keep changing the mapping function unt
207. configurations For this reason it is quite possible that in this manual functions will be described that are not contained in the software package that you use or that inversely certain functions that are available to you are not described here The software is protected by a dongle It is standard that an LPT dongle that has to be plugged into your computer s parallel port comes with the software USB dongles are required for laptops or computers without parallel ports e cell can neither be installed nor started without a dongle e The dongles are differently colored depending on their type USB Dongle LPT Dongle Scope of license black blue limited time dongle which only grants the user access to the software for a limited period of time network dongle e A network dongle can be plugged in to any one of a network s computers Please keep in mind that before cell can be installed the driver software for the network dongle has to be installed first The Setup menu includes an option for installing the driver software for the network dongle e f your printer is connected to the parallel port the dongle must be inserted be tween the PC and the printer cable First Steps First Steps First Steps The GUI User Interface GUI Menu Bar The graphical user interface influences the appearance of a program It determines which menus there are how the individual functions can be called up how and where files e g i
208. cquired or the live image freezes This command corresponds to the Acquire gt Snapshot command Magnification Table Shows magnification and calibration data Magnification Table x Magnification Pixel width Pixel height ur urm 00000 0 17007 0 17007 Cancel 50 00000 0 33333 0 33333 20 00000 0 83472 0 83472 Add 10 00000 1 65289 1 65259 5 00000 3 35570 3 35570 Delete All Help Tpetel width O 1 pisel height eh Magnification For each calibration the dialog box contains a row with the Magnification the Pixel width X calibration and the Pixel height Y calibration The calibration data are also displayed graphically If the X Y ratio is not fixed you will see two different calibration functions for the pixel width and the pixel height Click the Add button to copy the current calibration values into the list on the left hand side within the sheet If the list already contains a magnification value the same as the current magnification a message will appear notifying you that the list only permits one value for each magnification You may now directly replace the existing magnification if you wish Clicking the Delete button deletes any existing calibration data for the selected cali bration set To enable this button first select a row from the list Clicking the Delete All button deletes all existing calibration data in the list 105 Acquiring images Configure Input Magnification Configure I
209. cquiring images Multiple Image Alignment Note Meander Horizontal Meander Vertical For arranging the images by the Comb Horizontal method select the Comb and Hori zontal options Additionally you can exchange two images directly in the image area by drag ging amp dropping Select an image by clicking the left mouse button on the image area keep the left mouse button depressed and drag the mouse cursor to the new position In the Correlation group you prescribe the correlation that should be reached when aligning the images A correlation is the mathematical magnitude used to describe the degree of coincidence between the overlap region of two neighboring images It iS a positive real value in the range 0 to 1 The nearer the correlation is to 1 the better the congruence between both patterns In the Quality field you enter the needed correlation The maximum possible value is 1 0 the minimum value 0 25 If the value entered is not achieved a message will inform you of this when the images are assembled You may then either have the images assembled using the available correlation or return to this dialog box If the quality value is 1 the images will automatically be assembled at the greatest available correspondence there will be no messages In this case you will have no opportunity for determining the correlation the images are assembled with If you select the No Correlation check box the overlap region of
210. criptive term for the organization of your documents All data that you enter into the database must then be allocated to a project folder gt Your image analysis program suggests the selected descriptive term with an additional No as the folder name The folder name defined here reap pears later as identifying term in the insertion mask 7 Click on the Next gt button 8 Click on the Next gt button without defining your own database fields 9 Click on the Finish button 64 Archiving Images Define a database How to set up an express database The name of the new data This is the directory base is ExpressData base01 where the database files and all data inserted into the database are saved ExpressD atabasel1 Database files apll g VarchivesE xpressD atabasel New Database How would you like to organize your database Te ceemsemesecssced The Wizard generates a predefined database you may shore noo daciiments in the MaMia iments falder as well Select Finish to let the database assistant create a database with the following settings Name ExpressD atabasell Location of database files g sarchiveExpressD atabasel1 Location of images and documents g archrveSExpressD atabasell Organized by Folder Organizational IO Folder name ll Save settee as your database lt Back Cancel Help amp ExpressD atabaseD1 apl x
211. ct it Use Shift Click to select multiple objects You terminate or exit the edit object mode by rightclicking Select All Select None Selects or unselects all objects in the overlay Click the Select All button to select all objects of the overlay The button changes to Select None Click the Select None button to release all objects from selection The button changes to Select All 172 The Image menu Overlay Bar Object Properties Note Fill Line Arrows Displays the properties of a single selected object The exact look of the dialog box depends on the kind of object selected Some overlay objects cannot be edited These kinds of objects have a red selection marker Colors and Lines tab Object Properties Colors and Lines Fill Color f Line Color D Dashed Weight i Arras Begin style End style mea o The Colors and Lines tab is for the most overlay objects Use this tab to define colors and lines of overlay objects Fill effects really change how an object looks Filling an object means that you assign colors and a particular pattern to the object s surface If you do not assign any fill effects to an object or cancel fill effects the object will remain transparent You can choose single color fills and fill patterns as well Use the Color palette to choose what color you wish to fill the object selected with Closed figures such as rectangl
212. ctively in the Function group To do so first you select a function and then define those parameters that are definable in the A B and Offset fields Start with the offset and then enter the A and B parameters Another way to define parameters is by defining so called control points After you have selected a function you define two pairs which are respectively comprised of a Gray value and a related Intensity The corresponding A B and Offset parameters will be adjusted automatically Define gray values either by clicking on the arrowhead buttons of the list entering them directly or by clicking on specific pixels within the active image If you click on the Interactive button the cursor will move to the image selected in the Image buffer field The cursor will change into a circle with a radius defined by Spot 1 32 pixel Clicking the left mouse button will enter the arithmetical average of all of the pixels within the circle into the Gray value field In the Quantity field you enter the name of the geometric or physical quantity used for the intensity calibration e g the Height This quantity name appears in the measuring sheets and replaces the term Gray Value as header of a column when measuring the gray values of an image with a calibrated image intensity The following commands from the Measure menu are affected Pixel Value Pixel Map and Intensity Profile 108 Acquiring images Camera Configuration If you c
213. d Since the program allo cates space for every occupied image buffer using this command will relieve a considerable amount of memory For example a true color image with a resolution of 2576 x 1932 pixels requires about 15 megabytes of RAM MS Windows will then start swapping memory to disk If you are using up too much storage your disk will have to be active more and more often thus slowing down the reaction time of this and other applications Deleting images is how you can free up RAM and thus speed up the programs you re working with 191 The Image menu Protect Image Note You will not receive a warning message If the image has not been saved as a file in a database or on the hard disk it will be gone for good To delete multiple images all at once select them in the image buffer box Press Shift or Ctrl while selecting the images with the mouse Now the command in the Image menu will be called Delete Images If the Shift key is pressed during opening of the Image menu the Delete all Images command will appear instead of Delete Image The contents of all buffers as long as they are not write protected will be erased and the allocated memory will become available once again Write protected images will not be deleted by this command Protect Image Toggles the read only mode for the active image buffer on off Use this command to lock up the image to prevent overwriting or accidental deletion removal The conten
214. d as a single frame and inserted in the grid display in the position of the red cross The red cross will move to the position in which the next image will be inserted In the live image the overlapping areas of the neighboring image that is now applicable will be shown Click the Restart button to resume the acquisition process The live image will be inserted in the first position on the grid Images that have already been acquired will be overwritten Click the Align button to have the single frames combined into a composite image The last single frame will be acquired The Manual Acquisition dialog box will be closed and the Align Multiple Images dialog box will be opened Combines two or more single frames to make a composite image The Arrange command assumes that the first image of an image series is in the active image buffer For the creation of a composite image at least two images must be present For this reason when you use the Arrange command the system will also check the contents of the image buffer following that of the active image Only if both the active image buffer and the following buffer contain images will the combi nation procedure start otherwise it will be terminated with an error message Another 143 Acquiring images Arrange prerequisite for the creation of a composite image is that the images making it up must all be of the same type i e you cannot make a montage consisting of gray value a
215. database 4 Use the Database gt Administration gt File Storage command to call up the file storage wizard 98 Archiving Images Protect with a password 5 Select the Archiving of Documents option Select the Lock archived records check box In this case you can no longer store any more files in closed database folders after archiving Click on the Next gt button 6 Select the database you want to backup To do this enter the path name of the database in the field or click the button to select the directory Click the Next gt button 7 Select your CD DVD drive from the list Then click the Auto detect button to automatically select the size of the destination drive 8 Click on the Next gt button 9 You will get a list of all of the database folders of the database which have not yet been archived or which have been altered after the last archiving Select all the database folders which you would like to archive These should be finished procedures To do so use the operating systems default key stroke Ctrl left mouse key marks single records Shift left mouse button selects a row of records Ctri a selects all the records Click on the Next gt button 10 Before starting archiving carefully check the data in the File Storage dialog box 11 Click the Finish button to begin archiving 12 Follow the instructions of the database wizard gt After successful archiving all of the archived
216. dated this enables you to judge whether the image structures you re looking for are being covered by the thresholds you set There are 2 color displays available to choose from e Normally all pixels within the gray value range of the active phase will be dis played in the color you ve selected in the Color list e Select the Transparent check box to get a false color display of the phase s gray value range Select All to have the colors of all phases displayed simultaneously in your image Select Background to have those gray value ranges displayed in color that haven t been assigned to a phase There are 2 color displays available to choose from e Normally the background will be displayed red e Select the Transparent check box to get a false color display of the phase s gray value range 213 The Process menu Gray scale Images Select the Transparent check box to have the active phase displayed using a special LUT Dark image areas will be appear green to light yellow This type of coloring has the advantage of enabling you to look at both gray value range and image structure at the same time allowing you to define thresholds with even greater precision This check box is only available in conjunction with the Current and Background check boxes Click the File button to open the standard dialog box for the opening and loading of files This is where you can save threshold settings or load threshold settings you have alr
217. default name 337 The Stage Navigator Change Stage Navigator Properties Change Stage Navigator Properties Here you can change the Stage Navigator s default settings for instance the color of the image frames Stage Navigator Properties Background color Grapimage T E Color image Camera area Color Image frames Color E Set IY Show Interest area images Mame Interest area Max width 1000 5 as Max height fiooo Pixel Automatically update active image buffer T Optimization of the images Image overlap Stage Preferences fo z 5 Preferences Cancel Help Background color In this group you can determine in which gray value the background will be displayed for gray value images You will see this background when you start the Stage Nav igator and the vacant area of the stage will also be shown in this color When you use colored images you can determine in which color the background will be shown This enables you to make sure that the color of the background always contrasts with the color of the overview image To make changes in the background color effective you have to restart the Stage i Navigator and reload the overview image Area Determines the color in which an Interest area will be shown Depending on the color of the sample it may be advantageous to change this color to make the frame more easily recognizable Camera area Determines t
218. defined e g the Material or Order number field in the data base and in this case you want to have this information included with each image In this case you ll have to adapt the record objects to your database Adapting record objects to your own database is usually carried out by a report template Only then are newly defined record objects which are based on this template available 1 Select the File gt Report gt New command 2 Select the Normal report template in the General tab Select the Template option in the Create new group 3 Confirm by clicking OK gt Your image analysis program creates a new report template The header of the window displays the name of the new report template Template1 and the name of the active page template 3 image page 4 Open the image database containing the fields you wish to make use of for future standard reports 5 Use the Record button in the Report Objects button bar to insert an existing record object gt The inserted record object has already been selected 6 Click the Edit Object Template in the report window button bar to switch over to the edit object mode gt The edit object mode is where you can select and edit the separate elements of the record objects selected Any other objects defined on the page are displayed with a crosshatching pattern only for reference purposes 7 Select all field objects that you wish to replace with your own field o
219. dition subtraction multiplication or division the gray value range of the result s may be greater than that of the original image s When the 8 bit or 16 bit value range is exceeded that gray value will be displayed as 255 or 65535 respectively Negative values will be set to 0 To avoid this effect we recommend you reduce the gray value range of the sources first for example by dividing by suitable factor Boolean Operators The operators AND OR and XOR work on images bit by bit For each bit of gray value the corresponding logical operation will be carried out For gray value images the binary representation of 8 or 16 bit numbers must be taken into consideration Arithmetic Addition A Constant or the Source 2 image set will be added to the active image Source 1 Overflow clipping will be performed at 65535 for 16 bit data and at 255 for 8 bit data AND Pixel values in binary form are compared bit by bit If the bit is 1 for both images the result will be 1 otherwise it will be O Example 8 bit Source 1 170 10101010 Source 2 15 00001111 Result 00001010 10 Division The active image Source 1 will be divided by a Constant or by the Source 2 image set Digits after the decimal sign will be clipped Maximum The images in the Source and Source 2 image buffers will be compared with each other The resulting image contains the greater of the two gray values The resulting image will thus have maximum intensity
220. dition to that you will also find the buttons required for placing brackets The functions min and max have a special position since they use two operands instead of one The two operands are separated by the coma which can be found in the lower right of the function field Function gt Description exp A gt exponential function of A In A gt natural logarithm of A sqrt A gt square root of A 248 The Process menu Image Calculator Operand Box Scaling log10 A gt logarithm to the base 10 of A min A B gt minimum of A and B max A B gt maximum of A and B abs A gt absolute value of A You use the calculator s operand box to define the image buffer which you want to apply to the formula The buttons Source 1 and Source 2 designate the image manager s image buffers Source and Source 2 Operands which you attach like this to a formula do not yet mark any certain image buffer but are defined in relation to the image manager Only during the calculation are these operands replaced by the images defined in the image manager Click the Fix button if you want to make a calculation using a certain image buffer The list enables you to choose which image buffers are to be respectively acquired into the formula Image buffers are always described by a fixed character string Operand gt Meaning in the Image Manager Src1 gt Source Src2 gt Source 2 Img1 Img2 gt Image 1 Image 2 The result
221. does Automatic report generation report generator Use report generator to have multipage reports produced practically automatically do including images of a database or of the image manager Select a number of or lots of images from an image database and have them all added to a report using a single command Full database integrated access Along with the images themselves that you get out of a database you can have all additional information on the images contained in database fields of image data bases automatically included in a report Sheets with important measurement results can also be automatically filled in Working with images A particular focus of report generator is being able to work with images in an optimal way norm enlargements are followed detail zooms can be inserted appropriate image segments can be selected and more Texts Sheets Diagrams Graphs Most types of documents that you generate within your image analysis program can be inserted into a report Via report generator you can e g print out images along with related measurement sheets and diagrams on the same page Flexible Page Layouting Report generator provides you with the most flexible page layouting imaginable you set up your own template pages exactly the way you want them to be You generate your template pages only once These templates are the basis for your reports and ensure that the appearance of your documents is uniform MS
222. dows Bitmap BMP Windows Icon ICO and Enhanced Metafile EMF Object Properties for BMP objects Bits per Pixel Which properties of an inserted bitmap can be edited depends on the bit depth This is why the dialog box tells you what the bit depth is Color keying You can take a specific color within the bitmap and assign it a new color This color reassignment within a bitmap is called color keying You can also assign a bitmap color the color value which makes it transparent All pixels that have the selected color value will no longer be shown in the overlay The original image will then be visible at these pixels positions You can make use of this transparency option to keep the amount of image information covered up by an inserted bitmap e g a company logo to a minimum Foreground The Foreground palette is only available for monochrome bitmaps are black and white White is considered a foreground color and black a background color Choose any color you like from the Foreground palette All black pixels will be shown in the color you selected 174 The Image menu Overlay Bar Keying For bitmaps with a color intensity greater than 1 bit pixel select the bitmap color you wish to have replaced You select the color being replaced in the Keying palette The replacement color is selected in the Background palette The Keying palette is not available for monochrome bitmaps Background The background of a bit
223. e Next Page Last Page Add Page Delete Page E Set Zoom 26 aisle Teee al L Finish Object Editing Edit Object Template Leave Page Template Save Page Template Edit Page Template Redo New Page Template Save Report Document Undo You can only display one page in the report window Use the first buttons of the Report button bar to leaf back and forth through a report When paging the current report page will automatically be saved If you want to get to a particular page fast enter the page number into this field and confirm via Enter Click the Add Page button to add a page to the active report at any place After clicking the button select where the new page should be inserted You can add a new page either in front of the active report page or at the end of the report You have to indicate a page template for each page This determines the pages appearance s The newly added page will become the active page no matter what page was being shown before the new page was inserted Click the Delete Page button to delete the report s page which is currently displayed in the report window You have to confirm the deletion of a page Any and all image files and thumbnails in connection with the page will also be deleted Images inserted into a report as links will of course not be deleted Define the page format for the current report in the Report Properties dialog box Border and Format groups In addition dete
224. e belongs in the database During the image acquisition the image will be automati cally inserted into the database 75 Archiving Images Insert Images Multi dimensional You can insert multi dimensional images into a databank in exactly the same way as image you insert any other images You can recognize a multi dimensional image by the Multi Dimension entry in the predefined Image Type database field otep by step How to insert images from the image buffer box The step by step instructions apply subject to the following prerequisites You have set up an experiment database The database already contains a database folder You have acquired images during this experiment which are to be filed in the data base below the database folder 1 Mark the experiment for which the images are intended in the database win dow s tree structure gt In the database window you will find a preview window The preview window shows the record which is marked in the tree structure If a data base folder is marked then a folder icon is displayed 2 Mark all the images in the image buffer box that you want to insert e You mark a linked image buffer area by depressing the shift key and click ing on the first and last image e You can mark a random selection of images by depressing the Ctrl key and clicking on the images you require 3 You can use drag amp drop to move the images from the image buffer box to for example the preview
225. e fields are linked with AND You will only find the images which fulfill all the search criteria simultaneously e The query mask offers you a selection assistant for all date fields Click on the button next to the date field A dialog box with a predefined date opens Click on the arrow to open a calendar in which you can select the date that you need 3 Click on the Advanced gt gt button in the query mask Click on the Unselect All button Mark the check box in front of the Image data type to limit the search to images Other documents will not be found 4 Click on the Search button to carry out the search gt The images found will be displayed in a separate database folder with the name Query results The query results are displayed directly under the database icon in the tree structure gt Some information about the current query is displayed in the form for example the number of records that have been found gt The current marking jumps into the query results automatically 91 Archiving Images Load data The Query results ig Pe dete shows amp LifeScienceDemoS TAR apl all the data that has Ss a CSB Ele wala Al x3 been found The form contains information about the query Query results i Ey LifeScienceD emoS TAFE ms al Query results Query type Quer by example E C FISH SQL command select from Images where Reco E CJ Time Lapse fast Matching records 3 H CA Autofluorescence w
226. e image buffer box in the selected order If one of the image buffers is write protected your image analysis program stops loading at this point e Please note images in image buffers that are not write protected will be overwritten without warning 93 Archiving Images Load data If you mark several records the records in the gallery are sE numbered on the top right in the order of selection Select the Load Document command to load all the selected images into the image manager in the order selected amp ExampleD atabase apl cal lee Ee ele ala i a amp y at moe ra LE Choose View Set Wetrauiblser rela Velwer i a aa Insert b Spheres Skorodit Goto Record ExampleDatabase 4 Filtering unfiltered image Essel iitni Dieses tients Arrange Fields ony Tene Settings view Butan gar Define Contest Menu How to load all the data within a database folder 1 Mark a database folder in the tree structure 2 Select the Database gt Load Document with Subdocuments command if you want to load all the records within the database folder gt Images will be loaded one after another into the successive image buffers of the image buffer box gt Documents are opened in your image analysis program these are for example the sheets with the sfs file name extension and the diagrams with the sfd file name extension gt Other document files are loaded in the appropriate
227. e same folder as the overview image The consecutive numbering of the images will be continued Example The overview image is made up of the images lt Image name gt 1 tif to lt Image name gt 12 tif The first image with the higher magnification will be named lt Image name gt 13 tif gt In the overview image you will be shown which areas you have acquired with a higher magnification The image frames will also show these areas unless you have deactivated this display in the Properties gt Image frames field 340 The Stage Navigator Zooming in on an image An area of this overview image has been acquired a second time with a higher magnifica tion You can recognize it by the Grid in the grid fr ae soe go s fii Zooming in on an image 1 Click the Zoom in button to see an area in the overview image at a higher mag nification gt Ifyou have acquired this area once again with a higher magnification these images will be reloaded If not you will only see the data contained in the overview image zoomed in gt without any additional content 341 The Stage Navigator Adding Stage Navigator positions This ts the overview image shown at a high zoom level The areas that have been acquired once more with a higher magnification contain significantly more data E Stage Navigator Testi Z position 9815 00 Adding Stage Navigator positions If you want to memorize a particular area
228. e shift key and click the left mouse button 4 Click the right mouse button to finish the definition of the line Your image analysis program will automatically connect the starting point and the endpoint 5 Continue defining shapes by the active drawing method 6 Depress the middle mouse button or the Esc key to return to the Add Shape dialog box Ellipse This is how you define a shape with the Ellipse method 1 Click the Ellipse button The mouse pointer moves to the image window A circle or an ellipse is dis played in the overlay 2 Move the mouse to move the circle at the desired position 3 Move the mouse while pressing the left mouse button to distort the circle to an ellipse and or to change its size 4 To draw a circle use the shift key 5 Click the right mouse button to end the definition of the ellipse 6 Continue defining shapes by the active drawing method 7 Depress the middle mouse button or the Esc key to return to the Add Shape dialog box Rectangle This is how you define a shape with the Rectangle method 1 Click the Rectangle button The mouse pointer moves to the image window A rectangle is displayed in the overlay 2 Move the mouse to move the rectangle s position 3 Move the mouse while pressing the left mouse button to change the rectangle s size 4 Click the right mouse button to end the definition of the rectangle 5 Continue defining shapes by the active drawing method 6
229. e shown Select the Hexadecimal check box to have the gray values in the matrix displayed in hexadecimal format Clear it to have them shown in decimals Select the Color check box to have gray value numbers in the matrix displayed in colors shaded according to the gray value they represent With an 8 bit gray value or pseudo color image the color for all 16 gray values will change from black via green blue and red back to black and so on With a 16 bit gray value image the color of all 256 gray values will change in the same order of colors Binary image gray values at O which is black will be displayed in black those values at 255 white in red Image area structure is thus easier to follow in the matrix The Color check box is only available for 24 bit true color images if you have selected one of the three color values If two or all three are selected the color value numbers will be displayed in one color The three lower check boxes in the Options group are only available for true color images They are called Red Green and Blue if the RGB color model has been selected in the Color space group Hue Saturation and Intensity if the HSI model has been selected Select the check box needed to have the corresponding color displayed in the matrix Up to three color values can be displayed per pixel The Color space group is for determining the color model components to be displayed in the matrix only available for true color images
230. e BX IX steerage console and click the button 4 In the Acquire Z Stack dialog box click the Start button The current Z position will be saved as the maximum position the stage will travel in this direction The value will be shown in the right field next to the Start button 5 Now turn the focus wheel in the opposite direction and click Stop to define the maximum position the stage will move in this direction Again the current Z position will be saved and its value shown Note For the acquisition of the Z stack it does not make any difference in which order you define the two positions that limit the Z range The stage will always start the acqui sition at the Start position then travel through the whole Z range and stop at the Stop position 6 In the Z Resolution group define the number of images to be acquired in the Frames field The system will calculate the correct Z spacing between the frames and show the value in the Z Spacing field For example if the focus range is 50 um and you would like to acquire 10 images the Z spacing will be 5 5 um 50 um divided by 9 steps gt After every change the Z Distance field will show the complete range that the stage will move through 7 Click the Acquire button to start the acquisition Acquiring an image stack with the optimal Z Spacing To acquire an image stack with the optimal Z Spacing before you begin the acqui sition click the Z Spacing button to
231. e Equalize Contrast command changes the gray values behind the image s active look up table LUT The LUT has no effect on how gray values are adjusted The LUT itself remains totally unchanged The coloring of the resulting image may differ from the original image This is because the active LUT may assign different colors to the adjusted gray values which means that the coloring of the image onscreen will look different The Equalize Contrast command may alter the image s coloring A color that occurs frequently in the image will be spread meaning that the color will be displayed using a more varied array of shades of that color in the resulting image This means that coloring differences within the same color range will become much more evident so that specimen structures will stand out much better All in all dark images will become brighter and vice versa If you are however working with an image with strong contrasts and significant variance in brightness levels after using this command the resulting image will seem weaker in contrast because any strong fluc tuation in intensity has been balanced out equalized Optimize Contrast Performs an automatic optimization of contrast This command is only available for 8 bit or 16 bit gray value images and for 24 bit true color images however it is not available for binary and multi dimensional images This command is only available for false color images if the Special
232. e Phase Color Coding command Define Classification Defines the classification scheme Define Classification Classification LU rit New classification Sample Area s Classification parameters mm ran PEEP TEL ee Cancel Minimum Masmum 10000 Bins E Mode Linear 1250 00 1250 00 2500 00 2500 00 ar 50 00 ar 50 00 S000 00 2000 00 6250 00 6250 00 fo00 00 FO00 00 ar50 00 aro0 00 10000 00 Delete Unit Set colin CL b Compute Sheet l Show sample objects Help A Classification scheme is made up of a name a unit and a class division or clas sification Measurement values can be divided up into certain classes using classifi cation schemes A Class is a range of values within a classification scheme defined by a lower and upper value limit Each class is identified with its class ID this being one of a series of running numbers 1 2 3 Each class can be named and labeled with a color A classification scheme can be used to classify measurements in one sheet column Select the classification scheme needed in the Histogram dialog box in the Measure menu To open the dialog box click with the right mouse button on a sheet Select the Histogram command from that context menu The number of measurements per class can be displayed either in a sheet or in a diagram The Classification list displays the current classification scheme The
233. e Zoom Factor dropdown list or enter the zoom factor you want directly in the field e g 30 gt The test image will be reduced to 30 zoom The viewport will no longer be totally taken up by the image Where the patterned background starts in the viewport is where the image stops 5 Click the Zoom In button to double the current zoom factor gt The test image will now be displayed at a zoom factor of 60 6 Click the Adjust Zoom button to have the zoom factor adjusted to fit current viewport size e The length width ratio of the image will not change Unlike the automatic zoom factor this zoom factor is not linked to the size of a window i e even when you adjust the size of a window the zoom factor stays the same 7 Alter the size of the image window 8 Click the Adjust Window button to have window size adjusted to fit current E image size Saving GUI configuration Workspace You can save your graphical user interface in a file The current user interface is called a workspace A workspace includes the layout of all document windows and button bars as well as how viewport and image manager are positioned It can also include specific images and documents you wish to have loaded e Defining GUI layout You may want to define workspaces for each of the various kinds of tasks thus optimizing how the graphical user interface is laid out for each of these Sepa rate workspaces could be for image acquisition report gene
234. e automatically updated according to the chosen image number The size of the single frames will be determined by the XY calibration of the active input channel In the Horizontal field you enter the number of images that are to be arranged hori zontally The maximum number of image columns is 10 140 Acquiring images Multiple Image Alignment Note In the Vertical field you enter the number of images that are to be arranged vertically The maximum number of image rows is 10 In the Pixel Overlap group you specify the width of the overlap region between two adjacent images In the Horizontal field you enter the width of the region two horizontally arranged image should have in common The width is specified in pixels The maximum number of pixels for the overlap region is 512 the minimum is O pixels In the Vertical field you enter the width of the region two vertically arranged images should have in common The width is specified in pixels The maximum number of pixels for the overlap region is 512 the minimum is 0 pixels The image structures of the overlap region decide whether the automatic image alignment will be successful or not If you select an overlap region that is too large the correlation images may deliver more than one similar image region This ambi guity may result in an incorrect alignment An overlap region of 100 pixels is usually advisable In the Final Image Size group you specify the size of the overview im
235. e for the individual image acquisitions changes accordingly Click the Snapshot button to end the preview Snapshot Click the Snapshot button to acquire single images of the current fluorescence Your image analysis program acquires a single image of this fluorescence with the currently set parameters Any preview which may be running will be ended with this command Note Please note that images which may be in the buffer at the time of acquisition will be overwritten After you have acquired a single image the image will be allocated the name which is in the Fluorescence picklist For fluorescence acquisitions you will find the additional mFIP tab which contains all of the relevant information in the Image Information dialog box 122 Acquiring images Multiple Fluorescences Image Information General Dimensions mFIP Image 1 bg Fluorescence Target Myosine Flucroachrome TRITC Excitation BAF Emissiory man Acquisition Obs method MAB Cube position C Mirror cube U MNB Obj position F Magnification 20 00 Objective ACH PH 20K Exposure time SOO rg Integration 0 Mo of frames T Delay 0 2 Corection 0 OF Cancel Help By clicking the Acquire button you will start the acquisition of all of the fluores cences in the Fluorescences list This functions exactly the same as the Acquire Fluorescence Image command According to the settings in the Acquisitio
236. e image will be named MIA and will be written into the image buffer following the last image of the series Click on the Cancel button to return to the Align Multiple Images dialog box Repeats the last acquisition and alignment The acquisition functions require additional software that is not included in the stan dard packages Use this command to acquire a new image series using the old parameters and to align the individual images automatically For applying this command an automatic stage control unit is needed The command is only available if you have already acquired and aligned an image series using the Acquire command After calling the command the image series will be acquired without the Acquire Multiple Images dialog box having been opened The acquired images series is immediately aligned Using this command you will get an overview image without further settings Calibrates the shift system The acquisition functions require additional software that is not included in the stan dard packages 147 Acquiring images Calibrate Use this command to calibrate the position of the camera with respect to the micro scope stage axis Usually the camera is not aligned parallel to the traverse directions of the stage As a consequence the X and Y direction of the stage do not coincide with the sides of an acquired image An image recorded at a position X1 and a second image recorded after moving the stage to the posi
237. e into the Lower limit field Any gray values lesser than this value will be ignored for threshold calculation This value is an absolute value and is independent of the image histogram Enter the highest gray value for the calculation of your thresholds into the Upper limit field Enter the percentage of dark gray values that are to be ignored during threshold calculation into the Underflow field If you enter 1 here the program will ignore 1 of the darkest pixels when calculating your thresholds The lowest gray values to be ignored during threshold calculation vary from image to image and are thus calculated anew for each image based on its histogram Enter the percentage of light gray values that are to be ignored during threshold calculation into the Overflow field Click the Set RO button to define the portion of the image to be used for automatic threshold calculation Define the rectangular area interactively using the mouse This frame is only used for automatic threshold calculation Click the File button to open the standard dialog box for the opening and loading of files This is where you can save parameters for automatic threshold calculation or load previously saved parameters OK is not available when the Auto Seitings tab is active To set automatic thresholds you have to switch over to the Manual tab first and click on the Auto button there Then you can exit the dialog box by clicking on OK Set Col
238. e to be compressed You define whether or not 16 bit images are automatically convert ed to 8 bits and whether image overlays are burnt into the image be fore being sent Please note that the options for x Cancel ai saving images aent the same for all image formats Preprocessing e tomer G bit mages to ahit T Burn overlay into image Compression Quality es B OF Cancel Help Use the Report tab to determine the file format aiaullalabadadas hii Ez for the reports to be sent The RTF format has two advantages for the sending of reports you can considerably reduce the report s file size and the recipient can open the RTF file report in other applica tion programs e g MS Word Image Report Other Documents General Choose format Send report as a report container src ste Send report in Rich Text Format rtf Reduce image data Export images as thumbnails M Set image resolution 75 DPI I Use JPEG compression 75 OF Cancel Help 14 First Steps E mailing images How to e mail 1 Open all the documents and images you wish to send in an e mail e If you re planning on sending database images and documents open the database s and select the records desired 2 Use the File gt Send email command gt The Send email dialog box lists all images and documents currently loaded open in your image analysis progra
239. e to shear shifting will be filled in with a background color you can determine via the Fill Color button Click the Reset button to set all of the parameters for the transformations to 0 Please note that the background will also be reset Click the File button to save a parameter set for an image transformation A preview window designated for display of the image you re working with will appear in the foreground The image document window will be minimized if you do not have a dual screen system and the Dual monitor is active The preview window will display the image in the active image buffer at a zoom level of 100 independent of the zoom level currently set in the image document The preview window will be displayed at a maximum size fitting into the document area if the document area is not large enough for display of the whole image at 100 the zoom factor of the image will be adjusted accordingly The preview window will immediately display the current resulting image Shear Use this function to shear an image Enter the shear angle for the image into the Shear X and or Shear Y field Image calibration will be lost Rotation Use this transformation to rotate an image Enter the rotational angle you want in the Rotation field The image will be rotated around its center The possible rotational values are between O and 360 By default the image will be rotated clockwise to the right Select the Left
240. e values added to all color channels orto thousandth O does not alter the im only one age Channel parameter All Red Green or Blue Select e g A for all three color channels Histo Equal linearizes the number of pixels per intensity no parameters ize value This renders obscure details in darker im Stretch Inten sity Invert age areas more readily visible increases image contrast This is done by cen tering the image s intensity values and then re distributing them proportionally along the entire value range inverts pixels color values This transforms the original image into what looks like a photo graphic negative of the image no parameters no parameters Change Color Depth What will happen Use this command to convert a true color image into a false color image To do this you can select a palette and a dither option Use this command also for altering the palette of a false color image or for converting a false color image into a true color image Change Color Depth Method Windows System Palette 256 Cancel Help A preview window designated for display of the image you re working with will appear in the foreground The image document window will be minimized if you do not have a dual screen system and the Dual monitor is active The preview window will display the image in the active image buffer at a zoom level of 100 independent of the zoom level currently set in t
241. eady defined and saved Click the Auto button to use automatically calculated thresholds Parameters used for the automatic calculation of thresholds are taken from the settings in the Auto Settings tab To set automatic thresholds you have to click the Auto button before you exit the dialog box by clicking on OK Select a gray value range i e a phase from the Phase list and then click the Delete button if you wish to delete this phase This button is available as long as there is still at least one phase existent Auto Setting Sets the parameters used to automatically calculate thresholds Set Thresholds x banyal Auto Settings Dynamic Fired Lower limit 1141 H Upper limit 4095 5 Cancel Help The diagram in this dialog box displays a histogram of the image Histogram calcu lation is based on the portion of the image you have defined via the Set RO button Enter the number of gray value ranges i e phases you wish to have into the No of phases field When working with 16 bit images you will be limited to 2 phases plus background or 3 phases without background automatic threshold setting will then divide the image s whole gray value range for 8 bit images 0 255 into n 1 ranges n the number of phases desired The lower gray value range will not be assigned a phase it will be considered part of the back i E L PA M Preview SetRO File Undertow o When you first
242. eans that you can define special database fields for images e g the Microscope type database field These database fields contain information about the images and can also be used for search purposes For example you could find all the light microscopic images in the database Graph datatype For some program versions the image manager provides a third tab which contains storage for graphs Graphs are measurement curves which are displayed in their own separate document window In the same way as for images you can create data base field specially for graphs You insert graphs into the database from the image manager using drag amp drop Remember that you can insert graphs as records by drag amp drop from the image manager and also as documents Database gt Insert gt Documents You should always try to insert graphs as records because only then can you define database fields and thumbnail is available in the gallery view Data datatype You can insert a data record into a database folder which is not linked to a document You can use this possibility for example to structure the data within a database folder better e g several investigation methods have been used for an order but they must all be described by their own record Under each investigation method you can now insert the investigation results images or sheets otep by step How to arrange database fields Arrange the organizational field Customer so that the cus
243. ects Highlighted objects can also be framed Choose any color in the Line palette and select frame width in the Weight field Delete Overlay Deletes the overlay of the displayed image Note Note that the overlay will be deleted without further prompting Show Markers Shows markers in overlay Use this command to display the markers e g an annotation text that have been set in the image overlay Note Markers of the active image are listed in the Markers group on the Dimensions tab of the Image Information dialog box Use the Image gt Image Information command to open the dialog box containing the image information Define ROls Defines the regions of interest ROI for image analysis and automatic particle anal ysis 1 Enter a name for the ROI into the Label field e g Nucleus Any new ROI name will automatically be added to the Label list Use the names of this list later on for other similar ROIs e g other cell nuclei You do this by selecting the desired name from the Label list before you define a new ROI 2 Select the drawing method by which you wish to define the ROI in the Tools group See more about the Tools group and how to draw ROls with the respective drawing methods 180 The Image menu Define ROls 3 After having drawn the ROI rightclick to return to the dialog box The new ROI will be numbered and added to the Active ROls list Another color is automatically matched with ea
244. ecutive number will be proposed 2 Click the Add New Field button You will find this button immediately above the 66 Archiving Images Define a database 3 Change the standard name of the new user field in the Field Name field Enter for example Experiment Description as the new user field 4 The Data type list offers various types of data Select the Text entry 5 Mark the Required check box In this way the experiment description must always be entered when a new experiment is set up 6 Mark the Organizational field check box This means that the information regarding the experiment description belongs to all of the records that are stored under an experiment 7 Close the Add Field dialog box by clicking OK gt The new database field has then been set up and will be displayed in the Available Fields list a gt All of the database fields are characterized by an icon in the Define Fields dialog box The new database field will be prefixed by the icon for a user defined organizational field 8 In the Default group select the option next to the empty editing field Thus when a new experiment is set up the experiment description is always left free and must explicitly be reentered 9 In the Picklist group click on the Edit button to set up a picklist for the Exper iment Description field 10 In the Edit Picklist dialog box enter the name of a possible experiment descrip tion in the Value field e g Fluore
245. ed Most images are comprised of greater and lesser gray value modulations Greater gray value modulations are significant clearly visible gray value differences Lesser gray value modulations are minimal barely visible gray value differences The lesser gray value modulations are what define differential contrast The DCE filter Differential Contrast Enhancement separates the two image components in order to selectively enhance the lesser gray value modulations The situation is comparable for color values When true color images are involved the intensity component of color values is what defines differential contrast Minimal fluctuations in intensity will be enhanced such that the image s original coloring will remain unchanged Define DCE Bandwidth Enhancement g0 r T Quality Ramp optimize Preview Preview Window The DCE filter is defined by the parameters Bandwidth and Enhancement Bandwidth defines the range of gray values belonging to the lesser gray value modu lations The bandwidth accepts values between 0 and 100 When the bandwidth is narrow gray value modulations of a minimal dynamic range will be strongly enhanced This means that low bandwidth values result in greater image detail via a suppression of high amplitude contrast modulations Broad bandwidths result in greater contrast but also contain less image detail The parameter Enhancement indicates the factor by which lesser gr
246. ed 345 Loading 336 Save 335 Show 335 P Paste Objects Overlay 177 Perimeter ROI 297 perspective presentation of an EFI height map 131 Phase Analysis 254 301 Phase Color Coding 300 Pixel Map 292 Value 292 Point Value 321 Polygon LUT 163 Polygon method Add Shape 270 Positions Accessing 343 Adding 342 Editing 342 Storing 343 Posterize Filter 261 Preferences 63 Preset volume capacity 95 Preview 122 Preview window 85 Previous record 92 Print Field codes for headers footers 12 Images 11 To define multiple image page layouts 12 print spectra 310 Problem 343 Process menu 198 228 Processing 251 Protect Graph 317 Image 193 Pseudo Filter 231 245 Q qualitative EFI height map 130 Query 91 Query results 90 Ramp optimize 239 Rank Filter 235 247 rank filters 227 Readjust the stage how to 344 Rebuild the global overview 336 Record 63 Reimer 245 Repeat acquisition 147 Report Generator 25 Export Reports 29 File Format 27 New Reports 25 Report Templates 54 RTF Export 29 To export a report 29 To Generate a new report 28 To insert images from image database 43 To insert images from the image manager 36 To save a report 29 Report Objects 30 Adapt Record Objects 42 Align 32 Autotexts 47 Background Objects 56 Detail Zoom 37 Diagrams 54 field objects 40 Image Objects 34 Detail Zoom 37 Fit image into the frame 37 Move Image 37 Print magnification 35 Insert 33 Object Templates 59 Position 31
247. ed for display in the False Color mode The normal entry dialog box will open In it you can select one of the numerous predefined LUTs Click the Load button in this dialog box to load the selected LUT The application automatically switches to the False Color display mode Changes the Lookup Table LUT for the displayed image Lookup Tables LUTs or palettes make it possible to have gray value images displayed in color This is done in that the LUT assigns each gray value of an image three color values These three colors are mixed and result in a particular color in the monitor display Use the Edit LUT command to alter how an image is displayed on your monitor The image itself and its original gray values which are beneath the LUT remain untouched An image s LUT qualifies as additional image information It is considered part of the image and will automatically be saved along with the image itself as long as you use the TIF image format The gray values of an image s pixels comprise the LUT s input e g 0 255 for 8 bit images These gray values are listed according to their respective line number The values of the three colors are found under their three columns Red Green and Blue The numeric value listed here corresponds to the intensity of that color The three color intensities put together add up to the precise color that all pixels of that gray value are displayed in on the monitor If a
248. ed of image analysis functions a binary image has a data depth of 8 bits The two possible binary status values are coded through the gray values of either O black or 255 white Which gray values To transform a gray value image into a binary image it has to be known which pixels become white and of the original image are to be displayed in black and which ones in white This is which black determined via one or more gray value intervals Any pixels having gray values within the gray value interval s will be displayed white all others black Defining gray value Use the Process gt Set Thresholds command to define these gray value intervals intervals which is done by defining one or more phases If only one phase has been defined currently the Binarize command will automati cally use the phase s left and right threshold to generate a binary image If several phases are currently defined the Binarize command will open the dialog box of the same name This is where you can select one phase whose gray values are to be displayed in white or you can select all the phases Phaze Phase 1 OK i Cancel W Select all Help Select a phase whose gray values are to be binarized white from the Phase list Gray values of all other phases and the background will be binarized black Select the Select all check box to assign the color white to the gray values of all defined phases This is a way to have gray value areas t
249. ed standard record object only contains predefined fields which exist in every image database The content of these fields will be automatically included in the report 5 Now select several thumbnails in the database You do this by keeping the Ctrl key depressed and selecting the various images via leftclick 6 Drag the thumbnails from the database window directly on the first database object located on the report page gt If the number of records selected is greater than the number of record objects your image analysis program will automatically add the necessary number of pages gt Images are always added in the order in which they were selected in the database window He MABE bh Ble le eS ola Se 2 ele aie Report Heading angie line Subheading areorl up bo bo lre 54 Leam lS apl 2 Query results 3 16 Bit Display 3D Surface 3 Harp z lordene cod Borer fa irok Soon hegu cnnant rha Comet Automater Calibration Castlron Chain Color Adjustment Color Separation Color Threshold fi E iji i i i l u r tle ezed T ezldse gt Ose 22 ezed by zarae Graph Processing Image Acquisition 4 k tmh t Fage 1 1 Position 0 0 0 0 mm Size 2 2 LeamSlS 4 ADDA Fruitfly A 44 Report Generator Report objects Adapting record objects to your own database Your image database generally contains fields that differ from the sample templates included You have
250. edian e Variance a priori e Minimum It is possible to have user defined statistic functions displayed For this purpose you can use the Imaging C macro language to write an appropriate module The Available and Current lists contain all predefined statistical functions or param eters Any user defined functions from Imaging C module s that you have loaded with the Install button will be listed here as well The Available list contains the functions available for use but that are not being currently used The Current list contains the functions being calculated Select an entry from the list of available parameters Click on the Add gt gt button to add this function to the list of current parameters The button is only active if you have selected an entry from the list of available parameters Select an entry from the list of current parameters Click the lt lt Remove button to delete this function from the list of current parameters This button is only active if you have selected an entry from the list of current parameters Select an entry from the list of current parameters Click the Up button to have this function moved up one position in this list Click the Down button to have this function moved down one position in this list These buttons are only active if you select an entry from the list of current parameters and if this entry is not at the top no Up or bottom no Down of the list Select the Mean is known a
251. egion with regard to the size of the individual image On changing the parameter the sketch is automatically updated The final image size is displayed below the sketch Note that the actual size of the overview image can deviate from the value displayed here because the alignment of the individual images can shift images against one another During acquisition a red number indicates the currently recorded image After the snapshot is finished a thumbnail is displayed in the preview window In the Driving group you specify the acquisition sequence of the individual images Select the Meander option to take a winding course when acquiring the individual images Using this method minimizes the traversing of the microscope stage Select the Comb option to acquire the individual images column by column In the Shift type group you specify the shift device used for acquiring different object regions 141 Acquiring images Acquire Manual If you select the Specimen shift option the object will be moved while the camera stays fixed This shift type requires a microscope stage with a remote control The Image option is not relevant for your image analysis program The logical input channel is used to set all input parameters for image acquisition For example you can define a series of macro steps which must be performed before or after image acquisition In the Logical Camera group you specify how these pre and postprocessing steps
252. eld entry itself They can however also include all other kinds of report objects excepting placeholders Field objects can only be inserted within a record object 40 Report Generator Report objects Edit field objects You can only begin to edit field objects once you have clicked on the Edit Object Template button in the edit object mode moving you down a level within the edit object mode What make up field Field objects can in essence include all kinds of report objects Normally field objects objects contain at least the name and content of the database field Your image analysis program does not automatically adjust the length of the text object to fit the field entries or database fields When a field entry is longer than the text object the field entry will be cut off on the right hand side You should thus keep an eye on how long your text objects are Decide their length depending on how long the longest field entry or database field can be Three differently for matted field objects il Field name lu strat e th eva ri ety with ae 4 ag object created for database fields of the which field objects can appear in a report Field entry gt lt Field entry gt i Hl Image height 40 98 mm Available field object components Text vari placeholder for the The field name is dictated by the definition of the fields in the data able name of the database base When you insert a field object the con
253. en starts the burning software gt Your image analysis program checks to see if the files were properly burned The test can take some time based on the drive because it is checked bit by bit After the test has successfully been completed you will be asked to remove the CD and to label it with the provided identifier gt After the test has successfully been completed your image analysis program deletes the files from the temporary backup directory to create space for the next CD s files 15 Label the CD accordingly 16 Insert the next empty data medium and follow the database wizard s directions This is how you archive files on CD You have created the database on your local PC You need space on your hard disk and want to export older files from your database You have access to a CD DVD burner You have Nero Burning ROM software 1 Check the settings for the temporary backup directory Use the Special gt Pref erences command Select the path to which the data is going to be temporarily stored to from the Temporary storage directory field located in the Database tab 2 Check the directory name under which the database files of the database are stored To do this select the upper most entry in the database s tree structure located in the database window The database path is shown in the form in the lower right 3 Close the database from which you want to archive files Make sure that no other user is using the
254. ent color values comprising the active phase This will enlarge the color range the phase covers Click the Exclude button to reduce the color range of the active phase Use the options in the Preview group to check the thresholds you have selected To speed up your checking the thresholds will only be shown in a special window that must be defined for this purpose 217 The Process menu Set Color Thresholds Select the None option to turn off the preview None of the phases defined will be displayed in color within the image Select the Current option to have image areas belonging to the active phase displayed in the color selected The preview display will be continuously updated this enables you to judge whether the image structures you re looking for are being covered by the thresholds you set Select All to have the colors of all phases displayed simultaneously in your image To have image areas not yet assigned a phase displayed in color select the Back ground option The image background will then be displayed in the color of the phase currently active Click the Window button to define the preview window The mouse cursor will then automatically appear within the image and a frame will appear in the overlay Keeping the left mouse button depressed you can adjust the frame s size and posi tion as you wish To set the frame rightclick Click the File button to open the standard dialog box for the opening and
255. equently occurring gray values will be spread and intervals of more seldom gray values compressed This function corresponds to the Process gt Intensity gt Equalize Contrast command Logarithm logarithm function y log x Square quadratic function y x 2 Square Root root function y squareroot x Null null line y 0 The whole image will turn black Hyperbola hyperbolic function y 1 x Exponential exponential function y e x Gray8 a step function reducing the whole gray value range to 8 gray values For 8 bit gray value images 32 gray values are respectively reduced to and mapped as 1 gray value e g gray values 0 31 become the gray value O gray values 32 63 become 32 etc Gray16 a step function reducing the whole gray value range to 16 gray values For 8 bit gray value images 16 gray values are respectively reduced to and mapped as 1 gray value e g gray values 0 15 become the gray value O gray values 16 31 become 16 etc 201 The Process menu Modify Gray Values Inverse inverts the current mapping function The inverse function does NOT gen erally invert the original image Only if the linear mapping function is cur rently selected will the inverse function invert image contrasts XY Diagram The XY diagram in the dialog box shows the image s gray value distribution in red The current mapping function G resulting image f G original image appears in blue When working with gray val
256. er Graph range Components Channels Data type Version OF Cancel Help Comment How to open the Graph Information dialog box e Doubleclick on any graph buffer within the Image Manager to view information on that graph e Alt Return keys use these keys to view information on the active graph e Image Manager context menu rightclick on any graph buffer to open the corre sponding context menu then use the Graph Information command to see this graph s information without making it the active one displaying it e Graph Document context menu rightclick on the graph window to open the cor responding context menu then use the Graph Information command Define Graph History Defines the function groups of the history Define Graph History Ea Command groups Cancel QOEMD Extension Select all efi ExcelDDE Remove all Fast Fourier Transtormation Default File silane OFIS hi ELEk Help Record commands Record Graph History provides you with a tool which will record the changes done to the original graph These changes may be due to the execution of certain analyses functions or modifications of the appearance the Record Graph History commands opens the Define Graph History dialog box where you can select the command groups to be recorded if applied to the active graph 326 Graph Convert to Activating the recording adds an additional tab called Histor
257. er No or Customer No then replace the term Project with your organizational ID using the following step by step guide 1 Select the Database gt Insert gt organizational ID command The exact com mand is created dynamically and depends on the organizational ID of the active database If your organizational ID is called Project the command name is then Database gt Insert gt Project gt The insertion mask shows all database fields which you have defined in the Arrange Fields dialog box for the Insert view and the Project data type 2 Enter the required information in the Insert Project dialog box or select an entry from the picklist Fields which require an entry are marked by an exclamation mark 3 The automatically created entry Project lt ConsecutiveNo gt will be offered as the project name You can replace this entry with any other project name The database will however not accept a database folder name that has already been used in the database 4 Click on the Insert button gt In the database window you will find a tree structure on lower left The uppermost entry in the tree structure is the name of the database A new project will be created as a database folder below the database name You can now insert the project data such as images graphs sheets and other documents into this database folder gt Inthe database window you will find a gallery view on the upper right The new project will
258. ercentage is 50 Defines the Rank filter The rank filter is used for image smoothing whereby it primarily filters out isolated pixels with gray values which differ greatly from their immediate surroundings Constant gray value areas in the adjacent area of this pixel and edges are retained after using a rank filter In contrast to filtering with a Median filter the filtered image thus loses none of its original clarity 235 The Process menu Sigma Define Rank Filter Cancel Execute File Preview Window i Eeg Help Enter the diameter of the surrounding area in pixels into the Size field You can select diameters ranging from 2 to 50 at intervals of 1 The preset value is 3 The illus tration shows the exact shape of the surrounding area for diameters ranging from 2 to 6 The larger the diameter you select the larger the areas of identical gray values in the resulting image Radius 2 3 4 5 6 Pixel neighborhoods for different radii Enter the gray value the central pixel of the adjacent area is to be replaced by in the Rank field When rank is 0 central pixels will always be replaced with the lowest gray value of the surrounding pixel area when rank is 100 by the highest gray value At a rank of 50 the central pixel will be set to the median gray value of the rank sequence The preset rank is 50
259. ermined using the current image calibration For images not acquired via your image analysis software these fields will simply display image width and height in pixels Enter an arbitrary comment regarding the image in the Comment field Click the Audio button to open the Audio button bar Using the Audio button bar you may record and play an audio comment which will be attached to the image 195 The Image menu The Dimensions tab The Dimensions tab General Dimensions Dimensions Color Channels 2 Layers 31 ime rames unused Color Channels Color Attenuator a 100 00 0 0 C 100 00 0 0 OF Cancel Help In the Dimensions group you will find for every available dimension color channel Z level time the number of single images that the multi dimensional image contains in the corresponding dimension number color channels number of layers and number of points in time The Color Channels table lists the individual channels by name and offers you addi tional information about each channel Use the slide controls to gain access to all of the information Doubleclick the color field to access the Edit Fluorescence Color dialog box Use this dialog box to edit the color information to be used for the display 196 The Process menu The Process menu The Process menu Intensity Maximize Contrast Modifies gray values to maximize gray value dynamics The full range of values available for adj
260. ersus the gray value itself The graph will be written by default into the first graph buffer in the image manager To avoid overwriting an existing graph just click into an empty graph buffer before calculating the histogram Pixel Map Shows the pixel map of the selected image This command opens a dialog box displaying an X Y matrix containing the gray values of a particular image area Position and size of the image area can be adjusted The intensity structure of the current image area can be analyzed in this way 292 The Measure menu Pixel Map Pixel Map Close Move ddi Help Options T Hexadecimal I Color coded Red Greer M Ene f RGE The X Y matrix in the dialog box lists gray color values of the image area selected The image s area s position is shown in the upper and left border Click on the Move button to change the image area shown in the matrix The mouse cursor will appear Move it through the image and with it the image area the changing data in the matrix correspond to these movements The white crosshairs in the matrix show you the mouse cursor coordinates Use the directional keys on the keyboard to position the mouse cursor with pixel precision Leftclick to end move ment and to return to the dialog box Determine the format and color of the gray value numbers of the matrix in the Options group For true color images determine which color components are to b
261. ery to a narrow stripe gt Your image analysis program changes the size of the database window so that it takes up half of the document area The database window is posi tioned on the right of the image manager 3 If you are mainly working inside the database click on the Full view button in the EE button bar of the database document gt Your image analysis program divides the database window into four sections 4 In the tree structure mark the uppermost entry with the apl file name exten sion gt The form contains the following information about your database The fields shown here are predefined and cannot be edited using the Arrange Fields command File Name Name of the database S Path Complete directory name of the apl database file Opened Information about the status of the database You can only carry out exclusively operations which modify the structure of the database if the database is opened exclusively Read only Information about the status of the database A database can for ex ample be opened on a read only basis if you have created it on a me dium such as a CD ROM Database Clear identification of the database this identifier is only used inter identifier nally Converted from contain the information on whether or not the STAR database was 3 2 converted from an Archive mdb database Version number with which the STAR database was created 5 Mark the different database folders i
262. es 2 Load the acquired reference images into the image buffer 3 Click the Calibrate Spectral Unmixing button located in the Image Analysis iy button bar gt This opens the Measure Ratio Unmixing dialog box to define two ROIs The first one has to be within an area where the fluorochrome is visible while the second ROI is used for background correction 4 To define the ROIs for the calibration click the Define ROIs button Define ROIs 5 In the Define ROIs dialog box click the Delete All button if ROIs have already been defined 6 Select a drawing tool For example click the Elliose button to define a round ROI 7 Choose an image area with significant fluorescence signal 8 Click the right mouse button to confirm the ROI and return to the Define ROIs dialog box 9 Define another ROI that is situated on a dark background area 10 Close the Define ROIs dialog box gt The ratio of the average intensities within the first ROIs is computed auto matically and its value displayed in the dialog box Measure Ratio Unmixing Please define two ROIs ROT 1 should be an area where the Fluorochrome is visible ROT 2 should be a dark background area Define ROIs Computed ratios 4 77 Select or type label 11 Label the ratio e g GFP and then click the Save button This procedure has to be repeated for each of the fluorochromes used that is for all reference images 278 Spectral Unmix
263. es The command opens the Kinetics dialog box 298 The Measure menu Kinetic The Intensity Profiles tab Intensity Profiles Dimensions Direction i Time Kinetics Profile Parameters Select ROIs From Image 31 Muscle Cell Contraction 1 ROIs Available ROIs ROl2Z ROT 1 M Sheet I Highlight selected ROIs OF Cancel Help Select in the Direction group if the intensity changes are to be calculated over time if so select the Time Kinetics option or as a Z Profile if so select the Z Profile option Depending on the image type one option or the other one or both of them can be selected since the program recognizes the dimensions automatically e Simple time series Only the Time Kinetics option is available e Z stacks Only the Z Profile option is available e Z stacks in time lapse Both Time Kinetics and Z Profile options are available Select in the Parameters group from which data set the ROIs shall be taken and for which ones the analysis shall be performed Select the Sheet check box to generate in addition to the graph the corresponding table Select the Highlight selected ROIs check box to display the ROI outline with bold lines 299 The Measure menu Phase Color Coding The Dimensions tab This tab shows three groups Color Channels Z Layers and Time Frames Intensity Profiles Dimensions Color Channels Gray alaye Eram Moo E bor DE step E all
264. es ellipses and polygons are filled with the color selected If you do not want an object to be filled then select No Fill If you wish to have a bicolor fill pattern for the object selected then use the Fill Effects command from the Color drop down list The Color list is where you choose the color desired for the object selected Select No Line if you do not want the object selected to have an outline You are provided with five different kinds of lines solid dashed dotted dash dot and dash dot dot When using Windows 95 and 98 any lines that have a weight i e width that is greater than 1 pixel will be displayed as solid lines There are no such restrictions on line display when using Windows NT Define the width in pixels of the line type selected in the Weight field You can have large dots various kinds of arrowheads or other graphics placed at the tips of lines Enclosed objects e g rectangles and polygons do not have tips That s why you cannot assign any arrows to them Polygons cannot have arrows either Select the kind of arrowhead desired for the initial and or terminal points of the line selected 6 different kinds available Your image analysis program determines the size of the arrowheads based on the value in the Weight field 173 The Image menu Layer Layer Allows to select which overlay layers will be visible Click the Layer button to open a menu for the selection of the layers In the first grou
265. estination image buffer How to apply the filter to other images using the same parameters 1 Open the Filter submenu 2 Select the filter desired Define Filter User Filter Enables the definition of a filter matrix A user defined filter provides you with the opportunity to select all filter parameters yourself A user defined filter can be used e g for combining different filters this is done by adding the weight factors of the individual factors Another application is modification of existing filters this is done by increasing mean image brightness by adding an offset If you apply the user filter the following operation will be applied to each image pixel 228 The Process menu Define Filter Wicht tW i Wl ___ _ ___ __ _ _ __ offset Normalization Factor where Iz intensity of the central pixel in the original image I1 In intensity of neighboring pixels in the original image W1 Wn matrix elements or weight factors of the user filter Parameters for user defined filters are set in the Define User Filter dialog box Define User Filter x Sie C 3x3 5x5 7x7 9x9 Uk Cancel po po po Execute o o T Preview FP pp T l P Ho e e File A FF nm nm co cc cc a oo po les H Help Erith B T Normalization f B Offset 0 is IY Hex lattice To determine the size of the editable matrix field select one of the fol
266. ete function You do however delete the entire measuring object that was measured 4 Click the Delete Measurement button located in the measurement display gt The selected measurement value disappears from the tree view The respective measurement object is simultaneously deleted from the overlay e Please note You cannot delete the measurement values by deleting the image overlay The image overlay is recreated from the measurement files as soon as the image has been reactivated or new measurements have been added 5 It is sometimes easier to delete a value from a measurement by simply identi fying it on the image In this case click the Delete Measurement from Image button on the measurement button bar gt The pointer moves to the image window 6 On the image click on all the measurement objects that are to be deleted gt All deleted values disappear from the overlay and from the tree view 7 Depress the middle mouse button or the Esc button once you have deleted all the values you wanted to delete 283 Interactive Image Measurement Create measurement sheets This is how you delete an entire measurement 1 Select the image names from the tree view located in the measurement display 2 Click the Delete Measurement button located in the measurement display gt The selected entry appears from the tree view All measurement values which belong to this image are deleted e You only delete the measurement value wit
267. example To do so use the Alt 2 key stroke First Steps The GUI User Interface Document Area Image window Status bar Documents can only be displayed within this area Each document is opened within a separate window Your image analysis program supports the following document types a m Diagram Sheet Graph Report 3D Workspace oe meee The image window is a special window for viewing either loaded and or live images It is possible to view up to 25 images simultaneously For this purpose the image window will be divided up into numerous windows Such a window will be named a viewport in the text that follows Each viewport can display a single image To alter the image display within the image window e g zoom factor use the Image button bar The status bar contains among other things e a brief descriptions of all functions Simply move the pointer over the command or button for this information e name of the active input channel e position and size of the global frame Loading and saving images Loading images El Image buffer box icons You can load several images simultaneously Click the Open button in the Open Image dialog box to load all selected image files The image files will be loaded into successive image buffers The first image buffer is the active image buffer To select e a continuous group of images Then while pressing Shift leftclick on the last image e an arbitrary se
268. ext frame is indicated in the graph document by a dashed rect angle Clear the Centered text check box to left align the text in the text frame Select the check box to center the text Use the features in the Relative position group to add a label to a special X position of the graph In the Position field enter the X position that belongs to the label The dimension of the field depends on the current graph dimension Click the Set button to find the X position interactively A dashed line connects text frame and the current X position Leftclick to confirm the setting and transfer the value into the Position field To move the text label itself click the Set button next to the Label text field or click the Align button Click the Align button to update the position of the text frame The text will be shifted horizontally directly above the current X value in the Position field To move the text frame in the vertical direction click the Set button next to the Label text field Select the Draw line check box to connect text frame and the X position by a line Select the Draw arrow check box to connect text frame and the X position by a line Modify Modifies the identifiers Modify Labels x Labels Ti Delete Delete All Label text Pe se Anchor text W Centered text Relative position Foston i ZF Set l Draw line Align D Draw arrow Cancel Help Select the label to be modified
269. f calibration and the calibration length If you select the Horizontal option the calibration is carried out using a distance in the horizontal direction If you select the Vertical option the calibration is carried out using a distance in the vertical direction If you select the Arbitrary option the calibra tion is carried out using a distance in the arbitrary direction This option is valid only if you select the Fixed check box Before calibrating enter the Calibration length i e the length of the known distance you are using to calibrate with When you click on the Calibrate button a line is displayed on the screen The direc tion of the line depends on the option that is set in the Calibration group Define the first point of the calibration distance by leftclicking A second line will appear and you should position this one at the end point of the calibration distance Click with your left mouse button to return to the Configure Input dialog box Click the Unit button to open the Set Unit dialog box Here you define the unit of the scale bar 104 Acquiring images Camera Configuration Click the Save button to open the Magnification Table dialog box Here you can generate a table of different calibrations for the different magnifications Click on the Acquire button to have an image shown live onscreen This function corresponds to the Acquire gt Acquire command If you click the Snapshot button an image is a
270. f necessary check the magnification you have specified As a rule an objective with a small magnification will be used for the overview image 7 Click the Scan button gt The program will switch to the live image and start the acquisition A window will inform you of the probable duration of the acquisition You can cancel the acquisition at any time by pressing the ESC key gt After the last acquisition in your display the live image will be stopped and the overview image will be shown gt The Stage Navigator will switch to the Online Mode and only the area of the overview image will be displayed Create an overview image of the whole stage area If you have defined stage limits that are approximately the size of the sample this is the easiest way for you to create an overview image If however the stage limits have not been correctly defined you will acquire too many empty images outside the sample if you use this possibility 1 If necessary switch to the Stage Area Mode since you can only acquire over view images in this mode see also The Modi of the Stage Navigator on page 336 2 Click the Scan m x n images button gt The Create Overview dialog box will open 3 Click the Whole Stage button to create an overview image of the whole stage area In this case the current camera position is of no importance all stage positions that can be reached will in turn be included gt In the Count X and Count Y fields y
271. f you then create a report based on this template the placeholders are filled with concrete images or database information Diagrams sheets and single sheet cells can be 30 Report Generator Report objects Background Objects autotext Selecting objects Selecting several objects Object Properties The buttons of the Re port Objects button bar are for all report objects which can be inserted This button bar is part of report generator and only appears when the report window is active You will find the button bar on the right edge of the user interface by de fault Size and position Altering object size via a mouse defined as placeholders Use a placeholder s properties e g size and position to define the properties of images or texts you wish to later insert into a report Background objects are defined on the template page and appear on each page of the report which is based on this template page A company logo address or frame are common background objects AutoTexts are texts defined on the template page and which are updated for each new report page automatically Creation date and page number are typical Auto Texts You generally have to select objects first before you are able to edit them Leftclick once on the object to select it Selection markers indicate that an object has been selected If you keep the Shift key depressed you can select several objects All objects you select
272. files You can then edit these files using other programs and also print them out 92 Archiving Images Work in the database window otep by step How to load an image 1 Mark an empty image buffer in the image buffer box 2 Doubleclick in the gallery view of the database document on the image that you want to load gt The image is loaded into the image buffer and represented in the image window e Alternatively you can drag and drop the image either from the gallery view or from the tree structure onto the image buffer e Please note using these two methods will load the image but not the doc i uments which may be attached to them How to load several images in a defined order 1 If the images that you want to load are in different database folders carry outa query to find all the required images Search for example for all the images which have been inserted for the projects A and B gt All images found will be displayed in the gallery view 2 Mark the images in the order in which you want to load them To do this depress the Ctrl key and mark the images with the left mouse button gt All the marked images have a colored background The numbers in the top right corner of the images represent the order of the marking 3 Mark an empty image buffer in the image buffer box 4 Select the Database gt Load Document command gt The marked images will be loaded into the successive image buffers of th
273. font size within diagrams in Word Please note that font and font size may be altered simply by your opening the metafile in the graphic editing mode of Word Diagrams Click this button in the Report Objects button bar to insert a diagram into a report Report templates Creating saving new templates Background Information Report templates Report templates are a kind of report file in which you determine what your future reports are to look like Each report you create using report generator is based on a report template Component parts of A report template consists of page templates and object templates These are gener areport template ated within a particular report template and are not available for use in other report templates You cannot import page or object templates from other report templates 54 Report Generator Report templates You can of course modify existing report templates and save them under a different name This way you can use all previously defined templates for your new report template What are report Report templates determine what the reports based on them look like Defining report templates for templates is thus of quintessential importance as far as creating your own reports is concerned The work you invest initially creating a well thought out report template will pay off You will save time when you get down to actually creating reports with that template Altering existing If you alte
274. fore be made on a regular basis so that the data is as up to date as possible The original data remains unchanged in the database To make a backup you can either use the software wizard for file storage or also the backup software which you normally use to secure data Warning amp You should DEFINITELY make backups of your database at regular intervals Select the Database gt Administration gt Restore Database Backup command to use the backup when restoring individual database folders following data loss Database wizard Use the Database gt Administration gt File Storage command to activate the data base wizard The wizard guides you step by step through the backup and the archiving procedures CD DVD Burner It is possible to access a CD or DVD burner directly from your image analysis program The Nero Burning ROM software is a prerequisite for this Please note the following points e Due to data security data will be written to the temporary backup directory when saving or archiving data Even when directly accessing CD or DVD burners you still require available storage capacity The required size of the available stor age is equivalent to the capacity of the CD or DVD During file storage the sys tem automatically checks if there is enough storage space on the drive for the temporary backup directory e The database files with the apl mtb and tnb file name extensions are al ways burned on a separate medium e
275. g area has been set Click the Preferences button and the Stage Preferences dialog box will open You will find more detailed information in the chapter Alignment of Camera and Stage on page 329 339 The Stage Navigator Acquire parts of the overview image with a higher magnification Acquire parts of the overview image with a higher magnification Howto j z You can not only look at interesting parts of the overview image in the live image you can also acquire them with a higher magnification These more detailed images will be reloaded when you click the Zoom In button while you are in the overview image The procedure is the same as when you acquire an overview image Here too you can choose whether you define a number of images that are to be acquired or inter actively specify an Interest area How to acquire a part of the overview image with a higher magnification 1 In the live image go to the part of the sample you are interested in and adjust the settings focus exposure time etc to provide an optimal image 2 Click either the Scan m x n images button or the Scan Interest Area button gt If you have chosen the Scan m x n images option the Create Overview dialog box will appear In it you will be shown the number of images that are going to be acquired and the higher magnification gt Click the Scan button gt The area you have defined will be acquired The images will be saved in th
276. ge This can be either the actual image itself that is being corrected Source 1 or it can be a reference image from the source 2 image buffer Source 2 Define how the correction image is to be prepared in the Preparation group If the contents of the source 2 image buffer are to be used for the correction image it will not be necessary to preprocess the correction image In this case choose the None option This setting is quite useful when you are able to acquire a correction image from the imaging equipment itself a blank image not of a specimen If the option NxN average filter has been selected the NxN average filter group will appear in the lower left corner of this dialog box This group parameterizes the number of iterations in the Iterations field and the size of the NxN filter matrix in the Size field which will be used to filter the original image The larger the matrix the greater intensity fluctuation can be equalized If the option Interactive zero level has been selected the Define interactive zero level group will appear in the lower left corner of the dialog box You define the Hotspot radius from 1 64 here To define the zero level you have to select three areas within the original image These areas are circles containing the hotspot radius you have defined The average gray value of each of these three circles will then be used to determine a correction plane for the image 221 The Process menu Shading
277. ges that are all not protected Otherwise the Protect Image command will not be available If the Shift key is pressed when you open the mage menu via mouse this command will change to Clear Image Protection instead of Protect Image The protection of all buffers will be removed with this command If the active document is a text document the lock button in the Standard button bar acts as Protect Text command This command will write protect the text 192 The Image menu Define Image History Define Image History Defines the function groups of the history Define Image History Ea Command groups Ei a Cancel Hel T Record commands o Hep If you activate the image history a lot of commands can be scripted during the image processing The script is added to the Image Information dialog box of the images in the image buffer The Image Information dialog box will be extended by an additional tab the History tab If you use the TIF format to save the image the history is saved along with the image If you load a saved image with an already existing History tab the scripted commands are appended to the image history To avoid an inestimable amount of single records you can restrict the commands that are recorded The Define Image History dialog box offers functional groups i e Acquisition Every group comprises a set of commands Select only the important groups to get an expressive processing history
278. ges the contents of all the report pages containing this text variable It does not matter whether the report pages are based on the same or different page templates A text variable can also appear on the same page more than once Text variables are only linked as far as content is concerned and not with regard to the formatting This means that just because you alter the formatting of a text variable does not automatically mean that the other text variables of the same type will be updated in the report 47 Report Generator Text objects Method Record Mame 26 04 00 The report above contains various kinds of text These are described in the sheet below Texts that are text variables appear in square brackets Editable text objects have dotted lines within them as they are to be filled out with text Field objects that are automatically filled out when you insert a record object are put in square bracket i Dedie Particular properties o Object Properties dia Report e button bar o fi box Bey The Background Object check box in report alana only text Select and Move tab has been se lected Text variable The text object s name in the General tab starts with an symbol autotext On the Genera tab the text object re ceives the type AutoText The Report Object Properties dialog box now includes an additional tab Au toText Editable text The Background Object check box NOT in a record or field objec
279. gram supports multidimensional image types you will find an additional tab in the image properties which enables you to determine which dimensions are to be acquired in the report for an empty image object 34 Report Generator Report objects Print magnification Print magnification Print size Image size Rotate image The illustration shows the inserted image in three different available magnifications in the ob ject properties The im age object is the same size each time A frame indicates the size of the image object There are two different predefined magnifications showing different image seg ments An image s print size within a report depends on the image object properties selected and on image calibration The following is generally true print size print magnification x image size The print magnification is the absolute magnification at which the image is printed out on paper Your image analysis program determines print magnification via the print size and the absolute image size The print size is the size the image is printed on paper Without any automatic labeling the print size of the image will correspond exactly to the size of the image object If you have the magnification or the scale shown the print size of the image will be less than that of the image object The image size is the actual size of the image It is determined by image calibration An image that is 500 pixels in width and calib
280. graph docu ment The minimum gain factor is 2 Possible gain factors are 2 5 10 20 50 100 and so forth The display of the graph is automatically updated when changing the gain factor Modify Split Gain Modifies a split gain factor Modify Split Gain x calie Delete 2 x at 127 0 Delete All Split position i oF B Set Gain factor 2 is Cancel Help 316 Graph Protect Graph Change the display of the graph so that all split positions you would like to modify are visible For example click the Default size button in the graph document to display the whole graph The Splits list offers all of the split positions that have been defined for this graph Choose the split position you would like to modify All split positions that are currently not displayed in the graph document are grayed In the Split position field the currently selected split position s X value will be shown Click the Set button to move the split position to another X value The Gain factor field shows the selected split position s current gain factor Use the arrow buttons next to the Gain factor field to set the gain factor for all of the X values that are greater than the split position The display of the graph is automatically updated when changing the gain factor Click the Delete button to delete the selected split position Be sure to adapt the gain factors of the following split position Click the Delete All button t
281. gt Pref erences gt Images gt Allow operations on false color images check box has been selected This command ignores 3 of the brightest and 3 of the darkest pixels and spreads the intensity range of the remaining pixels linearly to cover the maximum available value range Very bright and very dark image areas remain unchanged The contrast within evenly illuminated image areas is increased however This command is effec tive e g if an image contains white or black pixels due to image defects which occurred during acquisition In this case the actual image information will be more clearly contrasted Using linear mapping the image structures are evenly empha sized The total appearance of the image structures is closer to the original than when you use the Equalize Contrast command 199 The Process menu Modify Gray Values Gray value images The gray value histogram is measured for gray value images The lower and upper 3 of the histogram area will be removed The remaining histogram will be mapped linearly to cover the maximum value range 0 255 8 Bit respectively 0 65535 16 Bit False color images 8 bit false color image are processed just like 8 bit gray value images False color images are in fact gray value images that are displayed on the monitor in color via a look up table LUT To look up the LUT used for the display of the active image use e g the Image gt Edit Image command The Optimize Contrast comma
282. gt View tab After insertion click the Object Prop erties button to define what colors are to be used for filling and for the frame If you wish for the corners of the rectangle you insert to be rounded then press Shift and click the Rectangle button at the same time Size and position of rectangles can be altered subsequently as you like To insert a rectangle 1 Click the Rectangle button A rectangle is shown in the image window 2 Pull the rectangle to the desired size by depressing the left mouse button Posi tion the rectangle by moving the mouse 3 Click the right mouse button to confirm the size and position of the rectangle Creates an ellipse object in the overlay When you insert ellipses they appear selected Their object properties are predefined red outline and no filling The preset line weight is defined in the Pen size field in the Special gt Preferences gt View tab After insertion click the Object Prop erties button to define what colors are to be used for filling and for the frame If you want the ellipse you insert to be a circle then press Shift and click the Ellipse button at the same time You can also decide to make your ellipse into a circle while you are still actually drawing it positioning it within the overlay Size shape and position of ellipses can be altered subsequently as you like To insert an ellipse 1 Click the Ellipse button An ellipse is shown in the image window 2 P
283. h this function not the image The image is continued to be normally loaded into the image buffer In the measurement display select what you would like to delete l S eee P Messung l scherl 5 3 Bild El Lae Ursprung Bild M anager x 0 mm O mam Linie Lange 0 14 m Lange 0 16 m ecen Liste ne Messungen This is how you identify a measurement on the image If you have measured a very large amount of values you will find it especially easy to find them on the image rather than in the measurement display 1 Click the Image Link button located in the measurement display gt The pointer moves to the image window 2 Click on any measurement object in the image overlay gt The respective measurement value is shown in the measurement display 3 Depress the middle mouse button or the Esc key to exit the image window Create measurement sheets Background Information Measuring on Before each measurement the images can either be newly acquired with your numerous images camera or you can load already saved images in successive image buffers In both cases please make sure that the image buffers list is always covered by the measurement display in the measurement environment You can change the views at all times and again show the image buffer list To do this click on the List or Gallery tabs at the bottom of the image manager If you are offered too many tabs in the image manager do t
284. hat are not next to one another displayed in white Binarize Color Image Converts a true color image to a binary image Use the Process gt Set Color Thresholds command to define the color value intervals which is done by defining one or more phases If only one phase has been defined currently the Binarize Color Image command will automatically use the 6 color thresholds of this phase to generate a binary image All color values within the phase s limits will appear white in the resulting image all other color values will appear black If several phases are defined currently the Binarize Color Image command will open the Binarize dialog box Here s where you select the phase s whose color values are to be displayed in white or all phases Define Shading Correction Defines parameters for the shading correction Shading correction is a background correction in an image meant to compensate for irregular illumination effects This correction is also known as unsharp masking Shading caused by the camera can be corrected directly at image acquisition To define online shading correction for the logical input use the Acquire gt Camera Configuration gt Input command 220 The Process menu Define Shading Correction Define Shading Correction Assumed deterioration Preparation of shading image nk Hone f Multiplicative C Additive Cancel NaN average filter Interac
285. hat the filter suits your needs The lower the value the more separation lines will be found Higher values result in minimal gray value fluctuations being ignored The result provided by the separator is an image containing the outlines of the objects to be separated The width of the separation lines is one pixel You can decide whether these lines are white 255 intensity or black 0 intensity You can decide whether the image background is either black or white accordingly meaning the resulting image will be a binary image Or you may take the image background from the original image In this case the image type of the resulting image remains the same When using white separation lines any pixels that were originally white will be reassigned an intensity value of 254 so that only the separation lines have an 240 The Process menu Define Filter Note Edit Lines Note intensity value of 255 When using black separation lines any pixels originally black will be given an intensity value of 1 This makes it possible to differentiate between separation lines and the actual image objects when setting thresholds After separating objects the resulting image is the ideal basis for automatic particle detection As the separation lines are only one pixel wide you must set fourfold connectivity in the Define Detection dialog box so that the separation lines will actu ally separate the particles For eight fold connectivity the partic
286. hat the object button located on the Report Objects button bar is now engaged 4 Defined the object on the report page For example keep the left mouse button depressed and drag the rectangle to its desired size e Please note Some objects might require numerous mouse clicks when de fining them End the definition of such an object by clicking the right mouse button gt The object is inserted as selected so that you can edit it right away Once again the Select Objects button which is the first button on the Report Objects button bar is engaged e However you can alter the position and the size of the inserted object any time 5 In order to format the object rightclick and select the Properties command Aligning objects in relation to one another 1 Show the Align button bar should it not be visible e To do this rightclick on any place within the report to open a context sen sitive menu All button bars having to do with report generator are listed be neath the Bution Bars entry Select the Align button bar 2 Position an object at the position desired within the report This object is to be the reference object for the positioning of the other objects 3 You now select the first object you wish to move in relation to another object gt Gray selection markers will appear around the object selected 4 Keep the Shift key depressed and select the reference object 33 Report Generator Image Objects gt
287. have dialog boxes that are different to those of 16 bit images Multi dimensional images A 16 bit gray value image can contain intensity values ranging from 0 to 65535 A multi dimensional image contains the same number of intensity values in every single image Most monitors can only display 256 gray values 8 bit which is already far beyond the number of gray levels the human eye can differentiate To be able to display a 16 bit image on your monitor a mapping function that sets how the inten sity values are to be displayed on the monitor has to be defined for every frame When the multi dimensional image contains two or more color channels each color channel will be displayed in its own color whereby in each case 256 different color values are available per channel for the displaying of the image The color channels can each be individually displayed or all simultaneously When they are displayed simultaneously the colors of the individual color channels will be mixed for the display Use the Adjust Display dialog box to set the intensity range that is to be displayed on your monitor in 256 color values Note The intensity range that is to be displayed on your monitor will be set individually for each color channel is then though valid for all of this color channel s images that differ in their time or Z dimensions Therefore before you access this command you should look through a Z stack or a time lapse image to find a typ
288. have to be treated when acquiring the image series Using the Preprocessing list you determine whether the preprocessing steps should be performed for every image of the series Always only for the first image Only first image or for no image of the series Never Using the Postprocessing list you determine whether the postprocessing steps should be performed for every image of the series Always only for the last image Only last image or for no image of the series Never Click the Close button to close the dialog box without acquiring an image series Click the Acquire button to start the acquisition of the image series A rotation of the camera with reference to the direction of the stage drive causes a displacement of the individual images This displacement is automatically corrected provided the configuration has been calibrated If there is no valid calibration for the active logical input channel magnification and the shift type you can calibrate the system right now A corresponding message is displayed before the acquisition starts Click on the Align button to be able to access the Multiple Image Alignment gt Arrange command directly via the current dialog box Use this command to compose a whole image from the acquired individual images Acquire Manual Records an image series with a non motorized stage Image scaling For the manual acquisition Full size must be set as image scaling in the input chan ne
289. he File gt Report gt New command gt The New Report dialog box offers you all report templates that are located in the current report user template and workgroup template directories 2 Select the report template in the New Report dialog box you wish to base your new report template on Warning 3 Select the Template option in the Create new group lt P gt Only via this step can you decide that you want to create a report template and not a report 4 Confirm via OK to create the new report template gt You can always edit only one report or report template at a time This means that if you still have a report or report template that is open you ll be asked whether you d like to save any changes made or not gt The report currently open will be closed and the first page of the report template will be loaded into the report window gt The Graphical User Interface GUI for editing report templates is some what different than the one used for creating and editing reports You ll know that you are editing a report template and not a report because the background is gray 57 Report Generator Creating saving new templates Adding a new page template for several images You wish to add a new template page that is to contain 6 images 1 Click the Select Page Template button in the report window button bar to open a list of all template pages defined in the current report template 2 Select a page template you ca
290. he Stage Navigator Sequence when switching on the devices Note G Calibrated input channel Alignment of Cam era and Stage How to j z To enable the image analysis program to correctly access your stage please observe the following sequence when switching on the devices 1 PC without starting the image analysis program 2 Control box of the microscope 3 Stage controller 4 Image analysis program 5 Initialize the stage please observe the following note Step 5 is necessary for all systems that have a separate stage control box In this case the stage must be initialized again every time the image analysis program is started As long as the stage has not been initialized the Driver not initialized mes sage will appear behind the name of the stage You will find the procedure for initial izing the stage on page 343 If on the other hand your system does not have a separate stage control box but is equipped with an Oasis 4i PC board from the Objective Imaging company the stage does not need to be individually initialized The input channel must be correctly calibrated If an input channel has been cali brated with incorrect values the stage will go to wrong positions Soft Imaging System GmbH Fire Wire Cameras will have been calibrated during their installation unless this option was declined at that time You can recognize an input channel that has already been calibrated by the X Y Calibrated entr
291. he Stage Navigator Properties dialog box and while keeping the shift key pressed click the Preferences button gt Then the Stage Preferences dialog box will open with the additional invert axis group 2 Select the Horizontal and Vertical options there to exchange the X Y axes The input has been incorrectly calibrated 1 Calibrate the entrance anew gt You will find additional information in your image analysis program docu mentation The leading pitch value entered in your image analysis program for your stage does not correspond to the real leading pitch 1 Access the Special gt Preferences menu and switch to the Stage tab 2 Select your current stage there in the Selection list 3 Click the Properties button 4 Check if the leading pitch value entered there tallies with the real leading pitch value 5 Close the Properties for lt stage name gt dialog box with OK 6 In the Selection list select the virtual stage gt If you select any other stage than the virtual stage at this point you will not be able to start the Stage Navigator 7 Exit the Stage preferences dialog box with OK 345 The Stage Navigator Trouble Shooting 346 Index Symbols m x n images 332 Numerics 1 Derivation 2 Derivative 321 3D presentation of an EFI height map 131 A A multi channel time lapse image 7 accumulative method EFI 129 Acquire 102 EFI 132 Multiple Image Alignment MIA 140 Acquire Manual 142 Acq
292. he Word Break check box so that your image analysis program cal culates wordwrap automatically This is the only way you can be sure the text will automatically adjust to any changes you make to the size and po sition of the text object e Clear the Word Break check box if you e g want to list single words one beneath the other Now if you need wordwrap you ll have to explicitly set the wordwrap within the text object via the Enter key Text format is now unrelated to the size of the text object 8 Define a frame surrounding your text object in the Line tab gt Clear the Transparent check box and determine line width and color A number of line types are only available for the thinnest line width 9 If you wish to have image comment highlighted in color go to the Fill tab to define a highlighting color gt Clear the Transparent Fill check box and determine the highlighting color via the Foreground Color button 10 Click OK to close the dialog box 49 Report Generator Inserting sheets Inserting sheets Background Information Sheets Click this button in the Report Objects button bar to insert a sheet into a report If there are any sheets open during insertion you will be informed via picklist about the open sheets Select the sheet which you would like to insert from the list If there are no open sheets insert a placeholder for the sheets The sheet object you insert when working in a report template in order to
293. he active database folder Drag amp Drop into the database drag one or more images from the image man ager into a database folder When you do this you can see the images in an en try in the tree structure The images will then be inserted below this entry You can also see the images in a preview window or as thumbnails in the gallery view The mouse pointer will show you by way of its form whether or not the Drag amp Drop operation you want to perform is allowed The table below shows the mouse pointers you may encounter when you carry out a Drag amp Drop oper ation from the image manager onto the gallery view in the database window The images will be inserted at the same level as the thumbnails that are on display No records can be inserted immediately below the first level In this case you will re ceive a message and you can set up a new database folder The images will be inserted one level below the thumbnails that are on display You cannot insert any data here That is for example the case when six levels al ready exist below the database folder Neither can any further data be inserted un der a document Insert snapshot An image can be inserted into the database directly after the image acquisition To do this select the Special gt Preferences command On the Image tab select the Database entry from the Image acquisition gt Sequence list Before you switch into live mode mark the database folder to which the imag
294. he color in which the Camera area will be shown The Camera area can only be seen in the Stage Area Mode and in the Online Mode Image frames Determines the color in which the Image frames will be shown You can choose whether you want the image frames to be shown or not If you clear the Show check box the frames will no longer be shown 338 The Stage Navigator Change Stage Navigator Properties Interest area images Name Max width Max height Automatically update active image buffer Optimization of the images Image overlap Illustration of an over lapping area a very large one was selected for this example Preferences All of the preferences in this group have an affect only on Interest area images i e on images that have been defined as Interest areas Switch into the Interest Area Mode to see the changes you have made in the Stage Navigator Determines the image name that will be given as default The image name Interest Area is preset Defines in pixels the maximum possible width of an Interest area image Even when you interactively draw the Interest area out beyond this width the image that will be loaded into the image buffer will not be wider than this value The height will be dynamically adapted to keep the proportions of the image the same Defines in pixels the maximum possible height of an Interest area image The width will be dynamically adapted to keep the proportions of the image
295. he complementary color of the paint brush sector This check box is only active at zoom levels that are greater than or equal to 4 At lesser zoom levels pixels are simply too small for you to be able to distinguish between them and the grid lines Select the Line check box to draw a line This line will be as wide as the whole pixel sector i e the ROI or brush Rightclick to end this paint mode Clear the Line check box if you simply want to work in the regular brush mode to assign pixels the color you have selected leftclick To paint over more pixels using the brush keep the left mouse button depressed while you move the mouse To select the color you are painting with place the mouse cursor on a pixel of that color and rightclick The Connectivity group is only active if the brush is the size of one pixel The Brush field will contain a value of 1 Select 4 to be able to draw lines of one pixel in width and whose pixels are touching at one of their four sides This is important for when you re drawing outlines around particles to distinguish them from one another in preparation for particle analysis This outline will reliably separate particles from one another also if you select the Include diagonals 8 option in the Connectivity group in the Define Detection dialog box Select 8 to be able to draw lines of one pixel in width whose pixels have to touch at least diagonally
296. he displayed part of the graph In this case the result comprises only the X range selected before and the new graph will consist of the corresponding number of channels only The remaining elements of the button bar refer to overlay functions The display of additional graphs in the graph window is termed overlay Use the Graph gt Overlay Selection command to define a graph as an overlay If one or more overlays have been loaded they are all shown with the actual graph at the same time If this switch is set the scroll bars and all buttons of the button bar concerning the axis scale apply to the selected overlay only This function is for the optical adjust ment of the overlay to the original graph for purposes of comparison For example this function may be useful in cases where one of the graphs has an X or Y offset After clicking on the Overlay Mode button the scale markings of the diagram remain those that are valid for the original graph The Log X button and Log Y buttons are however relevant to the whole of the graph on display 309 Graph Printing graphs In case there are several overlays use this list to select the active overlay The active overlay is affected by the overlay operations explained below The color of the graph in brackets will be allocated to the corresponding overlay Fit mz Click this button to adjust the Y scale of the overlay to the scale of the actual graph Fit This function is a quick and co
297. he field the name of the corresponding graph buffer will be changed accordingly The Date field will be automatically read in during the graph creation The Graph data group provides some useful information on the graph The informa tion given in this group are read in automatically during creation Markers and Labels Set Sets the identifiers Set Labels Ea Label text Pe se T Anchortest If Centered text Data position Position 127 TC Draw line TD Draw arrow Cancel Help In the Label text field enter the text for the annotations The amount of text is limited to 116 signs 313 Graph Modify Click the Set button to position the text frame in the graph document Note that the text frame can only positioned in the visible part of the graph You cannot scroll or zoom the graph while you set the labels Select the Anchor text check box to position the text frame at a fixed position of the graph document The position of the text frame does not depend on the currently displayed part or zoom factor of the graph Use this option for general descriptive text referring to the whole graph Clear the Anchor text check box to position the text frame at a special position of the graph When you shift the graph or change the zoom factor the position of the text frame will change accordingly Use the Centered text check box to format the text in the text frame When the dialog box is opened the t
298. he following hide either the List or the Gallery tabs Use the Special gt Preferences gt Image command Create Click the Create Measurement Sheet located in the measurement button bar to measure export the measured values to a table All measurement values are always exported ment sheet from one image to one sheet If you have done measurements on numerous images 284 Interactive Image Measurement Create measurement sheets Data in the measurement sheet Editing sheets otep by step decide whether or not the measurement values for each image are to be exported to individual sheets or if all the measurement values are to be exported to the same sheet The measurement object s type and the measurement values are listed in the measurement sheet All areas are for example written to one column even if they were defined with different methods If you export the measurement values from numerous images into one sheet the measurements of the same type will be written to one column e g all measured areas The unit of the measurement values in the measurement sheet corresponds to the unit in which the image has been calibrated To change this unit use the Image gt Calibrate Image command If you are exporting the measurement values from various images to one sheet the unit for all the images from the image calibration of the active image buffer is adopted The possibilities of editing measurement sheets which have already bee
299. he image document The preview window will be displayed at a maximum size fitting into the document area if the document area is not large enough for display of the whole image at 100 the zoom factor of the image will be adjusted accordingly The preview window will immediately display the current resulting image You are thus able to immediately see the effect different palettes or a different dither algorithm values have on the image and thus are able to obtain optimal conversion conditions Set the bit depth for the resulting image in the Method list The Colors field will only be active if you have selected the Optimized Palette method You will thus be generating a false color image Colors Ditheririg EEEE Colors in input image 210708 261 The Process menu Transform Image This is where you set the number of colors to be used by the palette of the resulting image for optimal adaptation to either a true color image or false color image having a different palette The higher the number of colors set the better your results Avail able values are from 1 256 The Dithering field will only be active if you have selected one of the following methods Optimized Palette Netscape Palette or Windows System Palette You will thus be generating a false color image You can select one of a number of dither algorithms from the list Floyd Steinberg Stucki Burkes Sierra Stevenson Arce Jarvis lt No dithering
300. he integer can be edited as you please Decimal places can only be edited one digit at a time Compute Classification Mininiwrn cc M asimum hoo Cancel Bins Booo Auta Mode Linear kd Help Criterion 4pes The lower class limit is still considered a part of that class The upper limit however is excluded from that class Classes can overlap A measurement is mathematically sorted into a particular class if the following is true From lt Measurement lt To Click the Sheet button to generate a sheet according to the current classification scheme This sheet will correspond to the sheet in the dialog box This sheet will contain the additional column ID Class which displays the name of the classification scheme The columns From and To indicate the unit of measurement of the classifi cation scheme Click the New button to have the name of the new classification scheme taken from the New Classification field and inserted into the Classification list 303 The Measure menu Define Statistics This button is only then active if the New Classification field contains an entry and this entry is not already on the Classification list If you wish to define a new classification scheme its name must first be entered into the New Classification field Then click the New button This name will now appear in the Classification field This is now the current classification scheme which can be edited as you ple
301. he scroll bars Reducing Brightness means moving the display curve down on the Y axis You are thus subtracting a constant from the current mapping function that you set using the intensity scroll bar The lowest intensity setting you can have is 0 0 At this setting all mapping functions will be the same the horizontal line G r 0 The resulting image will be totally black Increasing Brightness means moving the display curve up on the Y axis You are thus adding a constant to the current mapping function which you set using the inten sity scroll bar The highest intensity setting you can have is 100 At this setting all mapping functions will be the same the horizontal line G r 255 for binary and 8 bit gray value images The resulting image will be totally white Reducing Contrast means rotating the mapping function clockwise from the center of the diagram Horizontal segments of the mapping function assign the corre sponding gray value ranges of the X axis to a single gray value of the Y axis Use the contrast scroll bar to rotate segments of the mapping function into the horizontal and beyond The affected gray value areas in the image will first become solid in tone i e a Single shade of gray and then as you rotate further contrast will be reversed At the lowest contrast setting possible 0 0 every mapping function will look the same This setting corresponds essentially to the step function G r 255 for all darke
302. hem are written to a new data sheet For each following measurement a new line is added to the data sheet End the measurement sequence by rightclicking Select one column of the data sheet to append further measurements to it Horizontal and Vertical Distance Measures the horizontal and vertical distance between two interactively chosen points Integration Calculates the integral Use the Graph gt Measure gt Integration command to calculate the integral of a specific graph range The graph range can be defined interactively Change the display of the graph so that the interesting part of the graph is completely displayed in the graph document If you want to calculate the integral intensity below a specific structure in the graph to begin with use the Graph gt Calculation gt Background Subtraction command Use the background corrected graph as an input for the Integration command Define the lower and upper limits of the integration range by clicking them with the left mouse button The limits of the integration range will be indicated as labels in the graph document Use the Modify command to modify or delete the measuring labels A measuring sheet is created that contains the integration limits and the resulting integral intensity Click the right mouse button to finish the measurement or repeat the last step to measure another integral intensity It is possible to accumulate measurements in one output sheet To do so click
303. hing field The smoothing filter is turned off when the value O is entered The larger the value the larger the area with outliers however actual image information will also be suppressed Look for a smoothing value in which the faulty pixels are suppressed and the object becomes rounded whereby other objects are not influenced The four Color Space button are options with which you determine the color space by defining the tolerance The buttons are only relevant for 24 bit true color images The RGB color space is the default for true color images When using binary 8 bit gray value 16 bit gray value and 8 bit false color images the tolerance determines the gray threshold values When using the RGB color space the tolerance value is added or subtracted to the R G and B value of the starting point The RGB color space is effective if you have to detect one or more color tones with small saturation differences This is due to the fact that other colors are detected in the RGB color space before the saturation levels of one color have been exhausted When using the HSI color space the tolerance value is added or subtracted to the H S and value of the starting point HSI color space is effective to detect one entire color tone in all saturation levels except black white and added or subtracted from the color value of the starting point The Hue color space is effective to capture an entire color tone and all its saturation levels
304. ht phases can be selected For each phase a color for display is selected Gray value areas not assigned to a phase will remain uncolored 300 The Measure menu Phase Analysis Objects in true color images to be displayed in false color are defined with the Process gt Set Color Thresholds command Low and up thresholds for the three color parameters are set here and depending on whether you are working with the RGB or HSI system the six thresholds will be assigned a phase Thresholds can also be set interactively within the image To do this one or several circular image areas are selected Their RGB or HSI color values define a phase For each phase a color for display is selected Image areas not assigned a phase will be displayed in black Phase color coding will only be applied to the image area within a frame if a frame has been set Phase color coding will only be applied to the image areas which lie under the white areas of a mask if a mask has been set This command generates an 8 bit false color image in the destination image buffer All gray value areas assigned a phase will be colored according to that phase A color display of various gray value areas enables you to e g more easily visually distinguish between different materials or to have selected image structures accen tuated For true color images you can have objects located in selected color ranges displayed in any false color Phase Analysis Evaluates a
305. ia OK Generating a new report 1 Select the File gt Report gt New command gt The New Report dialog box offers you report templates that you can base your new report on 2 Select the report template named Normal in the General tab in order to create an empty report 3 The Report option is default in the Create new group 4 Confirm via OK to have the report generated gt The first page of the new report will appear within a separate window The first page s appearance is determined in the report template gt Your image analysis program will display a number of button bars to be used in making and editing reports These button bars are context sensi tive i e aS soon as you activate another document the report button bars will disappear Adding pages to a report 5 Click the Add Page button in the Report button bar to add a page to the report gt The Add Page dialog box will be opened This is where you determine where you want to add a page within the report 6 Select the Insert page option within the Add Page dialog box to insert the new page directly before the current report page This is the option you choose if you have to add a page to a report that is finished otherwise Select the Append page option to add a last page to a report no matter which report page is the active one or not 7 Confirm by clicking OK gt The Add Page Template dialog box is opened if the used report template contains more
306. ical image for which you would like to adjust the display To do this use the buttons on the Image Navigator button bar 152 The Image menu Image Display In the Adjust Display dialog box you can change the way the image appears on your monitor Any change you make in this dialog box will be immediately shown you in the live image _ Histogram Histogram display Mapping functions Adjust Display x Adjust Display Detail Mapping Js Intensit 52 51 Clip min 0 10 Fla Max 0 10 Ee 53 66 E At the top of the dialog box you will find a colored button for each of the image s color channels Click on one of the colored buttons to select the appropriate channel Click the white button to display all color channels at once In this mode you cannot change the display Thus all controls in the dialog box are disabled Should the white button be pressed you can click on one of the narrow gray buttons below a color to change the way the corresponding channel is to be displayed while all of the other color channels will also be displayed The diagram in the dialog box shows the intensity histogram of the currently active color channel The histogram or intensity distribution plots the number of pixels in the image against the intensity It shows of how many pixels of which intensity the image consists The histogram s scaling is by default set so that it encompasses only the range betwee
307. ideways shift will be compen sated that for instance can occur when you make image acquisitions with a stereo microscope or when the sample slips appreciably away during the image acquisi tion With the accumulative method such a pre alignment is not possible Height map Qualitative height You can also understand each single frame as an imaginary horizontal slice cut map through the sample s mountain landscape Because the sharply focused area of a single frame shows you where the sample s surface lies within a certain height range For this reason the gray value image mentioned above that the EFI algorithm sets up internally is at the same time a qualitative height map of the sample s surface This height map will though only show the height ratios correctly when the single frames have been saved in the correct order in the image buffers used The correct order means Images acquired at consecutive focus settings must be saved in corre spondingly consecutive image buffers For the calculation of the EFI image itself this order is of no importance When you use the standard method you will yourself have to take care that this order is maintained When you use the fully automated method the images will be auto matically written into the image buffers in the correct order It is not possible to create a height map when you use the accumulative method since the images used for this method will not be saved Apart from that EFI does not
308. il it does what you want it to do such that the new gray value image has the intensity and contrast you re looking for Gray value changes will always be shown throughout the whole image even if you have set a frame A frame will however be recognized as such in the resulting image The resulting image will then be the same size as the original image yet the image area outside the frame will be assigned a gray value of 0 making it appear black The Function list contains all mapping functions predefined and user defined Select the desired mapping function from this list The previous mapping function will be deleted from the XY diagram and the new one will be drawn in as a blue curve At the same time both scroll bars Intensity and Contrast will be set at 50 If you wish define a new mapping function right in the diagram and then insert it into the Function list To do so enter the name of the new function into the list field and then click the Save button The following are the available predefined mapping functions Function Meaning Linear linear function y x Original image is not altered Max stretch linear spreading or stretching of the existent gray value range to the maxi mum gray value range possible contrast will be enhanced This function corresponds to the Process gt Intensity gt Maximize Contrast command Equalization nonlinear contrast optimization of an image Intervals containing fr
309. ill be automati cally done for all other image formats e f possible compress the images to keep the size of the e mail to a mini mum Use the JPEG image format if the recipient wants to open the imag es with another application program since most application programs can not load compressed TIF images 7 To determine the file format for sending reports select the Report tab located in the Send email Preferences dialog box Select the Send report in Rich Text Format rtf option if the recipient wants to open the report in another applica tion program such as MS Word For RTF format you can reduce the resolution of the images in the report 8 Close the Send email Preferences dialog box by clicking on OK 9 Please Note Select the Custom option in the Send email dialog box to activate lt P the format settings your have just made 10 Click the Send button 15 First Steps E mailing images gt Allimage and document files selected will appear as attachments in a new e mail document Please keep in mind that as long as the e mail document is open all other functions in your image analysis program are not accessible All selected documents will appear in the e mail as attachments a Untitled Message Rich Text asa S eewo aR 4 E options AB File Edit View Insert Format Tools Actions Help ns i Sy gt i Mt i il Wl ii Diagrm1 sfd 8KB Sheetl sqd 6KB Text1
310. ill be calculated Ip R G B 3 e The color factor F for each color component of each pixel within the circle will then be determined based on this calculation e g the red color factor would be F R I R Take a look at this example a pixel has the following color components R G B 100 245 255 thus an average intensity of 200 and a red color fac tor of F R 200 100 2 0 e The three color factors will be averaged for all pixels within the circle meaning that a correction factor lt F R gt lt F G gt and lt F B gt will be determined for each color component e And now the color components of all the image s pixels will be multiplied by the corresponding correction factor s When you use white balance the colors of all the pixels within an image will be shifted This color shift will be less significant the lower the color intensity is of the pixels within the circle If pixels within the circle are not in color i e are some shade of gray then these pixels color components will have values as follows R G B Furthermore the three correction factors will be lt F gt 1 and in fact white balance will have no effect on the image The opposite of the above is also true color shift is more significant the higher the color intensity is of the pixels within the circle Let s assume your image doesn t have any color tinging If you then select an image area that is in color using the user defined
311. ill be displayed in the image overlay The commands of the RGB Studio gt Shape menu will then be available Creates a new image in the destination image buffer The shapes in the resulting image comprise all areas of the original image that were not covered by a shape The inverted shapes will be displayed in the image overlay 271 The Process menu Shape Copy Pixels Copies all image sections within a shape into the image in the destination image buffer Press Shift at the same time you select the command to be able to position the pixels within the destination image before inserting them This command is only available if the destination image buffer contains an image that has the same bit depth as the original image After calling the command the Define Border dialog box will be opened Here you define the width of a shape s border in pixels This border will be blurred to create a smoother transition from the shape content to the image background Note Please note The border defined here will also be taken into consideration by the following commands Crop Pixels Fill Shape Crop Shape Fill Border Crop Pixels Crops the pixels in the shape and creates a new image from them The Select Fill Color dialog box will be opened This is where you select the color or gray value for the image background This dialog box varies depending on the image type Select the color or gray value desired and click on OK The res
312. image analysis program offers a wide range of statistic parameters Click the Define Statistics button in the measurement display to select parameters which are relevant for measuring Only the selected parameters appear in the measurement display and in the statistics sheet You can always export the statistics of measured values to a sheet In doing so you determine which images are to be considered for the statistics in a separate dialog box The statistic values are always written to a separate sheet they cannot be written to a sheet together with the measurement values This is how you work with the statistics functions 1 Carry out an area measurement on numerous images gt The Statistics for Area group located in the measurement display is updated after each executed measurement By default it contains the amount of executed measurements the mean minimum and maximum area measured and the standard deviation 2 In the tree view select the Perimeter measurement value gt The data in the measurement display no longer correspond to the measured areas but to the measured perimeter 3 Click the Define Statistics button You will find the button in the measurement display above the tree view gt You will find all available statistic parameters in the Define Statistics dialog box You will find an explanation to each parameter selected All of the parameters which are currently selected are shown in the list on the right en
313. image from the Feasible image objects list and click the name of a fluores cence to have this fluorescence allocated to that image The name of the fluores cence and its color will be written next to the image name Select the check box next to this image to have this allocation used for the new multi dimensional image In the Available Fluorescences list you will find only fluorescences that you have previously defined by using the Acquire gt Multiple Fluorescences gt Define Fluoro chromes command Click the OK button to have a new multi dimensional image created from the selected and newly defined fluorescence images Edit Image Multidimensional Edits an image pixel by pixel This command is not available for multi channel data sets images After using the command a part ROI of the image is zoomed 8x in the left part of the dialog box A paintbrush is displayed within this image Move the mouse cursor onto a single pixel or onto several pixels to modify their gray or color values E dit mane Fisel information Position Value ROI Ongin 515 293 Size 346 446 Zoom Brush Donne cteity rel il Nore fka E IF rae Color selection Pixel 228 The Pixel information group displays the X and Y positions of the mouse cursor as well as the gray value or RGB and HSI values in case of a color image 189 The Image menu Edit Image The RO group displays the position of the zoomed image
314. in size than the designated image will be repeated and put beneath the designated image making it look like a watermark due to its repeated occurrence throughout the image 266 The Process menu RGB Studio The resulting image will be written into the destination image buffer The original image will appear to be on top All of its colors remain as they were The texture image will appear to be beneath the designated image The texture image will be converted into a gray value image before it is combined with the original image The texture watermarks are thus gray Separate Colors Merge Colors Shape Add Using the Separate Colors command you can have the color components comprising a true color image separated into color levels A gray value image will be generated for each color channel You have various color models to choose from Use the commands of this submenu to select one of these color models RGB CMYK CMY HSV or HLS The resulting images will be written into the destination image buffer and the subse quent image buffers Use the commands of the Merge Colors submenu to have gray value images combined into a true color image Each gray value image will be assigned a partic ular color level of a color channel You have various color models to choose from Use the commands of this submenu to select one of these color models RGB CMYK CMY HSV or HLS Load the three or four images you want to have combined in
315. in the cell are located you can overlay a fluorescence image with a transmission image in the viewport when you do this these images can be acquired by contrast enhancing methods You can only overlay them when both images have an identical X Y resolution A multi dimensional image can be overlaid either with a single image snapshot or with a multi dimensional image whereby the number of single images in the time lapse image or Z stack must be the same for both multi dimensional images To create an overlay image of fluorescence and transmission images 1 Activate the fluorescence image it can be monochromatic or multi channel 2 Open the menu of the Select Color Channel button and choose Select Trans mission A dialog box opens that lists the images that would fit for an overlay 3 Choose the desired image and click OK gt The viewport shows the resulting overlay image 4 Deselect the Overlay Transmission command to remove the overlay The overlay image is not a new data set it only exists on screen To create an overlay image as new data set use the Edit gt Copy and the Edit gt Paste command lt Ctrl c gt and lt Ctrl v gt and store the new image This image is a 3x8bit RGB image not an nx16bit image 20 Button Bars The Stack Navigator button bar Navigate Z Select Transmission Shows a dialog to select the transmission image Select Overlay Image Available overlay images GFF Cancel YFP Hel
316. in the image will be altered accordingly Click the Test button to reset the clipping frame back to its original automatically calculated position Click OK to confirm the proposed clipping region Click the Interactive button to define the clipping border interactively via the clipping frame above the mouse cursor Rightclick to save the current settings in the Clipping Border Pixel group and to return to the Clipping dialog box Click on the OK button to clip the image in the active image buffer Note that the resulting image is not transferred to the destination image buffer but will replace the original image in the active image buffer 151 The Image menu The Image menu The Image menu Image Display The commands in the Image Display menu are image processing functions that do not alter the image data Only the appearance of an image on the screen i e the image s display will be changed Adjust Display A multi dimensional image or a 16 bit standard image contains as a rule far more information than can be shown on your monitor Use the Adjust Display command to change the way an image is displayed on your monitor in such a way that you can see the interesting structures in it optimally Note The Adjust Display command only changes the way the image is displayed not the contents of the image The appearance and functionality of the dialog box depend on what type the image is Multi dimensional images
317. in the overview image you can save its position and subsequently return to it at any time To do so you must be in the Stage Area Mode or in the Online Mode Click the right mouse button Click the Add Posi Add Position tion option in the context menu Positions Scan m x n images Scan Interest Area Define Context Menu Moving to Stage Navigator positions and editing them To do so click the right mouse button Click the Positions command in the context menu 342 The Stage Navigator Trouble Shooting Going to a position Removing a position o Stage Navigator Positions Position information Name Position 1 k 35501 00 Y 39938 00 Z FOO0 00 Magnification 10 00 Previa Remove M Auto preview Cancel Help You will see all the positions that have been saved In the Position Information group you will find more precise details In the Name field you can change the names Fur thermore you will see the X Y and Z values of the saved position as well as the magnification with which this position was saved To go to a position choose a position and click the Preview button If the Auto pre view check box has been selected the Stage Navigator will indicate the area in the overview image as soon as you have chosen a position Choose a position and click the Remove button to delete a position Please note the positions are only saved as long as the overview image is loaded When you close the
318. indicate where the lower and upper limits of each channel are Each color channel can be located anywhere between the values 0 and 255 The easiest way to define color ranges is to go right into the image and select a portion of the picture containing the color desired To do this first click on the New button located in the Include pixel group Select a color for your active phase from the Color list This color will be displayed in the diagram s color bar indicating where this phase is Use the Preview to have portions of the image belonging to a phase displayed in the phase s color Click the New button to define a new color range i e phase After clicking on the button a new entry will be added to the Phase list This new phase entry comprises the standard phase name plus its number This new color range always includes the values 0 127 for all three colors Select a color range i e phase from the Phase list Then click on the Delete button to delete the phase selected This button is available as long as there is still at least one phase existent 216 The Process menu Set Thresholds Note If you are using the standard phase names the correspondence between an actual phase and its standard name may get changed around When you for example delete Phase 2 Phase 3 will then become Phase 2 Phase 4 will then be Phase3 and so on A Phase that has been deleted will not be entirely forgotten until y
319. ing Mew Folder No records available J Extended Acquisith 9 Multiple Image Alig 3 Phase analysis 3 TEM acquisition 3J Stereo images 3 Extended focal ime 3 Filtering 3 Particle analysis ExampleD atabase lt New Folder A New sort criterium is the creation date of the folder The latest folder is now inserted first 88 Choose View Archiving Images Work in the database window otep by step How to change the view of your database 1 Select the Database gt View gt Choose View command gt The Choose View dialog box offers 6 different displays of the database window Full View gt A table is displayed in the full view The database view selected under Table View instead of the gallery the Full View option is automatically m chosen by clicking the Full View Galery View ma oan ineualng galery button located in the Database but ton bar Click the Next View button Full view gt The gallery is displayed as a single to alternate between gallery and ta Horizontal row In this way the size of the gal ble views Gallery View lery section is reduced in favor of Narrow View The form view disappears and the gallery is reduced to a small strip Equates to the view which is selected by clicking on the Narrow view but ton Structure Strip Reduces the database window to the preview window and tree structure Gallery View Reduces the database window to the gallery
320. ing Spectral Unmixing Unmixing Based on the calibration for the different fluorochromes the unmixing of a multi labeled sample can be performed 1 Load the multi channel image of a multi labeled sample into the active image buffer In our example it would be a two channel image of a GFP YFP labeled specimen The image is a two channel image by which the color channels were acquired with the GFP and the YFP excitation filters respectively gt Your image analysis software automatically recognizes the number of color channels and the dialog will have two or three fluorochromes to be selected Dukpa i Single image One image per channel Background Subtraction Constant fo fo w ROI A Define 2 Click the Spectral Unmixing button located in the Image Analysis button bar Ratio data 1 Fluorochrome GFP 2 Fluorochrome IYFF 3 Select the ratio data for the different fluorochromes from the respective pop up lists Before performing the linear unmixing it is additionally necessary to define a ROI for background correction 4 To define a ROI for background correction click the Define button to open the Define ROIs dialog box 5 Draw a ROI as usual to determine the background intensity 6 To start the unmixing algorithms click the OK button gt The processed image is automatically loaded into the current destination image buffer Acknowledgment The images shown were acquired with a cell4
321. ing image is always written to the Dest image buffer which can be defined with the help of the operands box located in the image manager The use of the relative operands Source and Source 2 Src1 and Src2 is very helpful if you want to use the formula on various image buffers more than once Select the Intensity Scaling check box if you want to activate the consideration of the Z scaling When deactivated only the plain pixel values for the calculation are used When using an active Z scaling the contrast of the result image is additionally maxi mized which means that the pixel values are adjusted so that they optimally utilize the result image s range of value You have the possibility of avoiding the maximized contrast by keeping the Z scaling turned off To do this clear the Maximized Contrast check box 249 The Process menu Arithmetic Operations Arithmetic Operations Performs arithmetic operations on multi dimensional images Arithmetic Operations Arithmetic Operations Dimensions Input Source 1 1 HERZ Source 2 Constant Operation Source 2 Constant value i is Standard Image Multi Channel Image OF Cancel Help What happens if there s a pixel value overflow Commands execute the arithmetical operations one pixel at a time thus processing all gray values 8 bit channels have values between 0 and 255 16 bit channels have values between 0 and 65535 When applying ad
322. ing the acquisition and computation phases the image window will be divided up into 2 viewports The left viewport will display the current live image and the right viewport the preliminary EFI image as much as has been computed Adjust your microscope s focus setting manually little by little at the same time keeping an eye on the onscreen live image At regular intervals EFI will freeze the live image and compare it with the preliminary EFI image If the still or frozen live image contains image areas that are in better focus than in the preliminary EFI image the less sharp EFI image areas will be replaced The same image buffer is in continuous use for the live image meaning that stills of the live image are continually being overwritten Due to this fact this EFI accumulative method requires a mere 2 image buffers one for the live image and one for the preliminary EFI image continually being newly computed After initiating this command the EF acquisition dialog box will be opened It will remain open during acquisition and EFI image computation EFI Acquisition x Frames 0 Start Delay m ho B Cancel Append Help The Frames field informs you how many images have been recorded at present Enter a value greater than 0 into the Delay field if you wish EFI to wait a certain length of time longer before acquiring the next image This will give you more time to adjust your microscope s focus setting 132
323. ing the nodal point selected 2 Position the mouse cursor at this little circle and press the left mouse button The circle will turn green 3 Keep pressing the mouse button and then move the nodal point in either an X or Y direction 4 There are two predefined nodal points blue at the left and right borders of the diagram You can shift these with the mouse as well if you like To delete nodal points 1 You can smooth the curve by deleting unnecessary nodal points This is done by positioning the mouse cursor at the nodal point and while pressing Shift key you leftclick Count Colors Counts how many different colors or gray values an image is comprised of Set Thresholds Note Sets thresholds of one or more phases for the images A number of image processing functions are only available for use on binary images If your image isn t binary already you will need to convert it i e binarize it A binary image has only 2 gray values 0 and 255 The way this binarization takes place is dependent on the type of image i e whether dealing with gray scale color or multidimensional images Due to this the command and the dialog boxes differ accordingly Currently there is no mechanism for setting thresholds for multidimensional images If you need to binarize multidimensional images you have to convert the image into a set of standard images first This is achieved with the Image gt Convert gt
324. ion you wish to save along with the images Database field entries provide an unambiguous characterization of each image which allows you to locate each image in the database Archiving Images 63 38 Report Generator Report objects Predefined fields User defined fields Record object Database images from the image manager What make up record objects Inserting sheets 50 Inserting diagrams 54 Predefined fields are fields prescribed by your image analysis program They contain image data information that your image analysis program can automatically read out e g image calibration The predefined fields of each image database are the same If you only use predefined fields in your reports you will not have to adapt the record objects of your report to your database Set up your own user defined fields for each image database e g user project comment or instrument These fields are usually different for each image data base Record objects that refer to user defined fields can thus only match one particular image database You can insert images directly from an image database into a record object When inserting the image into the record object all image entries belonging to an image can be automatically included in the report Use record objects to have report creation based on existing image databases largely automated Your image analysis program recognizes whether or not an image in the image manage
325. is called for example Project gt The Available and Current list display all organizational fields which relate to all the information within the database folder The required fields are formatted in bold type 4 Mark the Customer entry in the Available list and click on the Add gt gt button 5 Doubleclick on the Form View entry in the View Type list to display the data types for the form view 6 Mark the Form View gt Image data type gt The Available and Current lists now contain all existing database fields 7 Mark the Customer entry in the Available list and click on the Add gt gt button 8 Repeat the last two steps for all types of data gt The customer will now always be displayed in the form view 9 Now mark the Insert view and the Image data type 10 Transfer if required the Customer organizational field from the Available list into the Current list gt The customer will now be displayed in the insertion mask when an image is inserted 11 Close the Arrange Fields dialog box by clicking OK 83 How to arrange data base fields Archiving Images Arrange Fields Arrange Fields View Type Available Current ae Database fields can be BEE nio window arranged differently HE Form view ES Table View to aim at different needs o Query by Example for example for the H Inser insertion mask or when printing This is the organizational ID of the database
326. isibly subdivides gray values up into respective intervals The filtered image can then be superimposed over the original image indicating the magnitude of the various gray values The parameters of the filter are defined in the Define Reimer dialog box Interval E H Cancel Preview Eocene Wind ee The Interval field determines the width of the gray value interval 1 254 The preset interval value is 32 The lower the interval selected the greater the image detail that will stay visible in the resulting image Define Connectivity Filter Use this command to set a Connectivity filter s parameter What s it for With this filter you can analyze in which way gray value areas are connected to one another Connected gray value areas can be shown in different ways 232 The Process menu Define Filter The illustration demon strates the Connectivity filter s effect For the Delete mode the Number of neighbors varies At the top right an enlarged segment is shown in which the ap propriate number of neighboring pixels have been selected All of the pixels that were select ed have been set in black the rest in white Description of the illustration Number of neigh bors Delete Delete Delete Number of neighbors Number of neighbors Original 7 6 5 Color coding Number of neighbors Number of neighbors Delete Delete The original image shows several nested rectangles with different g
327. it of time Creating a report template involves 1 Finding a relevant name Give each of your page templates names relevant to 2 3 w w their contents This ensures that each user will have no trouble recognizing which page template is which when inserting a new report page s Labeling text placeholders A user should be able to immediately recognize and without having to try out each blank space which report template text fields are to be filled out and which ones are background objects You should thus label each text objects of a report that is meant to be filled out by the user right from the start in the report template You can e g place text variables within square brackets and write a request in text placeholders Do not define any text fields within your templates that contain no text The user will have difficulty relocating these text fields within the templates You can t tell whether a text object is autotext background text a text variable or editable text by simply looking at it This is why it s a good idea to decide on some kind of indicator to help you more quickly distinguish between different types of text If you have several users making use of the same templates all of them should be able to tell which text objects have to be filled out and which do not You could use e g square brackets to indicate text variables in your report templates Editable text could be indicated by three dots or you c
328. it true color images are comprised of 3 color components red green and blue Each color component has its own value range of 0 255 The 3 color component are spread interdependently This means that the image s coloring will not be radically changed rather that each of the colors will become either lighter or darker Say that e g for all pixels the lowest value of all three color components is 10 This pixel s color component at 10 will then be set at 0 This pixel s other two color components will be have 10 subtracted from their values Color components of the other pixels will be adapted linearly True color images often have single pixels that have one color component that is very bright or very dark Because of this fact this command will have a minimal effect If you want to enhance contrast you would do better to use the Equalize Contrast command Equalize Contrast Modifies gray values to create optimally distributed gray value dynamics The Equalize Contrast command non linearly optimizes an image s contrast What happens is that intervals of gray or color values occurring frequently will be spread intervals of gray or color values that are less frequent will be compressed This means that some image structures will be emphasized while others will be repressed thus changing the general visual impression of the image 198 The Process menu Intensity Gray value images False color images True color images
329. ite arrow button on the Overlay button bar to switch to the edit object mode Select None A selection will usually only be removed if you explicitly cancel it To cancel all selec tions you can click on the button with the red X for example Font size You can choose one of two options for dealing with font size in the overlay This setting is also taken into consideration when printing e You can have your text zoomed along with the image thus ensuring that text size matches image display size If you display the image in the viewport at a lesser zoom level text size will be reduced accordingly The Object Properties gt Text gt Zoom with image check box is selected in this case e For zoom levels less than 100 you can have the text displayed at a constant size The Object Properties gt Text gt Zoom with image check box is not selected in this case Eqs Locked Data Layer The default setting has data layers locked This means that objects in a data layer can not be selected or edited Copy Overlay To copy an overlay use the buttons of the Overlay button bar you cannot use the Windows clipboard Instead of pressing Ctrl c and then Ctrl v you use the buttons in the Overlay button bar Edit Overlay Selects moves and edits objects in the overlay In order to manipulate existing overlay objects you first have to select them Use the Edit Overlay button to do this Click on an existing object to sele
330. ith the positioning Procedure A central function is the automatic adjustment of single frames that belong together For this purpose the images will be arranged in an image matrix You set the position of the single frames in the Arrange Multiple Images dialog box The images will be adjusted to match one another from left to right and from the top to the bottom That is to say that each image in the first row will first be aligned with the image to its right Subsequently the first image in the first row will be aligned with the first image in the second row and so on Image one is to be aligned with image 2 Image 1 will be called the reference image image 2 the search image For the alignment on the border of the reference image a reference pattern will be defined that is then to be found in the search image To define the reference pattern the image border s histogram is evaluated and the area with the greatest contrast used To save time for this search the size of the reference pattern is limited to 128x128 pixels The reference pattern will be searched for along the whole of the search image s border A human viewer would have no problem comparing two images to find a similar or the same pattern An automated search procedure however requires a mathema tical criterion for the similarity of two image patterns For judging the similarity a reference image will be calculated from the reference and the search images A correlation is
331. ithin the histogram The blue line represents the lower threshold the red line the upper threshold Thresholds can be adjusted within the histogram To do so simply move your mouse cursor over one of the two lines Once the mouse cursor changes shape into a double arrow simply pull the line keeping the left mouse button depressed to the position desired You have to activate one of the color channels before you can adjust any thresholds Leftclick on one of the phases in the color bar Keeping the left mouse button depressed you can move both thresholds simultaneously Binarizes the active image The Binarize command is context sensitive It changes to Binarize Color mage if the active image buffer contains a 24 bit color image At present you cannot binarize multi dimensional images If you need to binarize multidimensional images you have to convert the image into a set of standard images first This is achieved with the Image gt Convert gt Split into Standard command The resulting gray scale images can be handled in the same way as any other gray scale images Binarizes an image according to the specified phases Some image analytical functions can only operate with binary images A binary image is composed of two different gray values only i e each pixel has one of two possible values set white or not set black In order to improve the processing 219 The Process menu Binarize Color Image spe
332. ition 2136 50 pm Z postion 5922 79 pmi oo Sequence Frame 2 Setting Le oo Sequence 3 Frame 3 Setting C Eames Controlled by mFIF Micmecaope Controlled by mFIF tonto micrsecope illumination OF File Cancel Start Details dd Your image analysis program is able to automatically operate connected hardware components motor stage camera microscope This tab is for organizing how these settings are used and how individual settings are assigned to specific sequences 137 Acquiring images Define Image Sequence Macro Define Image Sequence Macro Defines preprocessing and postprocessing steps which are to be executed before or after each sequence cycle or process BS Define Image Sequence Processing Sequence a alal al a Info Flow Settings Macro Process Apply preprocessing Apply postprocessing Frame etting Ci Cycle EE Sequence 3 Frame 3 Setting C Apply preprocessing Define Apply postprocessing Sequence Apply preprocessing Define Apply postprocessing OF File Cancel Start Details Help ok es tee _ Alongside the actual image acquisition you may also define other automation steps for your image analysis program to conduct while it carries out a process Select the Apply preprocessing check box in the Process Cycle and or Sequence groups to activate preprocessing for the respective unit Select the Apply postproces
333. itive Should the results of a calculation fall negative they will either be replaced by O not Maximize Contrast or a number will be added to all the pixel values so that the smallest pixel value is 0 When using an active intensity scaling the pixels are converted and allocated to another range of values Thus formerly negative figures as well as values beyond the 8 or 16 Bit savable integers can be displayed Dealing with unde Should the result for a pixel not be defined e g logarithm of a negative figure the fined Results resulting pixel will be assigned the value of the original pixel Varying Image Sizes When you use images of varying sizes for a calculation the size of the resulting image conforms with the first operand Since the images are calculated against each other pixel by pixel the calculation only takes place on the respective overlapping rectangle of two operands In doing so the upper left corners of the images are located on the top of each other Formula Field Formulas can either be written directly into the formula field or can be created by using the calculator keys The calculator is able to memorize up to 20 formulas in the formula field These formulas remain even after the program has been exited thus enabling you to use them again at a later time Function Field In addition to the basic calculating operations the calculator also offers some scien tific functions which are located in the function field In ad
334. ively To do so you will need to have an image that has an image area where you know there shouldn t be any color rather this particular image area is supposed to be black white or gray Here s an example you acquire an image of a gray and white cup In the image acquired the cup looks yellowish This yellowish tinge the color tinge can be corrected throughout the whole image What do do The mouse cursor will appear within the original image Define a circle interactively within the image area where you know the pixels should be white black or gray but at present are tinged Now three correction factors will be calculated based on the pixels within the circle one each for the three color components These correction factors are defined such that the pixels within the circle will be gray on average the circular area will have no color at all Using these correction factors the whole image will be corrected 206 The Process menu Intensity Active Frame What will happen Saturation Mouse Functions If you ve set a frame the mouse cursor can only be moved around within the image area of this frame and the circle described above can only be defined within the image area delineated by the frame The resulting image will be the same size as the Original image Any image area outside the frame will be given a pixel value of 0 meaning it will be black e Average intensity for each pixel n within the circle w
335. k the Object Properties button to select the arrow style Creates a polyline object in the overlay When you insert lines they appear selected Lines color is predefined red The preset line weight is defined in the Pen size field in the Special gt Preferences gt View tab After insertion click the Object Properties button to define color and line weight Arrows are not available in conjunction with polylines The positions of polylines can be altered subsequently as you like The course of a line cannot be altered To draw a polyline 1 Click the Polyline button The mouse pointer jumps into the image window 2 Position the mouse pointer to the starting point and click the left mouse button 3 Set additional mouse clicks along the line In this way you can compose the line from numerous straight segments 4 Keep the Shift key depressed and with the left mouse button click to delete the last segment step by step 5 Keep the left mouse button depressed to change to a free hand drawing mode 6 Click the right mouse button to end the definition of a line Inserts a polygon object in the overlay When you insert lines they appear selected A lines color is predefined red The preset line weight is defined in the Pen size field in the Special gt Preferences gt View tab After insertion click the Object Properties button to define color and line weight Arrows are not available in conjunction with polygons Polygons c
336. l Use the Acquire gt Configure Input command and activate the Display tab In the Image scaling list select the Full size 100 option The Manual Acquisition dialog box will be opened The live acquisition will be started The Manual Acquisition dialog box Manual Acquisition Miel In the area on the left Parameters the live image can be E g 7 E E Cloze seen on the left border of which a transparent E Horizontal strip of the image that Restart Overlap fao az Transparency fo ele has already been acquired can be seen Help a H Ltr ji Teieti m 142 Acquiring images Multiple Image Alignment Live image Overlap Overview Image Parameters c E IU Vertical Overlap Transparency Close Acquire Restarting Arrange Arrange With the manual acquisition method you move the stage position between acquisi tions by hand It is therefore possible to use this method with microscopes that are not motorized In the area on the left side of the dialog box the current live image will be displayed The live image can always be seen in its entirety When at least one acquisition has already been made the overlap area that has been set for the relevant neighboring image or images will be displayed transpar ently in the live image You can adjust the live image in this area to acquire single frames that are as well aligned as possible
337. lass of filters employing a nonlinear method for edge enhancement This filter is comprised of a set of derivative filters Gray value modu lations that are either horizontal or vertical will be especially enhanced The Sobel filter generally yields the amount of the difference and the direction of the most signif icant change The results provided by this filter are comparable to those of the Laplace filters If you need a clearer accentuation of edges then select the Laplace II filter this latter filter however does not suppress noise Performs a gradient filter for edge detection with a fast 2 x 2 matrix Roberts filter The Roberts filter registers transitions from light to dark pixels The Roberts filter belongs to the class of filters that employ a nonlinear method for edge enhancement This filter is comprised of a set of derivative filters Two 3x3 matrices will be computed Only the difference in brightness of two values respec tively are taken into consideration for each pixel Due to the low number of computa tions this filter functions well as a rapid edge filter The results provided by this filter are comparable to those of the Roberts Sobel and the Laplace filters The latter ones however are somewhat slower though more precise For a rapid pre analysis of an image the Roberts filter is a good bet for more thorough analysis however you d do better using the Sobel filter for example Performs a gradient filter for pha
338. lay button to get the minimum and maximum x values currently displayed in the graph window The Channel value indicates the current x increment X Calibration Offset Performs an X axis offset calibration Calibration Offset x Reference Set Mew value 1127 Y Calibration Performs a Y axis calibration New Y old r il 0 Cancel Help Normalize Y Performs a Y axis normalization Normalize Y Normalize e Maximum Channel E 4 r fet Hew value i B Cancel Help 318 Graph Overlay Selection Overlay Selection Measure Defines overlay graph Overlay Selection x Graphs Overlay 2 Histogram of EFl_ MAP 3 Histogram of EFI MAP Add gt gt 2 REMOVE Ren owe all T Copy labels Cancel Help It is possible to display several graphs simultaneously in the graph document The graph in the active graph buffer is the main graph the other graphs loaded can be used as overlays In the Graphs list of the Overlay Selection dialog box all loaded graphs despite of the graph in the active graph buffer are listed Click the Overlay Mode button in the graph window to adjust the display of the overlay graph only This is how you display two graphs simultaneously 1 Load the two graphs 1 and 2 Display graph 1 in the graph document 2 Use the Graph gt Overlay Selection command 3 Selected graph 2 in the Graphs list 4
339. ld 69 Archiving Images Define database fields 6 The Organizational field check box should not be selected In this way you can allocate a different comment to every record that is added to a database folder 7 Close the Add Field dialog box by clicking OK gt The new database field has then been set up and will be displayed in the Available Fields list 8 gt All of the database fields are characterized by an icon in the Define Fields dialog box The new database field will be prefixed by the icon for a user defined organizational field 8 In the Define Fields dialog box click the Close button to complete the definition of database fields Click the button with a yellow star on it to set up a new database field Field properties Field list Define Fields Name Comment In the Available Fields list select a database Datatype Text field to view its proper ties in the Define Fields dialog box Input required a i i Picklist du Audia File x Edit fey Bits per Pixel 424 Calibration Unit dy Channel gt Expand pichist on input nent Creation Date pont espand picwist an maut p E Restrict mout bo pickiest entries nent Creation Time cument Files Size locument Insertion Date Default cument Insertion Time Value of last input dy Document Modification Date dy Document Modification Time 9 Ea m Arrange Fields 9 Use the Database gt View gt
340. lder you want already exists you can click it If it does not yet exist enter the folder name in the Folder name field The folder name will also be used as the image name gt The following files will be saved If you have created the overview image from separate images these will be saved in the lt Folder name sequential No gt tif format Additionally a lt Folder name gt _Overview tif file and a dat file will be saved If you have created the overview image from one image only it will be saved in the lt Folder name sequential No gt tif format Additionally a lt Folder name gt _Overview tif file and a dat file will be saved 335 The Stage Navigator Loading an overview image in the Stage Navigator Loading an overview image in the Stage Navigator Once overview images have been acquired they can be reloaded at any time also from other workplaces within your image analysis program 1 Click the Open Folder button and navigate to the folder you want Click the OK button to load the overview image You do not need to select a file supplying the folder is sufficient 2 If you have created the overview image from separate images you can select the Rebuild the global overview check box The lt Image Name gt _Overview tif file will then be recreated For this reason it takes considerably longer for the overview image to appear Therefore select this check box only if you have made changes in the separate images in the mean
341. lect the first command in this submenu 4 Drag amp Drop drag the image that you want to insert into the database from the image manager onto the database name in the tree structure of the database window You will be asked to set up a new database folder Confirm the message by clicking Yes The name of a data The standard name for a new database folder is defined in the database settings base folder Use the Database gt Administration gt Settings command to view the settings for the active database The database must be opened exclusively if you want to alter the settings You can use any fixed text and two wildcards e The lt organizational ID gt is predefined as a fixed text You can replace the lt or ganizational ID gt with a text of your own preference e lt Date gt is a wildcard for insertion date The date format depends on the current settings of the operating system Your image analysis program uses the short date format selected from the local settings of MS Windows e lt Cons No gt is a whole number which is increased by 1 Every number is issued exactly once If you delete the last record with the consecutive number 10 then the next record will be given the number 11 72 Archiving Images Insert data otep by step How to create a new database folder You have created a project database with the organizational ID Project If you have defined a different organizational ID for your database for example Ord
342. lection of images select the first image by clicking on it with the left mouse button Keep the Ctrl key depressed while you use the left mouse button to select all of the image files you want e all of the images in a directory simply use the keyboard shortcut Ctrl A The File gt Open command is context sensitive This means the Open Image dialog box only appears if an image window is active If a text document is active the Open Text dialog box will appear etc The Open button is in the Standard button bar To have a look at the dropdown list of all the various commands for opening click the arrow next to this button After you have loaded an image it will be displayed in the image manager The image type image name and resolution will also be displayed directly in the image manager The information displayed differs depending on whether you have set the list or gallery view in the image manager First Steps Available image types The image shown here is a multi channel image that is made up of three single frames Each frame is 1376 Pixels wide and 1032 Pixels high Each single frame has a 16 bit depth of information Image Numbe Image Name HER 2 Stack 236 x 246 x 16 2 Tice Resolution Saving You save individual images with the File gt Save As command You should save images your images as TIF files Only when using TIF format are the additional image attributes overlay i
343. lem can be corrected subse quently via the inverse of the nonlinear function thus generating a linear effect as a result This method will provide you with more realistic images or provide you with a more realistic monitor display This correction method is called a gamma correction The Gamma Correction increases or reduces image contrast selectively depending on the intensity values of gray or color images e Agamma value of lt 1 will result in the whole image becoming darker Contrast will be enhanced in bright image areas e Agamma value of gt 1 will result in the whole image becoming lighter Contrast will be enhanced in dark image areas e Agamma correction allows you to emphasize either the background or the fore ground of an image where G r is the gray or color values of the resulting image G o is the gray or color values of the original image When working with true color images you can set gamma correction for each of the 3 color components separately Varying gamma values result in a shifting of colors meaning that an image s coloring will change This transforming property can be used to compensate for color tinge s First try using the Process gt Intensity gt White Balance command to correct a color tinge because these results are more easily reproducible This dialog box is context sensitive You ll get one dialog box for gray value binary and false color images and a different dialog box for true c
344. les will be intercon nected via the pixels positioned diagonally to each other and as such will be detected as a single particle Select the Black option to have object outlines shown in black on a white background Select the White option to have object outlines shown in white on a black back ground Select the Burn black option to have black object outlines superimposed over the original image Black pixels within the original image are reassigned 1 as intensity value Select the Burn white option to have white object outlines superimposed on the orig inal image White pixels within the original image are reassigned 254 as intensity value Click the Edit button to be able to edit the resulting image interactively after separa tion This opens the Edit Lines dialog box Edits the result of the separator As soon as you click the Edit button of the Define Separator dialog box the separator will be applied to the image according to its current parameters The Define Sepa rator dialog box will be closed and the Edit dialog box will be opened You will see the result in the overlay of the original image If you have set a global frame you will only see the image area selected A global frame is set using the Image gt Set Frame command Do not confuse the global frame with the preview window you set in the Define Separator dialog box The area defined by the preview window has absolutely nothing to do with editing When editing be
345. lick the Diagram button a graphic of the actual function I G will be displayed You may vary the parameters without closing the diagram The diagram is continually updated which means that you can see how the I G function is influ enced by the parameters If you click the Snapshot button the live image is taken and stored in the active frame buffer This command corresponds to the Acquire gt Snapshot command Click the Unit button to open the Set Unit dialog box Here you determine the unit for the intensity The selected unit is displayed above the button in the dialog box Click the File button to open the standard dialog box for saving and loading files Here you can write a comment on the settings on the Image Intensity tab and save or load it Configure Input Macro Here you enter macros to be executed during pre and or postprocessing Configure Input Info sy Calibration Magnification Macro Input Display Format Preprocessing steps o Delete W Enabled Postprocessing steps Execube Delete W Enabled OF Cancel Help The macros defined in the fields Preprocessing steps and Postprocessing steps will be executed before or after each image acquisition respectively Select the Enabled check box to have the macro commands entered here executed at image acquisition Disable this check box to skip over macro commands at image acquisition Click the Execute button to test the functi
346. lick the Finish Object Editing button to save the object template for the field object 8 Enter a relevant name for this field object into the Finish Object Editing dialog E e i box and click the Save Object Template button gt This will return you to the record object editing mode 9 Click the Field button in the Report Objects button bar to insert field objects based on the object template you have just defined 10 Click the Finish Object Editing button to save the now completed record object 11 Use the Record button in the Report Objects button bar to insert the newly defined record object onto a template page Planning report templates Before creating a report template 12 Using existing templates If it s possible to make use of an existing report 13 a template then do it Empty pages mean lots of work as well as the fact that creating so many report elements is redundant Defining your basic layout If possible define all background objects of the report in advance Draft a basic layout including frame s company logo an address space and space for general information such as the department name and or order number If you have to make changes later on you ll have to adjust each template page separately Existing reports cannot be automatically altered to fit a new layout This is why any avoidable changes that you have to make later on i e after creating the template take up quite a b
347. ll be able to adjust its settings as desired 102 Acquiring images Camera Configuration Configure Input Info Defines general information about the channel Configure Input Info ser Calibration Magnification Macro Input Display Format Channel Colorview I Feat Description Colorview Il Pw y Calibrated Symbol TUTE OF Cancel Help In the Channel field enter the name of the channel This name is used to identify the channel especially when addressing the input channel using the macro language Use short channel names because you may want to use them within Imaging C programming For the same reason you should not use a channel name twice Note Usually you should not change the name of an already existing input channel All macros selecting this input need the channel name as parameter Therefore changing the channel name can cause errors when running macros Use the Description field to enter information about the camera or about special prop erties of the input channel To change the Symbol simply select the one which fits your needs Please remember that the symbol does not determine the type of hardware device you want to use Note It is up to you to ensure that channel info corresponds to the device and the other settings The change of channel name or symbol does not influence the physical channel settings On the other hand the information on the Info ta
348. ll be set to black 0 respectively white 255 The values for Left and Right can be set between 0 and 50 Manual If you want to stipulate the gray value range that is to be displayed yourself select the Manual option in the Adjustment group When you have selected the Manual option in the Adjustment group you can use the Min and Max slide controls to set the maximum and minimum values The smallest possible value is 0 the greatest oe 65535 Thus it is possible to define a maximum value beyond the maximum intensity value of the image Use this option to compare the intensities of two images on the monitor The group below the Overflow group will only contain additional information when the image intensity has been calibrated By using the Image gt Calibrate Image command you can assign an image s gray values a magnitude The gray values then are equivalent to e g the object s height The group s name corresponds to the magnitude e g Height The numbers shown are equivalent to the gray values in the Min and Max fields converted to the magnitude A minimum gray value of 22000 is equivalent to e g a height of 2 5 um When you change the minimum and maximum values the measurement value will also be automatically updated 156 The Image menu Image Display Auto Adjust Display Note Note Gray Scale Note Adjusts automatically the channel intensities of multi dimensional image sets The Adjust Display command
349. ll pixels which belong to this object gt The selected parameter has now been activated for the measurement and will be shown in the Selected Measurements list below 6 In the Selected Measurements list select one of the default parameters for 2D objects e g Radius Mean Click the Delete button gt The deleted parameter has now been deactivated for the measurement It is of course available for later measurements 7 Close the Select Measurements dialog box by clicking OK gt The measurement display is updated and shows all of the active parame ters which can be used on a 2D object The sequence of the measurement parameters is adopted from the Select Measurements dialog box 8 Create a measurement sheet gt The measurement sheet contains all the active parameters The sequence of the measurement parameters is adopted from the Select Measurements dialog box Image Manager x x el celles Area 1063 38 nm Mean Hue 195 30 Shape Factor 0 72 Orientation Convex 59 03 tgs Measurements of T 25 2D Object 2D Object Area Mean Hue pm 1001 51 1040 96 2D Object Shape Factor 2D Object Orientation Convex 195 53 0 06 195 39 0 74 103 64 5 List 291 The Measure menu The Measure menu The Measure menu Pixel Value Shows the intensities and coordinates for interactively defined pixels Each pixel marked will be denoted by a cross in the ove
350. ll three color components have the same intensity R G B pixels will be displayed in gray If a pixel s values in the table are unequal a colored pixel will be the result for onscreen display If all intensity values and combinations thereof are used each pixel can be displayed on the monitor in one of 256 256 256 16 777 216 colors Pixels of false color images in contrast to true color images are restricted to one of 28 256 different colors as only 256 colors can be displayed simultaneously These 256 can however be selected from all 16 777 216 colors A false color image can be changed back into the original gray value image thus altering its monitor display Actual gray values will thus become visible from under the LUT Click the Linear button in the Edit LUT dialog box to do so Lookup tables can also be used in conjunction with 16 bit gray value images A LUT for 16 bit images only contains 256 entries as well There s a reason the program generally reduces 16 bit images to 256 gray values for monitor display To influence this kind of display method use the Image gt Image Display gt Adjust Display command The LUT generated for display of a 16 bit image will thus actually be applied to the virtual 8 bit image generated from the 16 bit image An image s LUT only alters how an image s gray values are displayed onscreen The original gray values under the LUT remain unchanged There are
351. ll visible are the edges of the image structures Identical gray value ranges in the background and within the light structures have been set to a gray value of 0 black In contrast to the Laplace filters after application of the Differentiate Y filter only edges parallel to the X axis will be visible Any edges that are perpendicular to the differentiation filter are no longer visible The Sharpen filter increases the higher edge gray value and decreases lower gray values The intensity within the conductors and of the background will remain unchanged This sort of edge accentuation corresponds precisely to the way the human eye views transitions from light to dark The Smoothing filters class comprises filters for averaging gray values surrounding a pixel This eliminates minor gray value peaking as well as statistical noise At the same time any gray value differences that contain image information will be flat tened out The resulting image will thus seem somewhat blurry due to this type of filtering 226 The Process menu General Information on Filter Operations The Rank Filters comprise a filter class of their own An environment of the central pixel is defined here with the filter matrix specifying the adjacent pixels to be included in the rank operation All gray values of the area surrounding the central pixel will be sorted according to magnitude in descending order The various rank filters differ as to which element of
352. lly completed the Movements column displays the whole traverse distance in X and Y directions The absolute stage position in the Shift position group is continuously updated The Pixel in x and Pixel in y columns give the number of pixels image 1 was displaced in relation to image 2 Image 1 was recorded at position 1 image 2 at position 2 Click the Calibration button to start the calibration routine After calibration has been successfully completed an entry will be added to the list called Calibration List 149 Acquiring images Clipping Keep an eye on the calibration after you select the command the image window will be divided up into two viewports When you click the Calibrate button an image of the current stage position will be acquired The image will be displayed in both view ports The stage will be moved in the X direction an image acquired and displayed in the right hand viewport A certain structure of the sample located in the middle of the first image then has a frame set around it In the second image in the right hand viewport a frame will be displayed which defines the search area for finding the structure selected in the first image After locating it a second frame in the second image indicates the same structure at a slightly different position however The program reads in stage movement and pixel shifts and displays them in the Calibra tion data group Calibrating the Y direction is done in the same wa
353. lor image is displayed on the monitor exactly the same way like the false color image A binary image is converted into a 24 bit true color image Black image areas are given the color value R G B 0 White image areas the color value R G B 255 To Multi Channel To Standard Converts the image to the multi dimensional image format Use the Image gt Convert gt To Multi Channel command to change the type of the selected image After conversion commands which work on multi channel images only can be used Gray value images are not modified during the conversion However the Dimen sions tab is added to the Image Information Color images are split into three different bands Each of which corresponds to one color channel red green and blue Converts the current image to a classic image Note that only the current view is converted If you have selected an image stack for example only the selected frame will be used Split into Standard Extracts the different color bands of an image and make them available as single gray value images 185 The Image menu Separate Note Please note that only the currently displayed image will be converted If you for example convert a Z stack only the frame that is on display will be used Separate Color Channels Extracts the color channels This command decomposes a data set a multi channel image into its color chan nels The resulting Z layers will be wri
354. lowing options 3x3 5x5 7x7 or 9x9 This determines the number of fields in the middle matrix line where hexagonal matrices are concerned You enter the weight factors in the fields of the dialog box Positive or negative whole numbers are permissible The Sum field indicates the current sum of all matrix elements Use this sum as a scale for the brightness of the resulting image 0 will mean your image is quite dark Mean brightness will not have changed if your sum is 1 Enter a value into the Normalization field which determines to what extent the gray values of the resulting image are spread i e how large the intensity range of the resulting image s gray values are The greater the normalization factor the more the gray values will be compressed Positive whole numbers greater than zero are permissible A useful value in this case is the sum of all weight factors This value is shown in the Sum field For all predefined derivative filters if the sum of the weight factors is a value greater than 0 this sum is used as the normalization factor If the sum is 0 a normalization factor of 1 is used The value in the Offset field determines the gray value to be added to the resulting image When you select the Hex lattice check box the square matrix can be replaced by a hexagonal matrix The following diagram shows determination of neighboring pixels using either a square or hexagonal filter matrix 229 The Proces
355. ls into a virtual ROI multi dimensional Virtual ROIs are not available for multi dimensional images images From particle The outline of a ROI defined by this drawing method is based on a detected particle Perform Shift Allows to move the selected ROI If no ROI is highlighted in the list the first ROI displayed in the Active ROIs list is moved The mouse pointer turns into a four pronged arrowhead and switches to the image Move the mouse to place the ROI at the desired position Click the right mouse button to return to the dialog box Delete Deletes the selected ROI Select the ROI via mouseclick in the Active ROIs list 183 The Image menu Set Frame Set Frame Resizes and positions the current frame Multidimensional For image editing functions that effect multi dimensional images a frame is of no images relevance The frame defines a rectangular area within the image It is a type of ROI Region Of Interest which limits the region available for image processing functions The frame has a global character Size and position of the current frame are displayed in the status bar These values do not depend on having the frame activated or not Even if the frame is not active size and position of the last set frame are displayed If the frame is not enabled the frame icon in the status bar is grayed Note Be sure to deactivate the frame if you do not need it anymore Otherwise you may get some rather surprising operation
356. lter functions are listed in the Filter List field of the Apply Filters dialog box Filter Function Parameters Gamma modifies intensity value of the pixels via the The gamma value lies between 0 1 gamma function Non linearities of the camera 100 0 1 does not alter the image and monitor can be thus corrected Gamma is between 2 2 and 2 5 for most monitors Hue modifies pixels color value H by rotation of the The color value H lies between color circle A rotation of 180 results in colors 0 0 and 360 0 0 does not alter the being displayed in their respective complemen image tary color Saturation modifies pixels saturation value Negative val Saturation lies between 1000 and ues decrease saturation positive ones increase 1000 thousandths 0 does not alter it the image Intensity modifies the intensity value of the pixels Nega Intensity lies between 1000 and 1000 tive values decrease brightness positive ones _thousandths 0 does not alter the im increase it age Contrast increases or reduces image contrast Contrast lies between 1000 and 1000 thousandths 0 does not alter the im age Histo increases or reduces image contrast To do so Contrast lies between 1000 and 1000 Contrast average brightness is determined via the histo thousandths O does not alter the im gram age Sharpen increases or decreases image sharpness Neg Values vary between 1000 and 1000 ative values decrease sharpness positive o
357. lters 226 Snapshot 102 122 Sobel Filter 244 245 Software Protection 1 Solarize Filter 261 Specimen shift 142 Split Gain 316 SRC files 27 SRD files 27 Stack 131 Stack Navigator 18 Stage alignment 329 339 Stage Area Mode 337 Stage initializing 329 Stage Navigator Activate add In 343 Adding positions 342 Button bar 330 Button bar is not shown 343 Buttons 331 Dialog box 328 Modi 336 Preconditions 329 Properties 338 Start screen display 330 Stage not initialized 344 Standard button bar 17 Statistic display interactive measuring 286 Index Statistics 304 Status bar 5 Stevenson Arce 263 Stretch Intensity Filter 262 Stucki 263 Subtract graphs 324 Summary of Features 328 T Table View 85 Temporary storage directory 95 Test image 9 Threshold 211 215 253 Transform Image 263 Tree structure Database 85 True color image 7 U Underflow 215 221 239 250 Unmixing 275 User Filter 246 User Interface 2 V Viewport 3 Viewport manager 3 Views Database 87 W Watermark 267 White Balance 206 Workspace 10 Z Z Calibration 165 Z Correction 124 Z Layer 187 Zoom factor Change 3 Z Position 124
358. lues will be shifted towards less and less saturation towards the interior of the circle of colors At the minimum of 1000 a colorless gray value image is the result The absolute saturation value of all pixels is O Move the slider to the right The pixel values will be shifted outwards within the circle of colors towards greater saturation Define RGB Adjustment Performs an adjustment of Red Green and Blue Use the Execute RGB Adjustment command to apply the current settings in the Define RGB Adjustment dialog box to the image in the source image buffer Define RGE Adjustment x Preview IY Preview Window og E ecute nee 4 jo kl File Green 1 jo B aan Blue _ fo B Help 209 The Process menu Define Curve Adjustment The Red Green and Blue parameters vary respectively between 255 and 255 These are absolute values which are added to or subtracted from the corresponding color components Adjusting red for example will change the proportion of red within the image The other color components will remain unchanged Hue saturation and intensity of the HSI color will however be adjusted Move the slider to the left The pixel values will be shifted to the left along the corre sponding axis of the RGB color space The proportion of that color will decrease 255 means that this color component is at 0 for all pixels and that this color no longer occurs in the image The image will bec
359. luorescent proteins opens new possibilities for the modern cell biol ogist Multi dimensional fluorescence microscopy offers the possibility to visualize the spatio temporal behavior of cellular sub cellular and molecular structures In order to simultaneously visualize different structures labels with different spectral properties have to be used A major problem in live cell imaging arises from the use of different fluorochromes with overlapping spectra in one multi labeled sample impairing a number of applica tions The considerable overlap of excitation and emission spectra of different fluo rochromes is exemplified by the spectra of enhanced green fluorescence protein eGFP and enhanced yellow fluorescent protein eYFP two commonly used vari ants of the green fluorescent protein eGFP gt eYFP Absorption 450 500 550 nm Even with the use of high quality optical filters it is not satisfactorily possible to sepa rate the spectral information The consequence is the excitation and imaging of YFP labeled structures with a GFP filter set and vice versa 275 Spectral Unmixing Spectral Imaging and linear unmixing Images taken with YFP dichroic and emitter Left GFP exciter right YFP exciter The HeLa cells shown here are transfected with eGFP fused with H2B histone protein and with eYFP tubulin fusion protein Ideally eGFP should be exclusively distributed in the nucleus and eYFP should be localized in the cyt
360. ly created and given a standard name page template consecutive number Click the Save Page Template button to alter the standard name e Base your new page template on an existing one To do this you first click the Select Page Template button and save the page template via the Save Page Template button under a different name Deleting page In order to delete a page template from a report template open the report template templates and have the list of current page templates displayed The Delete button is located in the Select Page Template dialog box Editing page Page templates are defined within report templates You can however also create templates withina new page templates within a report or alter existing ones To do this simply click report the buttons of the Report button bar shown on the left m Please note that any changes you make to a page template within a report only apply to that report The report template that the report is based on remains unaffected The next time you create a report using this report template you ll have the same unchanged page templates Step by step Creating a new report template You can only create a new report template based on an existing report template If you haven t created any of your own report templates yet then go to the General tab where you ll find predefined report templates that are included in the installation and which you can modify for your own purposes 1 Select t
361. m e All the files in this document list are selected by default The above figure shows a list of all types of doc pa se ad ee i e ei Diagram SIS Diagram sfd analysis program as Graph 1 9 analyS S Graph sqd well as their respective Wlka Image 1 Tagged Image Format tif standard formats Report Rich Test Format rtf w FFA Sheet 15 Sheet fs wel Text Text bet oe Text Text tst Document EY DemoBase 3 To clear all selections simply click the Unselect All button Select the docu ments you re interested in by clicking on the corresponding check box in the document list e A warning message will appear if your attachment exceeds a maximum size 4 Select the Add attached database documents check box to send a record and any appended documents images sheets graphs etc e An entire image database cannot be sent via e mail 5 Click the Preferences button to set file formats for all images sheets diagrams and graphs gt The Send email Preferences dialog box will be opened 6 Select the Image tab in the Send email Preferences dialog box to define the image file format File formats are always defined for all the respective docu ments of one single type not for one single document e The TIF format is default Select the Burn overlay into image and Convert 16 bit images to 8 bit check boxes for this file format if the recipient will open the images with another application program This w
362. mage Sequence Define Image Sequence Info Monitors an image sequence Define Image Sequence Processing Tretes oo Sequence 2 Frame 2 oo Sequence 3 Frame 3 Enter a name of your choice for the current process in the Process field If a process name already exists ISP automatically saves all of the process parameters and makes them available the next time this command is used Enter any description of the process into the Description field 135 Acquiring images Define Image Sequence Flow Define Image Sequence Flow Makes the main settings for controlling the process and controls acquisition as well MS Define Image Sequence Processing Info Flow Settings Macra Shark acquisition mmediately At cats Cycles Camera oo Sequence 1 Frame 1 Setting L ooo Sequence Frame 2 Setting Le oo Sequence 3 Frame 3 Setting Co Sumber Repeat every f Database Folder C Marchive test_O90704 test_090704 ap E OF File Cancel Stark Details Help Determine the process starting time in the Start acquisition group Select the Immediately option to have the acquisition begin as soon as you have clicked the Start button Select the At option to begin acquisition at a point in time of your choice Enter a time of your choice into the box Your image analysis program uses the Windows Timer to determine the time The Cycles group enables you to determine how often a sequen
363. mage alignment the stage movement that is actually necessary for a hori zontal image shift is computed on the basis of the calibration data First of all your stage must be initialized Special gt Preferences gt Stage tab Before starting the automatic calibration routine set the magnification you wish to use for the image acquisition Move the stage to a specimen position showing clearly visible structures to ensure that the program recognizes the structures in the two images and assigns the positions properly If your stage is not initialized correctly you will have the opportunity to define the stage areas after selecting the Calibrate command Then the Calibrate dialog box will be opened The image window will be divided up into two viewports 148 Acquiring images Multiple Image Alignment Cahbrate x Camera settings Close Channel Dummy Digitizer Camera Magnification 20 0 Preprocessing Never Postprocessing Only last image Shift position Shift type SOO00 Y 000i f Specimen shift Calibration data Calibration List Help EEE ff mage shitt Movements Pixel in Pinel in y In the Camera settings group you are informed of the active camera settings The Channel field displays the name of the active input The logical input channel is used to set all input parameters for image acquisition The Magnification field indicates the magnifica
364. mage buffer box 2 Image Display button bar 24 Image frames Color 338 Image frames color 338 Image Information 194 Image Manager Measurements Tab 282 Image menu 152 image name 195 Image overlap 339 Image Sequence Processing ISP 134 Image Stack button bar 24 Image window 5 Activate image window 8 Adjust Window 10 Images Load 7 Print 11 Send 13 Info window Database 89 Initializing the stage 329 Input 109 Input channel calibration 329 Insert Database folder 73 Document files 79 Documents 79 Images 76 Integration 320 intelligent Exposure 113 Intensity 198 Intensity Profile 295 Interactive measuring Measuring distances and areas 281 This is how you delete single measurement values 283 This is how you measure numerous images 285 To define an object with the magic wand 288 To delete an entire measurement 284 To identify a measurement on the image 284 To select measurement parameters 290 To work with the statistics functions 287 Interest area 337 Interest area images 339 Interest Area Mode 337 Invert Graph 323 Image 204 262 Shape 272 Invert axis 345 J Jarvis 263 K Kinetic 298 L Labels 313 Laplace Laplace II Filter 243 Layer 175 Link to image interactive measurement 284 Live image 331 Load Document with Subdocuments Database 95 Documents Database 93 Images 7 Lowpass 246 LUT 158 200 204 Macro 109 138 Magic Wand 269 Magic wand Interactive measurement 288 Magnification 167 Magnificati
365. mage calibration channel data microscope data image comment saved together with the image Note Please note that even multidimensional images do not have their own file format but are saved as TIF files Do not use other file formats as the TIF format to save multidimensional images in order to maintain all the information Available image types Your image analysis program supports a large number of image types These can be divided into two groups of image types the multidimensional images and the stan dard images In the image manager different image types are represented by different colored icons Note Many functions of your image analysis program are only available for special image types Multi dimensional Your program has also been designed especially for working with multidimensional image images Multi dimensional images are made up of a number of images that have been acquired one after the other During the acquisition the Z position and or the wavelength and or the time will have varied from image to image In other words multi dimensional images are monochrome or multicolored images that are charac terized by having an additional dimension This additional dimension can be one of space the Z position has been varied and or of time the position has been varied on a time scale For displaying multidimensional images use the Image Navigator button bar A monochrome image consists of only one color channel
366. mage information will be lost since the resulting image will have fewer pixels than the original For example at a zoom factor of 0 5 every second pixel will be left out Translation Use this transformation to shift an image by a user defined number of pixels Fills an entire image or parts of the image with a certain color The Select Fill Color dialog box will be opened This is where you select the color or gray value you wish to color the image with This dialog box varies depending on the image type Image overlays are not affected by color filling 264 The Process menu RGB Studio Note Select Fill Color Basic colors Custom colors i ip i i i ip i a U g ip ag HE Hue feo Red 128 Sat fo Green f en ColorlSolid Lur f 20 Blue li Zo Cancel Add to Custom Colors If you wish to fill the rectangular image area within the frame then be sure to set a frame first A mask or ROIs in contrast will not be taken into consideration Please note This command will overwrite the original image i e the image will be lost This command cannot be undone If you wish to keep the original image then be sure to copy it into another image buffer before executing the command Edit Palette Modifies the colors in false color images The command is only available for 8 bit gray value images and false color images Edit Palette x TTT TTT Litt el EERE Cancel
367. mages are displayed and much more This chapter describes the basic elements of a GUI Please note The Graphical User Interface GUI in your image analysis program is fully adaptable to meet your own specific requirements OLX te Image Process Measure Graph Special Window 7 Button Bars vatraaa ga g Si ae fm M Ky 7 a ba ee da it Image Manage Ls a 2 M 2 E 4 i s s DO a UTN vport Manager r 1376x1032 x16 3 HER 24 bit 1376x1032 x 24 FITC 1376 1032 16 Texas Red Su 1376x1032 x16 HER2 Z Stack 236 x 246 x16 2 Deconvolved 236 x 246 16 2 Beetle Eye 2 h32 x 302 x 24 A GS Ce Og ae Database Document Y jim imj Dehua a a Bl mld image Button Bare gt 5 H F fle law g Query results T i Active Image Buffer XA Image Buffer Box 48 Graphs 1 ll Es dd LifeScienceDemoS TAR o GS Guery results FISH Time Lapse fast Autofluorescence Antibody Labelling Unlimited Depth of Foc Histochemistry Brightfield Microscopy HeLa celle with Fura Helate Unmising Deconva HERZ 24 bit Record Name Sample Preparation Comment re Document Creation Date Document Creation Time Image Dimension Color Channels Z Layers Time Frames Size Size Bits per Pixel Binning Exposure Time 2 Step Width Makers Regions OF Interest
368. map in an overlay can also be made transparent If this is what you would like then select the Transparent option in the Background palette When dealing with monochrome bitmaps you may choose any color desired or the Transparent option in the Background palette All black pixels will be replaced by the color you have chosen or simply replaced with the pixels of the original image this being the transparent option Line You can frame bitmaps you insert into an overlay You select the frame color in the Width Line palette Frames can be colored in one of the 16 classic Windows colors Define the width in pixels of the line type selected in the Weight field The line can be between 1 and 7 pixels wide Save Objects Saves all selected objects of the overlay in a file This function is excellent for creating templates of frequently used overlay objects It can be used for both pre formatted single objects or also for groupings of objects The SIS OVL sample file is installed in the overlay directory part of setup and can be loaded from there Cut Overlay Objects Copies and afterwards deletes selected objects in the overlay Click the Cut Objects button to transfer the selected objects to the overlay clipboard Use the Paste Objects button to transfer the objects into the overlay of the active image If you press Shift at the same time the selected objects will just be deleted and not copied into the overlay clipboard
369. me colored neighboring pixels The central pixel in a 3x3 environ ment will be set at the gray value that results from the following calculation When N Number of neighbors with the same or a greater intensity l2 Intensity of the central pixel in the resulting image The resulting image will then contain only the gray values 16 32 48 64 80 96 128 and 144 In this mode the Number of neighbors group and the Iterations field will not be avail able In this mode the filter supplies information about the distribution of local minima Gray value differences of neighboring pixels will be highlighted In the Iterations field you set the number 1 300 of filter passes Edge Enhance Defines the parameters for the Edge Enhance filter The Edge Enhance filter is for enhancing edges occurring in the original image The term edge refers here to an image area in which gray values either rise or fall sharply This filter can be used in preparation for particle detection When using this filter keep in mind that besides edges noise may also be enhanced as well In the original image the filter examines the gray values of all pixels of an image area comprising NxN pixels During filtering the central pixel of the matrix will be assigned either the minimum or maximum value of its neighboring pixels depending on which gray value interval the central pixel is located in The gray value interval is a percentage of the difference bet
370. measurement results are lost if you save the image in another format e g BMP or JPG A one time measured object can no longer be edited You can however delete objects with faulty measurements and remeasure them To do so select the name of an image in the tree view to switch to that image Select a measurement function from the button bar and measure the desired structure The new measurement will be attached to the already existing measurements Each interactive measurement also creates an image overlay which shows the measured object The measurement overlay is linked to the measurement values and is automatically recreated from the measurement data when loading an image or when continuing an interactive measurement Should you have edited or deleted the measurement overlay the respective alterations will be reversed This makes sure that the measurement overlay and the measurement files remain consistent Be sure that you cannot alter the measurement data by editing one of the measurement objects in the overlay This is how you delete single measurement values 1 Load the image whose measurement values you would like to delete 2 Use the Alt 4 key stroke to switch to the measurements environment 3 Select the value you want deleted in the Measurements tab e When measuring certain objects such as a circles numerous values are measured simultaneously Please note that it is impossible to delete indi vidual values with the del
371. mode of the camera intelligent Exposure analyzes the real time histogram during a live acquisition in order to optimize the exposure times The exposure times for the live acquisition and the snapshot are automatically set independent of one another in such a way that the dynamic range of the camera are fully taken advantage of The optimization of the exposure time occurs continually and automatically intelligent Exposure arranges linear correlation between the exposure time and the mean gray value for calculating the optimal exposure time In order to take advantage of this correlation the camera s dark current must be known as an offset of the linear function Thus the first step to working with intelligent Exposure is measuring this dark current via the acquisition of an image using a darkened camera So the first time you start intelligent Exposure you will automatically be asked to carry out this calibration intelligent Exposure Ed a m correction oe Ser S Ereview ol Options live Exp 100 0 ms Snap Esp 50 00 ms The exposure times calculated by intelligent Exposure are displayed in the dialog box s status bar The term Live Exp Live Exposure Time stands for the exposure time of the live acquisition while Snap Exp Snapshot Exposure Time stands for the exposure time of the snapshot Independent of the calculated exposure time for the snapshot the exposure time for the live acquisition cannot exceed 12
372. moothing width fi 150 Smoothing cycles f B Cancel Help 323 Mean Value Graph Mean Value Enter the desired Smoothing width in the corresponding field The filter operation is repeated a number of times corresponding to the Smoothing cycles field Performs a mean value smoothing Mean alue Ea Smoothing width E 150 Cancel Help Enter the desired Smoothing width in the corresponding field The Smoothing width may be a number from 2 to 99 including Savitzky Golay Arithmetic Addition Subtraction Multiplication Performs a Savitzky Golay smoothing Savitzky Golayp Ea Smoothing width D F 50 Cancel Help The Savitzky Golay filter performs a local polynomial regression to determine the smoothed value for each data point Enter the desired Smoothing width in the corresponding field The smoothing width may be a number from 1 to 5 including Adds the graphs in source and source 2 The contents of each channel of the graphs are added The resulting new graph is written to the destination graph buffer Subtracts the graph in source 2 from source The contents of each channel is subtracted The resulting new graph is written to the destination graph buffer Multiplies the graph in source with source 2 The contents of each channel of the graphs are multiplied The resulting new graph is written to the destination graph buffer 324 Graph Graph Information
373. motorized microscope you should watch the following Before you can acquire a multi channel fluorescence image the correct obser vation methods for the fluorescence microscopy will need to have been defined You will find more detailed information about this in your microscope s user manual With the observation method you choose the correct filter cube for the fluorescence acquisition you want to make at the press of a button 114 Acquiring images Multiple Fluorescences Defining a channel It makes sense to define a separate channel only for fluorescence image acquisi tions 1 Use the Acquire gt Set input command 2 Select the camera s calibrated default input channel 3 In the Set input dialog box click the Duplicate Channel button Click the Configure input button 4 Make the following settings in the Configure Input dialog box Tab Recommended settings on the tab Delete all macros Input Minimum or middle exposure not available for all camera types no sharpen filter not available for all camera types Display Clear the Automatic Gain Display check box Clear all of the other check boxes on the tab In the Image scaling list select the Adjust to viewport option Format Select the Multi Channel check box Only with the motorized microscopes of the BX51 61 or IX71 81 type do you set the remote connection on the Magnification tab 5 Exit the dialog box with OK 6 Close the Set Input dialog box
374. n base your new page on and confirm via OK Try to use a template page as the basis for a new page layout which already contains all of the important standard elements of a report page gt The report window will now display the template page selected 3 Click the Image button in the Report Objects button bar to define a new image pa object within the template page 4 Now you define a rectangular segment for the image object Define its exact size and position via mouse or in the Position and Size tab 5 Drag a representative image from the image manager or from an image data base and drop it on one of the image objects to better be able to assess how the images will look in later reports 6 Select the image object and click the Properties button in the Report Objects button bar to determine the properties of the image object 7 Check the settings in the Select and Move tab e Clear the Background object check box to define the image object as an image placeholder Objects that you define as background objects cannot be edited later on in the report e Select the Fixed Position check box to establish a fixed size and position of an image object for all reports These cannot be altered later on in a re port 8 Click the Clear Object button in the Report Objects button bar to remove the test image from the report object gt The lettering of the scale of length and the values of the automatic zoom will be replaced by number
375. n created is rather limited You can alter the headers of the individual columns and hide values with the auto filter You will find all commands which have to do with sheets in a sepa rate context menu You can open the menu by clicking on a sheet with the right mouse button It is not possible to delete values or edit single values In this case you delete the measurement values either in the image or in the measurement display and create a new measurement sheet This is how you measure numerous images Distances are to be measured on numerous images and then later exported together to a sheet 1 Load the images to be measured 2 Use the Alt 4 key stroke to switch to the measurements environment 3 Maximize the size of the image window so that you have the largest amount of space possible for displaying the image 4 Click on the List or Gallery tabs located in the image manager to show the image buffer with the loaded images 5 Load the image to be measured from the image manager 6 Measure the desired segments 7 Repeat the last two steps for all the images on which you would like to make measurements 8 Click on the Create Measurement Sheet button located in the measurement button bar gt The Create Measurement Sheet dialog box opens This is how you delete single measurement values 283 285 The Create Measure ment Sheet dialog box lists all the images on which measurements have been made The active im
376. n of the image in the active image buffer if the image buffer is not write protected Scale Bar Properties Configures scale bar style and visibility The Scale Bar Properties dialog box offers three tabs Scale Bar Properties Display Sets the method of display for the scale bar Scale Bar Properties Display Format Size Scale bar selection I Horizontal scale bar P Vettical scale bar Palette bar false color palettes only Show scale Bar for D Viewport W Clipboard Printer Cancel Apply Help Determine which scale bar Horizontal Vertical Palette you wish to have displayed in the Scale bar selection group The selection you make here will apply to all appli cation areas of the scale bar Select the Horizontal scale bar check box to have a scale bar displayed in the view port parallel to the X axis Select the Vertical scale bar check box to have a scale bar displayed parallel to the Y axis in addition to the above horizontal scale bar There will be no lettering on the vertical scale bar Its length corresponds to the length of the horizontal scale bar As soon as a viewport becomes smaller than a certain size the vertical scale bar will disappear This applies to printing situations as well i e if several images are being printed out the space for an individual image may be insufficient for the vertical scale bar to be printed out as well 166 The Image menu
377. n options group mFIP asks you to carry out the various settings step by step and to confirm by clicking or starts a completely automatic acquisition Click on the File button to open the File n Output dialog box You can save the settings of the Acquire Fluorescence Images dialog box or load previously saved settings In this way you can define separate image acquisition sequences for different sorts of samples In the Camera settings group you define the acquisition parameters for the individual fluorescences The actual options which are available depend on the type of camera in use Thus you can define the exposure time and binning for CCD cameras but only define the integration for the Sony DXC 950 video camera Note The settings in this group refer only to the fluorescence which has just been marked You enter the required exposure time for the fluorescence acquisition into the Expo sure time fields You can set the times separately for the preview and the fluores cence acquisition The shortest exposure time is 1 ms the longest is 160 s 160 000 ms By using the Binning selection fields you can activate your camera s binning func tion You can switch on the binning separately for preview and single image However all images which are to be overlaid should be acquired with the same binning setting 123 Acquiring images Define Fluorescence Acquisition When binning the camera integrates the content of neighboring im
378. n the Next gt button 6 Select the database you want to backup To do this enter the path name of the database in the field or click the button to select the directory Click on the Next gt button 7 Select the destination drive for the backup Your CD DVD drive is explicitly shown in the list gt e Please note Select the burner from the list If you click the button and select the burner via Select Directory you will get an error message You can directly access OD DVD burners directly from your image analysis software In Please choose the destination drive For the storage this case the burner is going to be shown as an E E PLEXTOR CD R PX 320A a individual entry in the list of destination drives Drive type CD DVD In order to store your data properly you need to specify the destination volume size For CD ROM and removable drives Volume size 703 is MB Auto detect Burning options Burning speed 20 x g lt Back Cancel Help 8 Click the Auto detect button to automatically detect the size of the destination drive gt The available burning speeds will be selected together with the size of the destination drive 9 For security reasons do not select the maximum burning speed but stay one level below it This reduces the possibility of errors 10 Click the Next gt button 11 Select the scope the backup is to have Select the incremental backup option for example D
379. n the dimmest and the brightest pixels Rightclick on the diagram to open a context menu with which you can alter the way the histogram is to be displayed Use the Show Grid command to hide or display the grid lines Use the Y Axis Logarithmic to switch from a linear to a logarithmic display Use the logarithmic display when for example your images contain bright structures on a dark underground In this case a great many pixels will be dark so that the histogram will have a very high peak with small values Possible structures in the histogram s bright range will be lost A logarithmic display will make them visible A linear mapping function is preset The parameters Min and Max situated beneath the diagram and Intensity and Contrast are interconnected When you change one of these parameters the two in the other group are adjusted accordingly In the diagram area two vertical lines are displayed These lines show the borders of the section to be displayed There are several ways in which you can alter the intensity range that has been set You can use your mouse to move the line directly in the diagram enter the values you want in the Min and Max fields or use the slide controls 153 The Image menu Adjust Display Underflow Define the part of the image s histogram to be used for display in the Clip group Enter the percentage of channels to be clipped from the histogram in the Min and Max fields These percentages of the l
380. n the tree structure e g the database folder TEM Acquisition in the example database gt The preview window displays the schematic database folder gt The gallery displays all records which are located one level below the data base folder gt The form contains information about the selected database folder 6 Mark different images in the tree structure gt The preview window shows the thumbnail of the selected image gt The gallery shows all the records which are behind the image gt The form contains information about the selected image 87 How to sort your records 5 Archiving Images Click the Sort Records button to display the ExampleD atabase_apl Mma A order data using a different gt ea C4 ulel N J Filtering Fluorescence Imac Eg Grain size analysis EA Micro hardness 3 Multiple Image Alig _ Paint on aluminum lt Fa Particle analusis The current sort criterium is the record name ort Records Sort by Mo fo Ei bae COt S SSY C Ascending pth AELE Date bi D di EY Ph g i eS r Bits per Pixel Se ee Se eA Calibration Unit Channel Ascending Clear All Document Creation D ate f Descending Document Creation Time Help Document Files Size Document Insertion Date Ascending Document Insertion Time Document Modification Date Then by Descending l Ascending not specified l Descend
381. n this empty page template After saving the report template your image analysis program will delete this page template You can however make good use of this page for editing record objects because there are no other report objects that could interfere with your editing When you are creating an object template or a page layout begin by inserting the elements that are to appear in the background e g a frame Every object you subse quently insert will thus automatically be in the foreground Altering page Any and all alterations you make to a page layout have to be re conducted on each templates individual page of a report template Make use of the Select Page Template button in the Report Template button bar to edit individual template pages Determining the first Open the report template and click the Select Page Template button Then select one page of a report of the page templates listed for use as a cover page or first page of your report Now save your report and close the report window once the cover page has been acti 56 Report Generator Report templates vated When you then create a new report based on this report template the first page of the report to be opened will be this one Inserting new page There are two ways to insert new page templates within a report template templates e Click the New Page Template button to insert a blank page template into the re port template A blank page template will be automatical
382. n value of all intensities is used for display 21 Button Bars The Stack Navigator button bar Minimum Intensity Projection For each pixel in the XY plane the intensities of the different Z layers are compared The minimum intensity is used for display Navigate Time In time lapse experiments the images are subsequently acquired according to the parameters defined in the time lapse properties page and stored as an image stack If the active image buffer contains an image stack containing a time sequence the Navigate Time button is available in the Stack Navigator button bar With the Previous and Next buttons in the stack navigator you can navigate frame by frame backwards or forwards respectively through the time sequence The First and Last buttons can be used to display the first and the last image respectively of the acquired time series The number within the field represents the number of the currently displayed frame You can go directly to a specific frame by typing the respective number in the Go to field and pressing the Enter key The menu offers the following projection functions Single time frame One selected time frame is shown Use the navigating buttons in the Stack Navigator button bar to select the image you want from the time lapse image Maximum Intensity Projection For each pixel in the XY plane the intensities of the different time frames are compared The maximum intensity is used for display
383. nalysis program automatically creates statistics for each interactive measurement which are shown in the Statistics group located in the measurement display You can have these statistical values hidden or displayed To do this click the Show Hide Statistics button below in the measurement display The name for the Statistics for group is dynamic and it shows the measurement value to which the statistics are displayed Should numerous values e g area perimeter and mean radius be measured in a measurement method click on one of the measurement values in the measurement display to show the statistics data of the selected measurement value instead The Statistics on all images check box is marked and is a default setting For this reason the measurement values of the measured images contribute to the statistics Clear the check box to view only the statistics of one image Please note that this check box only deals with export to the measurement display and not with the export to a sheet 286 Interactive Image Measurement Using Statistics Functions A total of 60 segments were measured on numerous images The mean segment length is provided The spread of the measurement results is provided by the standard deviation Select parameter Create sheet otep by step Statistics for Length Count BU H Mean 380 85 mm Minimum o5 mm Masimum TAF mm Standard Deviation 156 01 mm W Statistics on all images Your
384. nd changes the gray values of image which are beneath the image s active lookup table The LUT has no effect on how gray values are adjusted The LUT itself remains totally unchanged The coloring of the resulting image may differ from the original image This is because the active LUT may assign different colors to the adjusted gray values which means that the coloring of the image onscreen will look different You can adjust the color display by modifying the active LUT Select the Image gt Image Display gt Edit LUT command to do so Furthermore you can delete the active LUT and have the actual gray value image displayed using the Image gt Image Display gt Set Gray LUT command True color images The histogram for 24 bit true color images is calculated based on the intensity values R G B 3 of all the pixels The lower and upper 3 of the histogram area will be removed The new upper and lower intensity values will be transferred to the histo grams of the three color components as the new minimum and maximum values The remaining histograms of the three color components will be respectively mapped linearly to cover the maximum value range of 0 255 Doing this results in more vivid and cleaner colors If an image however has very little white in it some color values may become over saturated and some image structures may disappear Modify Gray Values Modifies a lookup table and maps the active image correspondingly This
385. nd color images To begin with load all of the individual images that are to be arranged into consec utive image buffers Before calling the Arrange command activate the image buffer containing the first image of the series For an image series acquired using the Multiple Image Alignment gt Acquire command the same parameters will automat ically be set in the Arrange Images dialog box as for image acquisition Otherwise this dialog box will be opened with the same parameters used during the last image montage completed After calling the command the Arrange Multiple Images dialog box is opened Here you determine the position of the individual images Arrange Multiple Images Ea Correlation Quality f 5 T No Correlation Arrangement Meander Horizontal f Comb i Vertical Image Tiles Horizontal 2 K Vertical WE Cancel Help In the Image Tiles group you specify the number of images you want to acquire for the overview image In the Horizontal field you enter the number of images that are to be arranged hori zontally The maximum number of image columns is 10 In the Vertical field you enter the number of images that are to be arranged vertically The maximum number of image rows is 10 In the Arrangement group you define the way of arranging the images The first image is assumed to be in the active image buffer The available arrangement methods are illustrated below 144 A
386. nds of the RGB Studio gt Shape menu are available now Magic Wand Options The Magic Wand is a method used to define objects It offers you the possibility to locally set threshold values on the image To do so select the starting point on the image with the typical pixel values of the object being searched for All pixels in the area of the starting point whose pixel values are within the threshold value belong to the object The value in the Tolerance field is added to the pixel value of the starting point and then subtracted by it The tolerance value thus determines the threshold value You can directly define the tolerance on the image with the mouse Keep the left mouse key depressed and pull the mouse to the left to reduce the tolerance value or to reduce the size of the object Keep the left mouse key depressed and pull the mouse to the right to increase the tolerance value or to increase the size of the object Due to the fact that the tolerance value is normally the same for similar objects you only have to set an image s tolerance level once You can smooth an image before you utilize the magic wand In doing so image errors in the definition will be suppressed and the object appears rounder for example The rang filter serves as a smoothing filter Thus noise and individual light or dark pixels shot noise are filtered out 50 is a fixed setting for the rank para meter Enter the size of the rank filter s neighborhood in the Smoot
387. ne Processing ES Image Input Processing Measurement Available functions Current functions File Save Sheets Gray Thresholds Phase Analysis Color Thresholds Phase Analysis Grain Size Intercept Test Edit Dn ULE 2 Seimei e OF Cancel All processing steps contained in the Current functions list are applied to each processed image The order of the processing steps in the list corresponds to the order for the application This is an example for a function available Use this command to measure a percentage of the area of a gray value phase This command is only available for 8 bit grayscale images The phase analysis clas sifies different phases of the material as belonging to clear gray value regions With the phase analysis you can analyze up to six phases simultaneously With the Edit button you open the Phase Color Coding dialog box Here you define the gray value ranges of the individual phases The result of a phase analysis is a measuring table which lists the percentage of the area of all phases For each evaluated image the table contains the area percentage of the individual phases The total of all area percentages may be less than 1 if not all gray values are assigned to a phase Execute Processing Use this command to start the current automatic process All parameters for the automatic process are copied from the current settings in the Define Processing dialog box 254
388. nes thousandths O does not alter the im increase it age Remove replaces the color value of each pixel with the The size of the neighborhood lies be Noise median color of pixels in its neighborhood Rank tween 1 and 128 pixels 1 does not al filter This enables you to diminish noise the ter the image enter 8 e g for an 8x8 larger the grouping of neighboring pixels you pixel grouping use the more effectively you will be able to re duce the noise Average replaces the color value of each pixel with the The size of the neighborhood lies be average color of pixels in its neighborhood You tween 1 and 128 pixels 1 does not al can cause the image to become out of focus ter the image enter 8 e g for an 8x8 the larger the grouping of neighboring pixels pixel grouping the less it will be in focus Solarize imitates the effect resulting from photographic Thresholds can be set anywhere be film being accidentally and briefly exposed to tween 0 and 256 256 does not alter light This effect is achieved by inverting the the image color values of all pixels whose intensity values are greater than or equal to the set threshold The lower the threshold the greater the solar ization effect Oilify gives the image the appearance of an oil paint The size of the neighborhood lies be ing the larger the grouping of neighboring pix tween 1 and 128 pixels 1 does not al els the more convincing the effect ter the image enter 8 e g f
389. ng You can work with the sheet more easily when it is grouped A sheet that has been grouped together can be e g positioned as a single object Sheets in record Use sheets in record objects if you want to insert additional information from an objects image database together with a sheet A sheet which has been defined in a record object has an additional tab located in the sheet properties The Document Link tab enables you to exactly define the sheet cells which are to be inserted into the report Unlike the sheet objects which are directly inserted into the report the acquired sheet cells are independent of the window size of the sheet Use this possibility if your measurement results are always written to a sheet in the same form in order to determine the sheet cells which are relevant for you Sheet cells as field Many special Add Ins deliver standardized measurement sheets which are always objects constructed the same way Use a special field function to acquire individual measure ment results i e the G value of a grain size analysis into the record object The Sheet Cell field is automatically included in the list of database fields This field has additional object properties You can exactly specify the desired sheet cell in the Record Fields tab 50 Report Generator Report objects A sheet is a group of in dividual text objects Therefore you can change the appearance of a sheet from sheet cell to sheet cell otep
390. ng to your specifications placing them in successive image buffers then using them to calculate the EFI image You can only use this method when your microscope has an automated motorized stage steering and the appropriate software has been installed The way EFI works How is the EFI image Standard and fully automated methods To be able to create an EFI image from a calculated focus series EFI goes from pixel to pixel more precisely pixel neighborhood to pixel neighborhood through the whole series of single frames For every pixel EFI deter mines which image is the best focused at this point This image is assigned a certain numerical value that is entered in the corresponding pixel position in a gray value image When this algorithm has been completed a gray value image has been created in which EFI can read out from which image which image segment in the sharply focused EFI composite image has been adopted Accumulative method Here there are precisely two images which are compared with respect to their focus the live image and the preliminary EFI image The preliminary image that is then newly calculated overwrites the previous one just as the next live image overwrites the one that went before it Pre Alignment Both with the standard method and with the fully automated method an integrated Pre Alignment matches the single frames before the actual calculation of the EFI image is made if that is what you want In this way any s
391. not select them on a report page Text objects and all graphic objects will be automatically inserted as a background object in a template Image objects sheet objects diagram objects and graph objects by default are not background objects Definition Select one or more objects on a template page Open the dialog box on object prop erties Objects you have selected can be changed to background objects in the Select and Move tab Background image If you do use an image object as a background object then you should always insert objects the image as a link Otherwise the image will be resaved with every newly created report Altering background Use the Edit Page Template button to alter page templates within a report Any objects within a changes made to background objects will immediately affect all currently existing report report pages Any changes you make to objects that are not background objects will only affect newly created report pages Page templates The separate pages of a report template are called page templates They re used to define page layouts that you can use later on in a report Each page layout is saved under a separate name in the report template When you subsequently create a report based on that report template you can select a page template from the list for each report page The empty page template is a standard component of every report template Please note that it is not possible to define objects o
392. nput Magnification Organizes the available magnifications Configure Input x Info At Calibration Magnification Macro Input Display Format Unit Shift Magnification mari Set Unit ser Shift W Automatic 100 O0000 Device None Remote Gn Resulting calibration Pixel 0 07678 mm Pinel ey ratio 0 01678 mm 1 00000 OF Cancel Help The currently set magnification can be read out via remote for some microscope types and thus used for automatically calibrating the acquired image Select the Magnification tab to define the necessary settings The new magnification is valid for all images taken with this input channel e Be sure to change the magnification on the Magnification tab if you change the magnification of your microscope If your program version allows macros you can program the system to automatically open this dialog box when you use the Acquire or Snapshot command e For correct calibration you have to define the X Y calibration If these definitions are not correct the magnification will be meaningless because the image will be uncalibrated Select the current value in the Magnification field If your program version came with remote support the Device list contains the model of your microscope If any other device than None is selected the Set COM port button appears Click the Set COM port button to open the Select Remote COM
393. nscreen for reference purposes The edit object mode is available within a report for editing individual record objects Field entries are not filled out in the edit mode when you drag an image from a data base into the image object This ensures that the labeling of the fields remains general when you insert a new record object Creating new object A rule of thumb for all templates you generate within report generator you can only templates generate a new template by editing an existing one To generate a new object template for a record object you first insert a record object edit it and save the template under a different name Object templates are listed alphabetically Use this to your advantage and pick a name for object templates you frequently use that starts with a letter near the begin ning of the alphabet Deleting object You now edit an object template and then terminate the edit mode immediately templates Select the object template from the object template list in the Finish Object Editing dialog box and click the Delete Object Template button Once you have deleted all the templates on the list your image analysis program will offer you a standard template as soon as you have inserted a new record object 59 Report Generator Object templates Standard object If areport template does not contain any defined record objects your image analysis templates program will add a standard record when you click the Record button
394. ntally and simultaneously observe within the preview window how different parameters either strengthen or lessen the filtering effect For example for the Gamma filter you can enter the g constant within a value range of 0 1 1 00 0 in the Value field Two fields will appear for the Emboss and Noisify filters respectively AS soon as you adjust a parameter you will be able to see its effects in the preview window The Histo Equalize Stretch Intensity and Invert filters are predefined For this reason no parameters will be displayed for them Click the Execute button to apply the filter selected with the current parameters to the image The resulting filtered image will be shown in the preview window Click the Back button to reverse the most recent filter operation if you re not satisfied with the effects on the image resulting from the last filter operation The image shown in the preview window will revert to the appearance of the image before the most recent filter was applied Click the Reinitialize button to have all filter operations reversed e g if the entire filter sequence has not produced the desired effect The original image will be displayed in the preview window once again Now you can recommence filtering and try out another filter sequence Click OK to have the image currently being shown in the preview window written into the destination image buffer The dialog box will be closed The preview window will also be
395. nvenient method for the direct comparison of two graphs whose Y ranges are very different The highest Y value of the overlay is adjusted to the maximum Y value of the graph in the displayed X range You should keep in mind that after clicking this button the labels of the Y axis refer only to the actual graph and no longer refer to the overlay Printing graphs Use the File gt Print command to print currently loaded graphs 1 Activate the image window 2 Use the File gt Print command gt The Print Image dialog box provides you with several general page layouts 3 To print images and graphs simultaneously choose the Multiple Images Graphs entry from the Page layout list 4 To assign images and graphs to the relevant frames click the Layout button gt The Define Page Layout dialog box is opened 5 Activate the Multiple Images tab gt The Preview group displays the predefined page layout gt There is an additional list at the right side of the dialog box The Documents list displays all available image and graph buffers 6 Click on the plus sign to display the image buffers in the Documents list gt When an image buffer contains an image the image name will be given 7 Use the left mouse button to drag the desired image onto one of the frames gt The image buffer number is now connected with the selected frame i e when printing the selected image will be printed at the selected position 8 Repeat the last two ste
396. o matically adjusted whenever you change an objective That is to say the stage will be raised or lowered by a fixed Z value and moved by a fixed XY value In this way the image will always remain sharply focused and in the same position You will have to carry out a new calibration whenever you exchange objectives or insert a new objective in your turret 107 Acquiring images Configure Input Image Intensity Configure Input Image Intensity Calibrates the gray values of the input channel Configure Input x Macra Input Display Format Info ety Calibration Magnification Image Intensity Snapshot A G O0ffset p None loglOf G 0ffsetj A Offset O alls File Guanti Gray value Intensity fae Val Di J jas S BE Interactive ps S Interactive Spat nh Image butter i irer OF Cancel Help This is where you can assign a more realistic scale or description to the gray values of an image e g 0 255 gray values of an 8 bit image This might correspond e g to the actual height information of the texture of the image if the maximum and minimum values are known The calibration of the gray values is given by a function f G The gray value G is assigned a new intensity value defined by the function f linear exponential etc You define the functional relationship described above via 6 different functions and two or three parameters respe
397. o remove all of the split positions from the graph Protect Graph Toggles the read only mode for the active graph on off A protected graph is save from unintentional modification and deletion Delete Graph Calibration Deletes the graph in the active graph buffer Use the Delete Graph command to delete the currently active graph document Alter natively an active graph document can be deleted by pressing the Del key X Calibration Stretch Hinweis Modifies the calibration of the x axis The command creates a new graph in the next free graph buffer Before you use the command Change the display of the graph so that the inter esting part of the graph is completely displayed in the graph document Calibration Stretch Reference New values Lower E B Set Lower a B5 Upper 255 B Sel Lpper 255 is 1 0 Channel 1 0 Select All Select Display Cancel Help 317 Graph X Calibration Offset The calibration of the x axis will be changed by a linear transformation The original lower and upper x values will be assigned to the corresponding x values in the New values group In the Reference group the current x values of the active graph are shown Click the Set Lower button to define the lower value by a line cursor in the graph Use this option when there are reference points in the graph Click the Select All button to ascertain the minimum and maximum x values Click the Select Disp
398. of the color channel will be displayed in the dialog box on the top right Click the white button on the left side of the dialog box to have all of the color chan nels displayed simultaneously Should the white button be clicked you can click on one of the narrow gray buttons below a color to change the way the corresponding channel is to be displayed while all of the other color channels will also be displayed The diagram in the dialog box shows the intensity diagram of the selected color channel and the current mapping function for this channel Click on the mapping function to have a small circle drawn on the curve Move this circle to edit the mapping function As soon as you release the circle the image display will be updated correspondingly to the changed display function Click the Close button to close the dialog box and change the way the image is displayed accordingly The display function defined here will be adopted for all of the single frames in a time lapse image or a Z stack Frames that are either much brighter or much darker than the image for which you have defined the display func tion may possibly then be displayed overexposed or underexposed _ Note After the dialog box has been closed the display function will remain active To save the display function simply save the changed image Click the Reset button to load the display function that was active when you opened the Adjust Display dialog box
399. og box will be opened gt Your image analysis program will propose the report directory called C Reports ProjectXYZ in the Save in list 5 Enter a content relevant name for your report into the File name field 6 Select the report container src from the Save as type list to save the report in a single file 7 Click Save to save the report Saving Exporting report Background Information Rich Text Format The RTF format enables you to transfer formatted text documents between various programs that can be run on various platforms You can save reports in an RTF format and then e g load and edit them in MS Word e RTF reports are optimized for MS Word MS Word versions 97 and later i e the report s layout remains unchanged when loaded into MS Word e RTF reports cannot be reimported into your image analysis program e Images are always inserted into an RTF file as copies and not as links This is always the case no matter how the images were inserted into the original report e In MS Word RTF files can only be displayed and edited in the Layout or Online Layout view In the Normal or Outline view only continuous text will be dis played in Word In terms of Word a report contains no continuous text otep by step Exporting reports You want to send a report e g by e mail to fellow colleagues that have no access to your image analysis program or to the image files involved In order to do this you need a
400. olor images 204 The Process menu Intensity Note Note Gray value binary and false color images Set Gamma LUT Gamma r OF Cancel i Map image Change LUT File HOOO Enter the gamma value into the Gamma field Values from 0 01 to 10 are available Select the Map image option to generate a new image with different gray values The new gray values will be calculated using the gamma correction The resulting image will be written into the destination image buffer The image type of the resulting image will be the same as of the original image This option is for correcting camera non linearities Select the Map image option to generate a new image with different gray values The new gray values will be calculated using the gamma correction The resulting image will be written into the destination image buffer The image type of the resulting image will be the same as of the original image This option is for correcting camera non linearities Select the Change LUT option to generate a new image using a different LUT main taining the same gray values however The new LUT will be calculated using the gamma correction The 3 color components are the same meaning that your resulting image will be displayed on the monitor using gray pixels only no color pixels The image s original gray values behind the LUT remain the same This means that the original image behind the LUT is not l
401. omatic check box to enable your image analysis program to takes over complete control of the acquisition In the case of a manual microscope your program asks you to carry out each adjustment whilst in the case of an automatic microscope all the working steps are carried out automatically In the Z Correction field you define the required Z correction for the marked fluores cence For the acquisition of this fluorescence the stage corrects the Z position accordingly The Z Position field contains the current Z value for the stage Click the Focus button to adopt the current Z position as focus plane Click the Read button to calculate the offset for the current fluorescence 124 Acquiring images Acquire Z stack Note Click the Reset button to delete the set correction values once again The Reset command causes the correction values of all fluorescences to be deleted Acquire Fluorescence Image Acquires a fluorescence image The acquisition parameter is that of the current setting in the Define Fluorescence Acquisition dialog box Before you begin with the acquisition select a free image buffer You can also configure your software in such a way that your image analysis program will automat ically select the next image buffer for every acquisition you make To do this select the Special gt Preferences command In the Images tab select the Image buffers All entry in the Image acquisition gt Sequence list When you
402. ome black if you set all three parameters to 255 Move the slider to the right The pixel values will be shifted to the right along the corresponding axis of the RGB color space The proportion of that color will increase 255 means that this color component is at 255 for all pixels i e the color is satu rated The image will become white if you set all three parameters to 255 Define Curve Adjustment Adjusts image contrast on true color images You do this by modifying the color image histograms Use the Execute Curve Adjustment command to apply the current settings in the Define Curve Adjustment dialog box to the image in the source image buffer What are true color image histograms Each color component of the RGB color space can have up to 256 values i e each color component has 256 possible values The distribution of the values of a color component can be shown in a histogram the frequency of the pixels versus the channel number is plotted The HSI color space s intensity parameter is the mean value of the three color components R G and B Intensity distribution is shown via the frequency of the pixels plotted versus the intensity values Adjust Curves x XY Diagram The XY diagram of the dialog box contains both the histogram of the channel selected as well as the current mapping function When you open the dialog box for the first time the linear mapping function is preset meaning that the image will not be
403. on if you switch from either the Polygon or Formula method to the Sheet tab the LUTs you had defined there will be inserted into the sheet in the Sheet tab This exception enables you to e g have a whole LUT generated using a formula and then edit individual values in the sheet Please note this switch to the Sheet tab deletes the sheet values of the previous LUT You really should save any LUT you ve defined via the Sheet tab using the File button before you switch over to another tab A LUT can be saved as a separate file independent of its image You can then reload this file at a later time and use the LUT on the same image or transfer it to another image To do this click on the File button which all three tabs have 160 The Image menu Image Display Edit LUT Sheet Defines LUTs via sheet Edit LUT x Sheet Polygon Formula Enter values for lookup table 7 E E Linear Blue Interval E Ad l piid Sheet Oo OO Fe to Poa Oo o 07 0 Fe fo Pao co OF Cancel Help You can edit sheet values manually Inputting can be terminated by the Tab key moving you on to the next horizontal field or by the Enter key which will move you to the next row Click directly on the row numbers if you wish to select one or more rows You can copy the contents of the rows using Ctrl Insert and Ctrli Paste Use the clipboard to copy these contents into o
404. on 106 XY Calibration 104 Connectivity 232 Context menu 336 Contrast 198 199 208 Control box 329 Convert 327 Convert Image 185 Copy Image 192 Labels 315 Objects Overlay 176 Pixels 273 Shape 272 Correct Aspect 257 Count Colors 211 Create Mask 274 Create measurement sheets 284 Create one image from thumbnails 333 Crop Image 257 Pixels 273 Shape 273 Curve Adjustment 210 Cut Objects Overlay 176 Database 63 Database fields 66 Arrange 82 Data types 69 Default value 67 Define 66 Picklist 67 Database folder 64 Create 73 Database password 100 Database window 85 DCE 238 247 Define Classification 302 Curve Adjustment 210 Filter 224 228 Connectivity 232 Fluorescence Acquisition 116 120 Fluorescences 115 119 Fluorochromes 115 117 Graph History Graph history 326 HSI Adjustment 209 Image History 194 Image Series 253 Processing 251 RGB Adjustment 209 Index ROls 181 Statistics 304 Define Fluorescence Acquisition 117 Define grid 332 Defining fluorescence 115 Defining the fluorescence acquisition 116 Defining the fluorochrome 115 Definition of stage limits 333 Delete Delete Measurement from Image 283 Graph 317 Image 192 Labels 315 Measurement 283 Overlay 181 Shape 272 Depth of focus Illustrations 129 Derivative 321 derivative filters 225 Dialog box Create Overview 332 Differentiate X Differentiate Y 242 display graphs 308 Display Shape 274 Dithering 263 Divide graphs 325 Dongle 1 Draw
405. on table 105 Markers 313 Matrix Filter 224 Index Maximize Contrast 198 Mean Filter 244 Meander 141 Measure distances 319 Measure menu 292 Measurement button bar 280 Measurement Display 282 Defining statistics 287 Delete Measurement 283 Link to image 284 Selecting measurement parameters 289 Statistic display 286 Median Filter 244 Menu bar 2 Merge Colors 268 Methods EFI accumulative 129 fully automated 130 mFIP 114 122 Acquiring a multi channel fluorescence image 114 Mirror image 256 Modi of the Stage Navigator 336 Modify Gray Values 200 Labels 314 Split Gain 316 Monochrome image 6 Mosaic Filter 261 Multi Channel 110 Multi channel image 6 Multi channel Z stack image 6 Multi channel Z stack in time lapse image 7 Multi dimensional images 187 Multiple Image Alignment MIA 139 Multiply graphs 324 Next view 86 Noise 323 Noisify Filter 259 NxN Filter 230 O Offline Modi 336 Oilify Filter 261 Online Mode 337 Onscreen layout typical example 328 Open database 69 Open database exclusively 69 Index Optimization of the images 339 Optimize Contrast 199 Organizational fields 66 Organizational ID 63 Overflow 165 Overlap Area 146 Overlapping area 339 344 Overlay 171 319 Burning 173 Overlay transmission 20 Overview Image Acquire 331 Acquire parts with a higher magnification 340 As one image 331 Define the size 332 Deleting individual images 336 From separate images 331 Incorrectly compil
406. onality of the macros entered into these tabs Click the Delete button to delete any text contained within either the Preprocessing steps or Postprocessing steps fields Configure Input Input Sets various options for the channel Elements options and layout of the Input tab depends on the actual input device Please use the Help button on the Input tab to access the help page for your input device 109 Acquiring images Configure Input Display Configure Input Display Set various options for display during acquisition The functions offered options and layout of the Display tab depend on the input device that is being employed Please use the Help button on the Display tab to access the help page for your input device Configure Input Format Determines the XY resolution of the image and clips your image around the borders at acquisition Configure Input Info se Calibration Magnification Macro Input Display Format Image format Active area Horieontali Vertical Clipping border pixel ie lo I ace lo FI F Multi Channel Top jo B Bottom po B Interactive No Clipping OF Cancel Help The Image format list contains a series of predefined standard image formats The first time you use this tab the entry shown will be the resolution of your logical input This value is the standard value images are not clipped using this value This list con
407. only changes the way the image is displayed not the contents of the image Its optimal appearance will be calculated for every color channel on the basis of the intensity histogram For the calculation of this histogram all of the images that belong to a color channel will be taken into account that is to say the complete time lapse image or the complete Z stack The intensity range displayed will then have been accordingly changed to enable all of the intensity values that occur in the color channel to be shown This will ensure that no single frames will be overexposed However the calculation needed for a multi dimensional image that contains a large number of frames can take some time The current values for the overflow will be taken from the Image gt Image Display gt Adjust Display dialog box The other setting such as brightness and contrast will not be taken into account Using the Auto Adjust Display command will produce results that are different to those you achieve by clicking the Auto button in the Image gt Image Display gt Adjust Display dialog box There the intensity range for the automatic display function will be calculated from values in the active image When you close this dialog box all of a color channel s images will be displayed with these limits When you save a multi dimensional image in the TIF format the current appearance of the image will be saved with it Note that the Auto Adjust Display command w
408. option when you wish to separate particles distinguish able from the background due to their gray value The image can be pre processed using a mean filter before separation A sigma value of 0 means the mean filter is a binominal filter the mean filter is a sigma filter for sigma values greater than 0 Use the Smooth scroll bar to define the extent of smoothing for the binominal filter Set a value of 0 to use an untouched original image as the basis for separation The greater the value you set the more the image will be averaged The influence of any local brightness fluctuation will be greatly reduced by smoothing thus increasing the probability of finding true separation lines You may also use the sigma filter as a mean filter In the Sigma field you enter the half width value s of the Gaussian distribution used The greater the half width value the greater the averaging effect i e fewer separation lines will be found A sigma value of 0 means that the sigma filter is turned off i e is not applied If the sigma filter is in effect i e the sigma value is 1 or greater you can change the diameter of the pixel neighborhood in which averaging takes place via the Smooth value The greater the smoothing value the greater the area in which the sigma filter averages A smoothing value of 0 means that no averaging takes place at all The Sigma field will not be available Adjust the Fine Coarse scroll bar such t
409. or Thresholds Sets color thresholds using the RGB or HSI color space Thresholds cannot be set for a single image or image buffer They apply to all images you currently have loaded To convert a color image into a binary one you must define a range for each of the channels of the image either Red Green and Blue or Hue Saturation and Inten sity During binarization a pixel will be assigned the gray value 255 if all of its components fall into the valid range The Set Color Threshold dialog box offers two tabs 215 The Process menu Set Color Thresholds RGB Sets thresholds based on the RGB model RGB nsi Preview Red 0 127 i g i None o New Greer 0 B 12 Ooo Delete Blue off BAES e All C Background File Pa ere Include piel Bedo New Include Exclude OF Cancel The Phase list contains all color ranges that have already been defined e Select a phase from this list that you wish to edit e You can alter the standard phase name as you like To do so simply click on that phase s field and then enter the name of your choice Note Be sure to give each phase a specific name If you do not then when you delete a phase the standard phase name will be automatically modified That is to say the system changes the name of the individual color phases Color ranges are defined separately for each color channel The Red Green and Blue fields
410. or an 8x8 pixel grouping Posterize makes the image look like a poster containing The number of color levels lies be few different colors This is done by reducing the tween 1 and 64 1 does not alter the image color values to a specified number of image color levels for each color component Mosaic generates a mosaic pattern appearance for the Tiles can be between 1 and 1000 pix image This is done by dividing up the image into tiles of a defined size Each pixel within a tile is assigned the mean color of all pixels of that tile The larger the tiles the more evident the mosaic effect 260 els in size 1 does not alter the image enter 8 e g for tiles to be 8x8 pixel in size The Process menu RGB Studio Emboss gives the image a three dimensional effect Ob The Depth parameter is between 0 jects that are dark low in intensity will appear and 1000 0 does not alter the image as ridges and lighter objects as hollows The The Depth parameter is between 0 higher you set the Depth parameter the greater and 360 At 0 the fictive light source the 3 D effect The image will appear as if itis is situated beyond the upper image being illuminated by a light source The Direc edge tion parameter indicates from which direction this virtual source of light comes Noisify adds random pixels to the image in such a way Depth parameter number of added that the image becomes noisy You can have pixels is between 0 and 1000 per nois
411. ore J Statistics on all images BF ASR ON GOO CoO be ws EH a JIG lr Me Felis ES LiteScience MITET TI CC 12 Fw Exporting The measured values are displayed in color in the image s overlay In addition all measure measurement functions create an entry in the measurement display These ment measurement results remain linked to the image even after the image has been results saved You can export the measurement results to a sheet any time To do so click the Create Measurement Sheet button located in the measurement button bar Measurement All the measurement functions can be found on the Measurements button bar There button bar is one button for each function Begin a measurement by clicking on the button with the desired measurement function You can measure as many values on the image as you like You end the measurement by either depressing the middle mouse button or the Esc key 280 Interactive Image Measurement Interactive Image Measurement 3 Points Angle 4 Points Angle Rectangle Rotated Rectangle 3 Points Circle m Circle with Center and Radius Ellipse Measurements x l Takes ALA Clo olola alela rE l Create Measurement Sheet Delete Measurement from Image Polyline Move Origin Arbitrary Line Magic Wand Horizontal Line Free Hand Polygon Vertical Line Fitted Polygon Touch Count Interpolated Polygon Point Closed Polygon
412. orks on the currently shown display only not on the whole image Uses a gray LUT for the single color channel display This does only change the display not the image itself Thus you can simply switch to another display mode Note that the display mode is part of the image information If you save the image its current display mode is saved as well LUT Name of the LUT Note Uses a color LUT for the single color channel display This does only change the display not the image itself Thus you can simply switch to another display mode Use the Image gt Adjust Color gt Load LUT command to select the LUT to be used for display Note that the display mode including the LUT to be used is part of the image infor mation If you save the image its current display mode is saved as well Fluorescence Color Uses wavelength LUT for the single color channel display 157 Note Load LUT The Image menu Load LUT This does only change the display not the image itself Thus you can simply switch to another display mode Note that the display mode is part of the image information If you save the image its current display mode is saved as well Use the Edit Fluorescence Color command to modify the color information to be used for the display Editing the fluorescence color dialog box Edits fluorescence color of the single color channel Changing the representation color e g change for a more obvi
413. osol However it is evident from the images in the above figure that there is a significant contribution of the YFP labeled structures to the GFP image Similarly you can see a relatively strong but undesired fluorescence of the nucleus upon YFP excitation This phenomenon known as bleed through strongly reduces color resolution and constrains scientific conclusions Spectral Imaging and linear unmixing Using the technique of Spectral Imaging and Linear Unmixing it is possible to sepa rate and resort the contribution of different fluorochromes to the total signal in each color channel The principles of the method can easily be explained considering the example shown above To unmix the spectral information of the fluorochromes with strongly overlap ping emission spectra it is necessary to determine the spectral properties of the indi vidual Sunder the same imaging conditions used for the multi labeled samples the system has to be calibrated for each fluorochrome This is performed by taking refer ence images of single labeled samples with these fluorochromes using the same filter set excitation filters dichroic mirror and emission filter as for the later images of double or triple labeled specimen This means in our example that GFP and YFP reference images have to be taken for both the eGFP and the eYFP samples 276 Spectral Unmixing Spectral Unmixing Spectral match between eGFP fluorescence and optical filters H
414. ost The resulting image will be written into the destination image buffer No matter what image type the original image was the resulting image s type will always be false color image This option is for correcting monitor non linearity The Sheet tab in the Edit LUT dialog box contains the LUT of the resulting image such that you can reconstruct the imaging properties of the gamma correction Binary and gray value images become false color images because the resulting images have a LUT that is different than the linear standard LUT Use the Image gt Image Display gt Set Gray LUT command to assign the linear standard LUT to a resulting image In this way you regenerate the original image False color images remain false color images they will however lose some coloring on the monitor as the 3 color components of the new LUT are now the same The Original image s LUT will be lost if you haven t saved it in the Edit LUT dialog box When working with gray value images and you apply either the Map image or the Change LUT options you ll be getting resulting images that seem to be the same onscreen In fact these resulting images are quite different they have differing gray values a different LUT and sometimes they even have different image types 205 The Process menu White Balance True color images Set Gamma LUT ES Cancel File Preview Window Help Set the gamma values for the 3 color components red
415. ot suitable for exact measurements of gray color values The Keep X Y ratio check box determines whether the X Y ratio of the source image is to be maintained or not If you select it a link will be established between the X and Y fields Click the Resize button to execute the transformation This button is only active if one of the zoom factors is not equal to 1 0 255 The Process menu Rotate Rotate Rotates image from source image buffer into destination image buffer Properties Dimensions Angle a5 is Degrees 1 359 Set Angle Direction i Left Right Options Interpolation IY Keep iY ratio OF Cancel Help Enter the Angle and select the Direction of rotation Left or Right The Interpolation check box determines whether the neighboring pixels of a parti cular image point are to be used for averaging or not Interpolating really only makes sense and is in fact only possible for gray value and true color images it is not available for binary or false color images The Keep X Y ratio check box determines whether the X Y ratio of the source image is to be maintained or not Click the Rotate button to execute the transformation If you wish to define the rotation angle interactively click the Set Angle button You can then define the angle by using the mouse to define the two lines comprising the angle Mirror Mirrors the image along different axes from source
416. ot the text itself You can alter the size of the text object by pulling at the selection markers If you alter the size of a text object you are thus altering the size of the text object not of the actual text Font type and size are not affected Multi line text automatically fits itself to the altered size of the text object Text flow When in the text entry mode you can enter as many lines of text as you like The only lines that remain visible are those that fit within the text object All the lines that do not fit within the text object remain hidden from view This non visible text remains however existent it is simply not shown onscreen You then need to enlarge the text object correspondingly This is why text cannot be any longer than a page Insertion of Click this button in the Report Objects button bar to insert a text object A new text text objects object automatically contains the word text Keeping the left mouse button depressed draw a rectangle by moving the mouse The area of the rectangle represents the space reserved for text within the report Release the left mouse button to have the text object inserted into the document Text proper Click the Properties button in the Report Objects button bar to format the text You ties can also select several text objects and alter all their properties at the same time e Text color is a font property You can set it in the Font tab e Background color is set in the Fill tab The
417. ote that there are many functions which are only accessible via a button bar e g the functions required for editing an image overlay Use the Special gt Edit Button Bars command to make button bars look the way you want them to and include what you need A viewport is a window in the image window where each of the loaded images or the live image is displayed You can divide the image window into numerous viewports thus displaying numerous images simultaneously You can set both the number of viewports to be displayed and their arrangement by using the Arrange Viewports button in the Image window s button bar This button will open a field made up of 4x4 icons each of which represents its own viewport Simply move your mouse pointer over the schematic viewports to select columns and rows The maximum number of images that can be shown at one time is 16 FQ Images Afel On the image in the viewport manager you will see a red rectangle The rectangle shows the segment of the image that is currently on display in the image window s active viewport That naturally only applies when the image in the viewport is being displayed at a larger size than the viewport itself is The rectangle is interactive It can be freely moved within the viewport manager to display different areas in the view port It can also be resized by mouse drag to change the zoom factor in the viewport display
418. ou can determine the print template for different document types To do so use the File gt Define Page Layout command The template contains the page layout of single images multiple images database images and other documents e g sheets and diagrams A page layout consists of header footer definition and the position and magnification of images The report generator gives you many more possibilities as well as very complex page layouts The report generator which is integrated into your image analysis program enables you to design a page independently You define your own 5 sondado en for printing out single itiole i J Header Footer Header Footer Header Footer MHMP 3 naga an ua Single Image Multiple Images Other Documents tabase images and for printing out text sheets Border Preview Fit image size to diagrams and graphs as i Page borders well C Width 7 height Ton z5 El Bottom 25 Left 2 0 Right 2 0 A Header fio Footer fio Center M Horizontal IM Vertical 20 1 4 28 8 em Print Fie C Magnification Original 766 00 576 00 Fiel W Fixed image ratio Print area width hei Height ha E Magnification 0 0209 B 11 First Steps Printing images The illustration de Border Left Border Right scribes terms which are lt lt lt used in the Define Page Layout dialog box Distance Header
419. ou close the dialog box Until you do that you can retrieve any deleted phases by simply clicking on the New button The diagram in this dialog box displays the histogram of each of the color channels The red curve for example represents the distribution of red color values in the image The whole image is used for histogram calculation no matter whether you ve set a global frame or not Use the Y scroll bar to spread a histogram on the Y axis This enables you to evaluate the progression of the curve even when the number of pixels at a particular point on the curve is quite low The color bars below the histogram represents each of the color channels Color bars display phases that have already been defined Each bar displays its corre sponding color range in the same color as in the Preview Activate any of these color channels for a particular phase by simply leftclicking on the color bar of that channel The Red Green and Blue fields display the current thresholds for the active phase Two lines demarcate the active color channel within the histogram The blue line represents the lower threshold the red line the upper threshold Thresholds can be adjusted within the histogram To do so simply move your mouse cursor over one of the two lines Once the mouse cursor changes shape into a double arrow simply pull the line keeping the left mouse button depressed to the position desired You have to activate one of
420. ou will see the number of images that are going to be acquired You can still reduce this number then only part of the stage area will be included 333 The Stage Navigator How to acquire an overview image Create an overview image corresponding to the Interest Area 1 If necessary switch to the Stage Area Mode since you can only acquire over view images in this mode see also The Modi of the Stage Navigator on page 336 2 Go to the area in the live image that you want to use as your starting position 3 Click the Scan Interest Area button gt The Stage Navigator window will be activated By default an area that is somewhat larger than the current camera position will be defined as an Interest Area 4 Now you can use the mouse to adjust the Interest Area to the size you want E Tischnavigator Unbenannt D oa ee wage e 2 acquisition grid camera frame 2Pasitior 5988 00 gt You can still correct the position of the Interest Area To do this click in the Interest Area with the left mouse button then keeping the button pressed move the mouse 5 Click the right mouse button to move into the Create Overview dialog box gt You will see the number of images that are going to be acquired gt Additionally at this point you can specify whether you want to acquire the overview image as one image or as an image made up of separate images see page 331 6 Should you want to change the size of the
421. oup is set by default Your image analysis program then calculates the most ap propriate magnification based on the calibration data for the size of the im age object 4 Change the magnification option and select the Use fixed magnification option gt The edit field suggests a magnification for each image with which the image can be completely displayed in the image object 5 Enter various constant magnifications into the field and confirm by clicking Apply gt The size of the image object is what determines the maximum print size of the image The lower left segment of the image is all that will be shown because the image is larger than the image object e To move an image around within the frame click the Move image button in the Report Objects button bar 6 Select the X check box located in the Scale group to include a scale bar under neath the image being printed Select one of three possible scale bar types from the list The length of the scale bar is calculated from the pixel calibration data of an image and automatically adapted for printing The automatic scale bars are only accurate for images that are correctly calibrated Adding zoomed image segments Detail Zoom 1 Insert an empty image object 2 Drag a large image either from the database or from the image manager onto the image object 3 Select the image object and click the Detail Zoom button located in the Report Objects button bar gt The pointer will a
422. ous color can be useful to highlight structures and to differentiate structures details To open this dialog box load a multi channel image To open the image information simply doubleclick the image in the image buffer On the Image Information gt Dimen sions tab doubleclick the color field next to a fluorescence The Edit Fluorescence dialog box opens It allows you to select the three parameters relevant to the HSI color model Hue Saturation and Brightness Modifying the fluorescence color only applies to the image located in the active image buffer Edit Fluorescence Color Ea Hue 112 4 Saturation 1000 Brightness 1000 cancel To do so modify the values in the three numeric fields or move the three respective sliders The single color image is automatically updated according to the representation color The currently active color and its wavelength are displayed in the dialog box Click on the OK button to close the Edit Fluorescence Color dialog box S20 nm Loads display LUT for a single color channel Bi RGE Lookup Tables of Color Chann Miel E Cyan T ad Cold armH ot E 158 The Image menu Image Display Edit LUT Note Monitor Display of Gray Value Images in Color Possible colors Displaying the orig inal gray value image 16 bit gray value images LUTs for image oper ations Use the Load LUT command to set the LUT to be us
423. ow will show the current blended image directly Blend Image Series Creates an image series by blending two images at increasing alpha values The two images are taken from the source and the source 2 image buffer The Range group is where you set what increments and within what limits the weighting of the two images are to vary A Start value of 0 and an End value of 100 means that both the source and the source 2 images will appear unchanged in the series of blended images The Incr increment value determines the percentage that the relative proportions are altered in each of the following images For example at an increment of 20 blended images of the following weightings will be generated 0 100 20 80 40 60 60 40 80 20 100 0 Click the Preview button to view the blended images The blended images with the various weightings will be shown one after the other Press Esc to stop the preview Click OK to generate the blended images These will be written into the destination image buffer and subsequent image buffers Watermark Use the commands of the Watermark submenu to have a texture applied to a true color image Write the original image into the source and the texture image into the source 2 image buffer The Watermark gt Adapted command zooms the texture image to the size of the designated true color imaged The Watermark Tiled command does not change the size of the texture image A texture image smaller
424. owest and highest intensity pixels will not be displayed Click the Auto button to have the image shown on your monitor in such a way that all of the intensities in the image are on display The values that are excluded by the overflow will naturally not be shown Please note that when you use the automatic display function the contrast and inten sity settings will also be altered The Gamma value will be set at 1 Note Please also note that only the histogram of the single image on display will be eval uated for the calculation of the Min and Max limits The limits calculated here will be adopted for all of the single frames in a time lapse image or a Z stack Frames that are either much brighter or much darker than the image for which you have defined the display function may possibly then be displayed overexposed or underexposed At the same time the relative contrast within a multi dimensional image will be retained Use the Image gt Image Display gt Auto Adjust Display command to have every single frame within a time lapse image or a Z stack displayed optimally You should also click the Auto button to have changes in the Clip group adopted and the display function correspondingly adjusted The borders of the section to be displayed are set in a way to disregard pixels as defined in the Clip group Use the Min and Max fields beneath the diagram area to define the mapping range The smallest possible value is 0 the greatest 6553
425. own in color A true color image or RGB image is made up of 16777216 colors 24 bit A Fourier image is a 32 bit image made up of real and imaginary numbers of 16 bits respectively Loading images stored on the hard drive 1 In the Image Manager click on the image buffer you wish to load the image into with the left mouse button Activate for example image buffer 5 gt The image buffer selected will be color highlighted and assigned to the active viewport 2 Select the File gt Open command to load an image gt The Open Image dialog box will appear Dialog boxes for loading files are based on stan dard MS Windows Look ir E Images dialog boxes The dialog a box for loading images i also has a preview func tion Open Image History hy Computer A aa mg e My Netw File name Aulabel ti Files of type Tagged Image Format tif Cancel 3 Select Tagged Image Format tif the standard image format in the Files of type list Warning Please note that since multidimensional images do not have their own file format iy they have to be saved as TIF files You should only use the TIF format to save multi dimensional images since only in this format will all of the information be saved with it First Steps Available image types The Files of type list is present in all dialog _ Files of type Database mdb boxes used for loading Database f md
426. p Select the desired overlay image from the Available overlay images list This list contains all of the overlay images that are possible All of the images in the image manager that are of exactly the same size as the active image The active image s individual color channels also belong to the possible overlay images Click the OK button to have the selected image superimposed on the active image In z 3D experiments stacks of images are acquired at different focal planes If the active image buffer contains a Z stack the Navigate z button is available in the Stack Navigator button bar With the Previous and Next buttons in the stack navigator you can navigate frame by frame backward or forward The First and Last buttons can be used to display the first and the last image The number within the field represents the number of the currently displayed frame You can directly go to a specific frame by typing the respective number into the Go to field and pressing the Enter key The menu offers the following projection functions Single Z Layer One image of the Z stack is shown Use the buttons of the Stack Navigator button bar to select a Z layer Maximum Intensity Projection For each pixel in the XY plane the intensities of the different Z layers are compared The maximum intensity is used for display Mean Intensity Projection For each pixel in the XY plane the intensities of the different Z layers are analyzed The mea
427. p of commands select whether you want to show the Image Only the Overlay Only or Image And Overlay This selection will effect all images loaded In the second group of commands select which layers of the overlay will be shown Select Annotation Layer to show the annotation layer only The annotation layer contains all elements added using the functions of the Overlay button bar Select Data Layer to show the contents of the data layer The data layer is used by your image analysis program to store data resulting from functions performed on the image Select All Layers to show both annotation and data layer Unmark the Lock Data Layer entry of this menu to make the data layer editable Burn Overlay Click the Burn Overlay button to transfer the information from the overlay into the image directly Note that image information beneath overlay contents is lost This command is not available for multi channel data sets Delete Layers Deletes all objects in the visible overlay layers Load Objects Loads overlay objects from an existing file into the overlay of the active image When you click on this button the standard dialog box for loading files will be opened This is where you select the file desired to have it shown in the overlay There are four different kinds of files provided in the Files of type list the special format for overlay objects OVL along with image file formats as well The image formats available are Win
428. pl A Cg Experiment 1 i Experiment 2 Experiment No Experiment 3 Fixed no A periment 3 Time 1644 Twd 1 Date 31 01 2006 Client Production T52 Tw 3 TFOWE Expermet OA Z Experiment Desciption Fluorescence In Situ Hybridisation 78 Archiving Images Insert data Insert documents Background Information Documents Differences Document files Inserting document files otep by step Documents include all those texts diagrams sheets and reports which are loaded in your image analysis program You can insert documents directly into a database folder or also into another image or data type record Differences between documents and records of the image graph and data type e No further data can be inserted under a document e You cannot define database fields for documents which provide further informa tion when inserting the documents e There is only a limited number of database fields which you can use to search for documents These include record names file names or insertion date and insertion time Generally you will find the documents by looking for the higher ranking record Document files are data that has already been saved in a file These may also be files that cannot loaded in your image analysis program but which belong to other appli cation programs e g CorelDraw Excel Origin PDF AVI or Word files You can insert document files into the database as follow
429. play the overlap region between two images In the overlap area the gray or intensity values of two images have to be adjusted to each other There are three different methods offered e When using the Side by side method the overlap region is composed using the original gray values of the two individual images For the first half of the overlap area the values of the first image are taken for the second the values of the second e When using the Linear weighted method the value is weighted linearly accord ing to the distance of the value from the border of the own image e When using the Gauss weighted method every value is weighted using a gauss distribution Select the Original Size check box to display the overview image with a zoom factor 1 After selecting the check box the top left corner of the image is shown on the monitor Using this feature you can observe the whole image with the same high resolution as the individual images Use the scroll bars of the image area to display the interesting part of the image on the monitor One click on the scroll bar will center the next single image on the monitor 146 Acquiring images Multiple Image Alignment Note Repeat Note Calibrate Note Select the Cut Margins check box to cut the composite image to a rectangle shape All of the borders of individual images that stick out after the image alignment will be cut off Click the Background button to adjust the gray value of
430. positioning the mouse you will only be able to move the mouse vertically to be able to move only horizontally keep the Ctrl key depressed while positioning the mouse Select the color you want to change in the Color group The polygon representing this color will be placed in the foreground not covered up by the other polygons In the Polygon points gt Count field you define the number of points the currently displayed polygon is to have Then click the Define button Your image analysis program recalculates the number and position of the polygon points for the current curve All polygon points will be plotted equidistantly Up to 32 points can be defined for a single polygon If you select the Monochrome check box all changes will be applied to all three colors simultaneously This function enables you to define a monochrome gray value LUT In this mode the polygon will become yellow Click the File button to open the standard dialog box for the opening and loading of files Files will be saved using the LUP format 162 The Image menu Image Display Click the Linear button to delete all the points of the active color The edge points will be set to the coordinates 0 0 and 255 255 and joined by a line If the Monochrome check box has been selected this will be done for all three colors Edit LUT Formula Defines the LUT using three formulas Edit LUT x Sheet Polygon Formula o Linear W Red E W Limit W
431. ppear within the report window You are now only able to move the mouse pointer within the image object 4 Now define a rectangular segment within the image e Keeping the left mouse button depressed move the mouse to form a rect angle within the image Release the mouse button once the rectangle is the size desired gt Your image analysis program will select the image segment selected and will automatically add another image object showing the image detail selected gt Auxiliary lines provide a visual guide from the corners of the detail image to the corners of the image segment making it easy to see the visual corre spondence between the image segment and the detail image 5 Changing the size and position of the detail image 37 Report Generator Record objects e In order to position the detail image you move the mouse pointer onto the detail image As soon as an arrowed cross symbol appears at the mouse pointer you can press the left mouse button to pick up the detail image and move it to the spot you want to have it In order to alter the size of the detail image you first select the detail image Then pull one of the selection markers while keeping the left mouse button depressed gt The zoom level of the detail image will change according to the size of the image object The image segment being shown remains unchanged The auxiliary lines are automatically altered to fit the new position of the image segment detail im
432. ppears behind the stage name Since the last time that the image analysis software was started the stage has not been initialized 1 Select the stage you want to use in the Special gt Preferences gt Stage menu and click the Limits button 2 Set the stage limits then close the Define Stage Limits dialog box 3 In the Preferences gt Stage dialog box select the Virtual stage option in the Selection list again and close the dialog box The overview image looks as if it has been tiled The images are not evenly illuminated across the view field 1 Set the Shading correction gt This is only possible with Soft Imaging System GmbH s FView Il ColorView II and ColorView III cameras 2 Access the mage gt Configure Input menu and switch to the Input tab 3 In the Shading correction group select both the Offset and the Gain control boxes gt If you have further questions concerning the shading correction you will find the answers in your camera documentation 4 Exit the dialog box with OK The images are misaligned in respect to each other The overlapping area selected was too large Reduce the image overlap see page 338 The camera has not been positioned parallel to the stage movement directions Readjust the stage To do this follow the step by step instructions for setting the automatic rotations correction see page 329 then acquire a new image Should the result still not be satisfactory you will have to reset you
433. ppermost entry contains the number of the image buffer and the name of the image The current position of the image origin is automatically displayed for each image In the tree view the object and the values beneath the object are expanded after measurement A single entry is added to the tree view for each image which contains measurement values The measurement display has its own button bar Use the buttons for example to delete measurement values or to alter the presettings for the interactive measure ment Image Manager Ei Delete Measurement x iz fil ba Image Link pee 1 Image Show hide Statistics E 2 magez e e 3 Image3 xele EF Origin T 0 0 Select Measurements Define Statistics Length 523 mm Measurement Properties Length 600 mm Length 577 mm Length 593 mm 282 Interactive Image Measurement Save Load and Edit Measurement Results Save measurement results Warning l Editing measure ments Measurement results and image overlay Step by step All measurement files are linked to the image This link remains if you save the image in TIFF format Either use the File gt Save As command or insert the image into a database If an interactive measurement is linked to the image the measurement results are automatically listed in the measurement display when the image is reloaded The measurement files are only then saved with the image if you use the TIFF image format The
434. ps to assign up to six images or graphs to the frames 9 Click the Print button gt The system returns to the Print Image dialog box 10 Click OK to start the printing task What will happen e The selected images and graphs are fit into the frames e Note that only the graph range currently displayed in the graph window is print ed e In this mode the graphs are always printed without the additional graph informa tion 310 Graph Graph To print images and graphs simultaneously choose the Multiple Images Graphs entry from the Page layout list You may of course use this feature to print out multiple graphs onto one page Assign images and graphs by a simple drag amp drop operation from the Documents list to the page preview Printing graphs To print the graph of the active graph buffer activate the graph document and use the File gt Print command You may print the graph information together with the graph Note that only the graph range currently displayed in the graph window is print What will happen Print Image Printer Mame 206 HP LaserJet 4050 Series PS Properties Statys Default printer Ready Type HP Lagenwet 4050 Series PS Wher SSlntranet 206 ps Comment Laserdrucker HP 4050 PostScript Page layout Multiple ImagesGraphs Single Image Multiple Images Pririt imaa EAE EEEIEE he k Print to file Convert to AGE Frame OK e H
435. quired e documents are loaded e g an image and a report and e you have installed a MAPI supported e mail program and MAPI DLL file 13 First Steps E mailing images Sending work Select the Add a workspace for the selected documents check box in the Send email spaces via e mail dialog box to include a Workspace wos file along with the other documents you re e mailing The recipient can thus open the workspace along with all images and documents and display these in their original onscreen arrangement To do this the recipient will have to save all attachments in a separate directory To open a workspace along with all other documents select the File gt Workspace gt Open command The size of your e To receive a warning message when the size of your e mail attachments exceeds a mails certain limit which you may set yourself go to the following tab File gt Send email gt Preferences gt General The following are possibilities to reduce the size of your e mail e Leave out some documents e Compress images Go to the Image and Report tabs in the Send email Prefer ences dialog box to do this rh i ch a el the file format for all im Semi Sees IEEE E ages that you e mail Image Report Other Documents General The Save Image Op tions dialog box enables Image format l you to determine for the Save Image Options TIF format whether or Compression None Options not and how the images Ue ar
436. r Matrix 00 0 2 20 00 0 242 The Process menu Filter Image noise should be rather limited for application of this filter as this filter is suscep tible to image interference Results can be improved when a larger pixel area is taken into consideration This filter uses the matrix to compute a value called a The resulting pixel value is calculated via the absolute value of a This filter differentiates the image on the X axis This means that gray value modulations will be enhanced horizontally Smaller structures will however be emphasized as well The Differentiate X filter is one of the derivative class of filters If you wish to filter out edges on the Y axis then select the Differentiate Y filter If you wish to extract all edges of an object then select either the Laplace or Laplace filter Differentiate Y Laplace Laplace Il Performs a gradient filter for vertical edge enhancement Filter Matrix 020 0 20 000 Image noise should be rather limited for application of this filter as this filter is suscep tible to image interference Results can be improved when a larger pixel area is taken into consideration This filter uses the matrix to compute a value called a The resulting pixel value is calculated via the absolute value of a This filter differentiates the image on the Y axis This means that gray value modulations will be enhanced vertically Smaller structures will however be emphasized as well
437. r a report template these alterations only affect reports you create subse report templates quently Existing reports based on the now altered template cannot be automati cally updated Altering templates You can also alter or redefine page and object templates within a report These within reports templates are then however only available within that report and not for other reports that you create based on the unaltered report template The report template left contains three dif ferent page layouts in the example The report right consists of four successive pages The 1ImagePage is the only page template that was used of those avail able within the template Report template 1lmagePage 2imagePage 4lmagePage The following sheet lists the differences between the reports and the report templates ie Report templates gA A report consists of multiple successive A report template consists of multiple pages pages All report pages can be but don t with different appearances and functions have to be based on a single page layout e g a cover page and a page for 1 2 or 4 The 1ImagePage page template defines images Besides these page layouts report what all report pages look like in the exam templates can also have object templates for ple shown record and field objects Document window s white gray background The first field of the status bar indicates the The first field of the status bar is
438. r has been loaded from an image database You can insert this image directly from the image manager to a report whereby all of the database fields are correctly filled in Use this possibility to edit an image before inserting an image for better print results For example you can correct a tinge or conduct a sharpen filter on the image Record objects are complex report objects comprised of several separate compo nent objects Record objects usually consist of at least one image object and several field objects You can however make use of all other kinds of report objects within record objects text AutoText images and graphic elements Placeholders for text and more images are not permissible Sheets and diagrams in contrast serve as placeholders for documents which are stored below an image within the database 39 Record object structure see sheet on facing page field objects 5 can only be used within record objects They are usually comprised of the field name and field en try Variously formatted templates for field ob jects can be defined in a report template Report Generator Record objects Measuring Results Sheet Cell lt Sheet Cell gt image object placeholder for database All image properties described above apply to images image objects as well i e you insert images at varying magnifications either as links or copies into a record object text standard text to be printed Texts are usuall
439. r solid color image areas They are most noticeable when broad bandwidths and strong enhancement are in use Select the Ramp optimize check box to completely eliminate image artifacts resulting from the DCE filter This check box can be used as a kind of supplement to the Quality check box What will happen however is that all contrast either large in area or strong in intensity will be completely suppressed Enter the percentage of dark pixels to be set to black and of light pixels to be set to white into the Overflow field The last step involved with DCE filter calculation is a spreading of gray color values applied to the whole gray color value range This is why you can enhance image contrast in the resulting image by simply clipping off gray color values located at either the higher or lower extremities of the gray color value range and setting them to 0 or 255 respectively for 8 bit images These gray color values are avail able for spreading the remaining gray intensity values Percentages between 0 05 and 50 00 can be entered When you select 50 a gray value image will look very similar to a binary image colors will be either white or black Defines the separator for separating parts of an image Use this command to define parameters for a separator filter e g a filter for sepa rating objects such as connected particles or such as various intensity regions The separator is adjusted to special image propertie
440. r stage once more from scratch see the following instructions This is how you adjust the camera parallel to the directions in which the stage oper ates 1 Mount the camera onto your microscope as parallel as possible to the stage movement direction 2 Switch on the live overlay for the active input channel to do so mark the Image gt Configure Input gt Display gt Live overlay check box 3 Position a specimen that shows vertical and horizontal lines or structures under the microscope The edges of the microscope slide glass will serve this pur pose 4 Switch into live image 5 Paste a rectangle into the image To do so click the Overlay Button Bar in the Standard button bar and then click the Rectangle button 6 Use the joystick to move the object along the horizontal object line The object must move exactly parallel to the displayed frame 7 Correct the camera position as necessary by loosening the camera screws and turning the camera a little 344 The Stage Navigator Trouble Shooting Problem Cause 1 Correction 1 Cause 2 Correction 2 Cause 3 Correction 3 8 Repeat the last two steps until the camera and the stage are correctly adjusted The overview image has not been correctly compiled from the separate images The camera has been mounted back to front on the microscope 1 Turn the camera through 180 and readjust it See page 329 Alternatively you can invert the axes 1 Todo so go to t
441. r to black gray values in the original image G r 0 for all lighter to white gray values in the original image for binary and 8 bit gray value images The resulting image will resemble a binary image where one half has been set at white and the other half at black Enhancing Contrast means rotating the display curve counterclockwise from the center of the diagram Use the contrast scroll bar to rotate segments of the mapping function into the horizontal and beyond At the highest possible contrast setting 100 every mapping function will look the same This setting corresponds essen tially to the step function G r 0 for all darker to black gray values in the original image G r 255 for all lighter to white gray values in the original image for binary and 8 bit gray value images The resulting image will resemble a binary image where one half has been set at white and the other half at black Click the Change button to change the original image s gray values The resulting image will be written into the destination image buffer Binary images will be mapped to 8 bit gray value images 8 bit gray value images will be mapped to 8 bit gray value images and 16 bit gray value images as 16 bit gray value images The Cancel button will become the Close button as soon as you save a mapping function Click the Cancel or the Close button to exit the dialog box A resulting image will not be written into the destination image buffer
442. ranges is to go right into the image and select a portion of the picture containing the color desired To do this first click on the New button located in the Include pixel group The diagram in this dialog box displays only one curve at a time this is less confusing The diagram plots the number of pixels versus color saturation or inten sity The saturation curve corresponds e g to the histogram of the image after you have saturation has been separated The whole image area is used to calculate these curves no matter whether a global frame has been set or not Curve Color used in diagram Hue magenta Saturation light blue Intensity black Use the Y scroll bar to have the curve displayed spread on the Y axis This enables you to evaluate the progression of the curve even when the number of pixels at a particular point on the curve is quite low The color bars below the histogram represents each of the color channels Color bars display phases that have already been defined Each bar displays its corresponding color range in the same color as in the Preview Activate any of these color channels for a particular phase by simply leftclicking on the color bar of that channel If you switch back and forth between channels the curve s progression displayed in the diagram will of course change accordingly The Hue Sat and Int fields display the current thresholds for the active phase Two lines demarcate the active color channel w
443. rated at 0 5 um pixel has an absolute image width of 500x0 5 um 250 um Your image analysis program determines absolute image size via image information The report generator enables images to be rotated 90 Rightclick in a report window You will find the Rotate button bar under the Button Bars command Mark the image object and select one of the two buttons to rotate an image 90 clockwise or counter clockwise Fit image into the frame Use optimized magnifications we 40 50 60 ee 30 40 50 60 70 80 uaa ai io 100 1 200 um 50 wm Use fixed magnification we 40 50 4 sii 200 i me 100 um 300 Use fixed magnification 35 step by step sy b Report Generator Image Objects Inserting images from the image manager into a report 1 Load the images that you want inserted into the report into the image manager 2 Press F8 to open the Preferences dialog box and select the Report tab 3 You ll be making two fundamental decisions concerning how images are treated in the report in the Image group e Create image copies you decide whether images are to be saved along with the report or whether you d rather insert them as a link to an existing image file e Use thumbnails instead of full images Select either a high resolution im age display or thumbnail This selection only affects the display of an im age on the monitor and not the image quality in the actual printout 4 Click OK to close the
444. ration and image analysis Having separate workspaces gets you the onscreen layout you need and fast e Reloading images documents The path names of currently loaded images and documents can be saved in a workspace Saving the current GUI in a workspace at the end of your workday makes it totally easy for you to continue where you left off the next morning Any and all images sheets diagrams database s report s that were loaded when you saved the workspace will be right where you left them Warning Be sure to save all your images before shutting down your image analysis program lt P gt Any unsaved images will be deleted without prior warning Configuration The Special gt Configuration command enables you to individually determine versus Workspace elements on your user interface as well Please note that the configuration and work space contain different elements of the user interface Configuration refers to what 10 First Steps Printing images commands have been defined for menus button bars and keyboard e g user defined button bars A configuration saves what functions are available on your GUI A workspace however actually saves what the GUI looks like including specific documents The information saved in workspaces and configurations is totally different Please not that there is a default workspace for working with reports Use the Ctrl 2 key stroke to load this workspace Printing images Print Templates Y
445. ray values In the Color coding mode a pixel s gray value is proportional to the number of brighter or equally bright neighboring pixels In this way the areas of the image with a constant gray value will be displayed as one single gray value The transitions between the different gray value areas will be differently colored The color supplies information about its neighborhood s gray values This can be especially well recognized on the corners of the individual squares The benchmark has the most bright neighbors In the Delete respectively Extract modes the constant gray value areas are coded darkly respectively brightly Only the transitions between different areas will be reproduced brightly respectively darkly If you choose 7 neighbors only the corner pixel in this example has precisely 7 bright neighbors You define the Connectivity filter s parameters in the Define Connectivity dialog box The filter determines the number N of the gray values in a 3x3 environment that are Delete greater or equal to the central pixel s gray Extract Cancel value The result will be compared to the Color coding Execute value that you have selected in the Number Iterations of neighbors group Depending on whether or not the number found is the same as one h windo Help of the values you have selected the central pixel will then be differently handled You can select several check boxes If for example you want to delete
446. rea fractions according to the threshold settings This command is only available for 8 or 16 bit gray value images and for binary images as well not for 24 bit true color images It is available for 8 bit false color images only then if the Special gt Preferences gt Image gt Allow operations on false color images check box within the Image tab is selected Phase Analysis is the quantitative analysis of the area s of various gray value ranges To define an object for measuring its area determine the phases that make up the gray or color value range s of the object Objects in gray value images are defined with the Process gt Set Thresholds command Up to eight phases can be selected Areas of gray value ranges not assigned a phase will not be calculated Select the All option in the Manual tab to check and see via phase color coding whether gray value phases have been opti mally defined Objects in binary images are defined with the Process gt Set Thresholds command Select one phase for the white and one for the black objects in the Manual tab Objects in true color images are defined with the Set Color Thresholds command in the Process menu Lower and upper thresholds for the three color parameters are set here and depending on whether you are working with the RGB or HSI system the six thresholds will be assigned a phase Thresholds can also be set inter actively within the image To do this select a set
447. records are marked by a lock in the database window The table shows icons used to identify records within the database window s tree structure 7 read only record aa No data can be inserted into this folder archived record a Additional data can still be inserted underneath this record 2 TE archived and read only record i gg No additional data can be inserted into this folder The folder was locked during archiving Protect with a password Background Information Database password Session password You can assign different passwords for every database The database password protects the entire database against unauthorized access The database cannot be opened by a user who does not know the password Remember however that a database password does not protect against access or manipulations through Windows Explorer Make a note of the password and store it If you forget the password you will no longer be able to access the password protected database Even the vendor of the database has no possibility of accessing the database without knowing the pass word A session password is not related to a single database but to a workstation Assign a password to prevent unintended and unauthorized manipulation of the structure of your databases All commands which affect the structure of the database are only 99 Archiving Images Protect with a password available to the user who knows the session pas
448. red You can lock the database folder when storing Insertion of data into a locked database folder is then no longer possible Always use the wizard to secure data when archiving Never transfer data from a database in Windows Explorer This directory is used for temporary storage of the files when archiving the database Set up the directory for the database files and the temporary storage directory in the same partition on the main drive In this case the system checks the available space when inserting the data into the database As soon as less space is available in the temporary storage directory than defined in the Special gt Preferences gt Database gt Backup volume capacity no more data can be added to the database In this case you will be asked to archive data To do so select the Database gt Administration gt File Storage command Both single images and the entire database can be deleted without too much effort And no storage medium provides 100 data protection For this reason you should protect your database against unauthorized access and data loss as a result of accidental deletion or damage of the data medium e Protect your database with a password 95 Archiving Images Archive data e Make backup copies at regular intervals Backup Security When making a backup or security copy you copy the database data so as to use the copy backup to reconstruct the database in case of data loss A backup copy must there
449. remove text from the text object Select the check box to have the text lines automatically wordwrapped so they fit the width of the text object If you alter the width of a text object the text wordwrapping will be adjusted to the new width Please note that texts may be wordwrapped slightly differently onscreen and when printed out Select this check box to have the text size adjusted to correspond with the zoom level at which the image is displayed If you display the image in the viewport at a lesser zoom level text size will be reduced accordingly The position of the text object within the image remains unchanged This may result in overlay text becoming poorly legible or even illegible especially as far as unusually large images are concerned Clear the check box if you wish your text size to remain the same at zoom levels less than 100 In this case the text object will cover up a segment of the image that is larger or smaller depending on what the zoom level is It will however remain legible even at low zoom levels Please keep in mind that as far as low zoom levels are concerned the text object may be clipped 177 Rectangle Ellipse Line The Image menu Rectangle Creates a rectangle object in the overlay When you insert rectangles they appear selected Their object properties are predefined red outline and no filling The preset line weight is defined in the Pen size field in the Special gt Preferences
450. ret Even very small differences make themselves noticed as a different focusing position This fault will be corrected by the Z shift Click the XYZ Correction button to open the Shift Correction dialog box Shift Conrecton Devices Remote Olympus Dummy Bs 2 Stage Stage Simulation Correction 2 Parfocality sY Centering M Enable Objectives to calibrate Add UPLFL 4x 4 00 1 2 UPLAPO 10x 10 00 Remove 3 UPLFL 20 00 Edit 4 UPLFL 40 00 Calibrate EE Close First enter the objective you want to use To do this click on the Add button to open the Edit Objective dialog box Here you should specify the objective with its name its position in the objective turret and its magnification When you use a fully automatic microscope the objectives will be adopted from the Control Panel You can switch the Shift Correction on and off You should in any case always employ the Shift Correction when you are using either the Multiple Image Alignment or the Extended Focal Imaging acquisition process to acquire a series of images that are to be put together to form a composite image Select the Correction check box to employ the correction Click the Calibrate button to define the necessary XYZ shift after an objective has been changed A software wizard will then conduct you through the individual steps When a calibration has been successfully carried out the focus position will be aut
451. rlay For gray value and false color images the pixel s X Y coordinates and gray value will be listed in the sheet For true color images the pixel s X Y coordinates and RGB and HSI values will be listed The status bar displays current mouse cursor position and the respective gray value False color images will have the RGB values of the active LUT displayed as well True color images RGB and HSI values will be displayed Pixel values will be displayed on the left and mouse cursor position on the right within the status bar Select the Show labels gt Numbering option in the Special gt Preferences gt Measure tab to numerate each pixel measured This way the pixels in the overlay will clearly correspond to the values in the sheet If an image s gray values contain height data e g in images acquired by an atomic force microscope these height values can be measured directly and inserted in the sheet You will need to calibrate the intensity values accordingly This can be done in the Image gt Calibrate Image gt Image Intensity tab The height Z function depen dent on the intensity value is defined here Histogram Calculates the histograms of the selected color channels Use this command to have an image s gray value distribution calculated and plotted on a graph Note The histogram is an x y diagram in which an image s gray value distribution is displayed in a graph i e number of pixels per gray value v
452. rmine some of the properties of the Graphical User Interface GUI Grid and Ruler groups You can open this dialog box by rightclicking on any part of a report on which no object has been placed and selecting the Properties command from the context menu The file formats SRD and SRC are available for saving reports Both formats are exclusive file formats of your image analysis program and cannot be opened with other application programs Select the SRC file type to place all files which belong to the report in a single container file If you insert a report in an image database the report is automatically inserted in SRC format When using the SRD file type the report is not saved in a single file Similarly to the saving of a database there are several files and directories involved Any files that are part of a report will be automatically placed in a subdirectory named after the report When making backup copies the easiest thing to do is to copy the whole report directory 27 Report Generator Creating reports otep by step Set report properties 1 Leftclick anywhere within the report on the background gt Now none of the report objects is selected 2 Rightclick and select the Properties command 3 Select the desired properties for the report e g page format For example clear the Snap to grid check box to be able to position all objects as desired via mouse 4 Close the Report Properties dialog box v
453. rn into a cross within the diagram Position the cross at an X Y coordinate you want the new mapping function to run through If you want to draw a totally new function then start at the beginning positioning the cross to the left of the diagram where you want the function to begin where x G 0 To define the beginning point of a curve leftclick on it A curve is made up of line segments at whose end points you leftclick on You can draw the curve freehand if you like by simply keeping the left mouse button depressed while you use the mouse to draw the curve To end the drawing mode just rightclick The previous curve will be deleted at all X values where you draw a new Curve As you draw the new curve any remaining sections of the previous curve will remain visible A mapping function always comprises the complete value range of the X axis and will be made up of both new segments you have drawn as well as previous curve segments if you haven t redrawn the whole curve You can adjust and correct the new curve as much as you need or like to The line representing a mapping function may be reduced to the individual points it s comprised of This happens when steep curves are being drawn The reason for this is that the X and Y axes are made up of whole number gray values and when a curve is too steep a continuous line of that steep of a curve cannot be reproduced Another way to adjust an image s intensity and contrast is via the
454. ro level is assigned a value of 128 This means that gray values between 127 and 128 are available The Pseudo filter is a member of the derivative class of filters Other filters belonging to this class are the Sharpen and II Differentiate X and Y as well as the Laplace and II filters Define pseudo filter parameters in the Define Pseudo dialog box Contrast 2 B Cancel W Preview aoe Wir Help Define a contrast factor 0 01 100 in the Contrast field this will be multiplied by the weight factors of the filter matrix The preset value is 2 The greater the contrast factor the greater the 3 D effect Defines the equidistant intervals for the Reimer filter The Reimer filter can compute an equidensitometric image of the second order based on the original image Gray values occurring within a particular gray value interval in the original image will be displayed in a single gray value for that interval in the resulting equidensitometric image If in an equidensitometric image only the borders between the various gray value intervals are shown this is known as an equidensitometric image of the second order The Reimer filter is able to visibly subdivide continuous gray value ranges within an image which the naked eye alone would never be able to distinguish from one another 231 The Process menu Reimer Original The unfiltered image shows continuous gray value behavior The filtered image v
455. rs The sequence defined here is adopted by the measurement display and the export sheet If the Show labels gt Measurement result option is selected in the measurement settings measuring results are also written into the overlay Here it is where you determine which parameter is displayed if you are measuring more than one param eter If you want to display measurement results in the overlay the program always shows the first measurement parameter This is how you select measurement parameters You measure 2D Objects You are interested in the area and the color of the object 1 Measure a 2D object for example with the Magic Wand measurement function 2 Click the Select Measurements button You will find the button in the measure ment display above the tree view gt The parameters are classified based on measurement types in the tree view located in the upper left hand part of the dialog box 3 In the tree view select the 2D Object entry to limit the measurement parameter list to the parameter for 2D objects 290 Interactive Image Measurement Measuring Arbitrary Structures There are more measurement parame ters available for inter active measurements than those shown by default 4 Mark the check box of a measurement parameter in the Measurements list to activate it for the measurement 5 For example mark the Mean Hue check box to measure the hue of a 2D object An object s hue corresponds to the mean hue of a
456. rt Stage Navigator gt The system creates an image at the current stage position X0 then moves the stage one short segment in the direction X to the position X1 and cre ates an image there too The images created at the positions X1 and X0 are then compared and the amount the images are twisted to each other deter mined The same measurement is subsequently made for the twist of the stage in the direction Y gt When the adjustment has been made the Rotation field displays the rotation angle that the system will take into account with each stage movement Start Stage Navigator In the Stage Navigator button bar you will find the button that is shown on the left click it Should this button not be displayed please read the appropriate note in the chapter Trouble Shooting on page 343 The Table Navigator lt Unnamed gt dialog box will open The display on your screen when you start E Stage Navigator Unnamed the Stage Navigator CO be bd w A 3 f Tele position 0 00 me The image section will remain blank until an overview image has been acquired or loaded The current camera area will be identified by a yellow frame The size of the frame will be determined by the magnification you have set If you click the mouse in this frame and move it to another location you will change the X Y position of the stage If your stage has a motorized Z drive To move the stage along the Z axis you can either use the
457. rview image to move within the sample and to go to interesting areas that you can then look at and acquire in the live image with a higher resolution Choose this option when you only want to use the overview image as an aid to orientation but wish to look at the interesting areas in the live image Option Get separate images of high quality Alternatively you can also create an overview image that is made up of separate images In this case you will save all of the acquired separate images in their original resolution and additionally the overview image in a reduced resolution Obviously you will be using a lot more of your hard disk s storage capacity when you choose this option The advantage is that you can zoom in on these overview images When you do this the separate images will be reloaded making new data available to you Choose this option to be able to use overview images offline as well i e even when a live 331 The Stage Navigator How to acquire an overview image image is no longer available e g when a sample can only be preserved for a limited time or when it has to be returned Furthermore this option is the more suitable one when you want to pass the images on to other users of the image analysis program How to define the size of the overview image The size of an overview image can be defined in several ways Based on the current camera position specify how many images are to be acquired around thi
458. s e Mark the record in the tree structure to which you want to attach the document e Click the right mouse button and select the Insert gt Document File command e Select the correct directory and mark the required files e Click on the Insert button How to insert documents You have created a database An image is already in the database folder Now you want to file further result documents in the database folder 1 Load the documents that you want to insert into the database folder gt Documents are diagrams texts sheets and reports 2 In the tree structure of the database window mark the database folder into which you want to insert the documents 3 Click on the right mouse button to open a context menu Select the Insert gt Documents command gt The Insert Documents dialog box lists all loaded documents The check boxes in front of the documents are marked 4 Clear the check box of a document if you do not want to insert the relevant docu ment 5 Mark the Prompt for file names check box 6 Confirm the document selection by clicking OK gt The insertion mask for documents opens In contrast to the insertion of images you cannot define database fields for documents The insertion mask therefore contains only limited information about the document and an input field for the file name 7 Check the file names in the File name field If necessary change the document names 79 Archiving Images In
459. s Insert data gt The preview window on the upper left now contains the thumbnail of the image folder The information regarding the image which you have selected using the Arrange Fields command for the Form View and the Image data type is displayed on the lower right 77 Archiving Images Insert Images How to insert images ange MSE amp 1F0106 apl a A Am Mle ee a 438 a gt 43 Ge fe Dest Ive No records available a 1280x 1024x i Tw Et 1280 x102 _ p Iv4 10 1 28 0 cae Experiment 1 Expernment Mo Experiment 3 J Experiment 2 Fixed no Wy Experiment 3 Time 16 44 Date 31 01 2006 Client Producton Expenment Desciption Fluorescence In Situ Hybri T O 4 cem ok a O 5 HER O 6 YFP_on The images O 7 creyr The list offers selected in the O 8 Tv2 all images i 9 Tv loaded image manager Pe a oaded F are already a 11 Tv5 checked C 12 T 6 x Help Prompt for data input Always Once Never Insert Image The record name can be created automatically l i cymes m Hybridisati tu Hybridisation TE Insertion mask when inserting images Fields 1 3 Record Mame Experiment Desciption Comment Enter your comment here Help Database fields corresponding to the image amp IF0106 apl of x esac The image is inserted into the project folder z a
460. s drawing method if you want to transform an image object into a shape This is how you define a shape with the Magic Wand method Background information about the magic wand 1 Click on the Magic Wand button the pointer will move into the image window 2 Click on a typical color value in the object you would like to make into a shape The point selected in the object will be marked by a blue point in the overlay The image analysis program immediately looks for the object and highlights it with a red outline in the overlay 3 Should the desired object not have been found correctly do the following Click once again on another point on the object Sometimes the object can be found more easily with a new gray value or color value Change the size of the object found Keep the left mouse button depressed and pull the mouse to the right to increase the size of the object You can view the result as soon as you release the mouse button 4 Click the right mouse button to end the definition 5 Continue defining shapes by the active drawing method 6 Depress the middle mouse button or the Esc key to return to the Add Shape dialog box Deletes all shapes from the active image Transfers all shapes of the image in the source image buffer into the image in the destination image buffer This command is only available if the destination image buffer contains an image that has the same bit depth as the original image The copied shapes w
461. s menu NxN 06 ps4 Se OC Ooe NS SCC 6X XO 09 Square Hexagonal lattice lattice Image row 2n Image row 2n 1 Defines the iterations and size of the NxN filter Use this command to set parameters for a mean value filter using a square matrix of arbitrary size The NxN filter enables you to reduce statistical noise within your image The NxN filter averages the gray values of all pixels surrounding a central pixel and assigns them to that central pixel The name of the filter refers to the size of the image area whose pixels gray values are averaged the size of this image area comprises precisely NxN pixels When determining filter size N you should keep the following in mind generally the smaller the matrix the finer the details you can edit These details can include arti facts or interference Larger matrices suppress these effects but yield blurrier resulting images mean value filters smooth out gray value peaks The higher the number of iterations the greater this smoothing effect will be You may end up blur ring edges Comparison to other The NxN filter is a member of the smoothing filters group The NxN filter is most filters closely related to the Mean filter The NXN filter permits you to determine the size of the averaging area and thus the extent averaging is to take this is not possible with the Mean filter Define the NxN filter parameters in the Define NxN dialog box Iterations E
462. s or in none You cannot have a scale bar shown for one particular image Display of the scale bar is not a part of overlay display i e you cannot alter this scale bar using the Overlay button bar commands Overlays belong to their respec tive images and e g when the zoom factor of that image is changed they also increase and or decrease in size Select the Clipboard check box to have the scale bar burned automatically before you copy an image or image segment into the clipboard Use this option when you wish to export images along with the scale bar into another application program e g MS Word Please note that when working with false color images the color of the scale bar can change when you copy the image into the clipboard Select the Printer check box to have the scale bar s printed out along with the image If you print out the scale bar you should clear the Print scalebar check box located in the Header Footer tab in the File gt Define Page Layout dialog box Otherwise two scale bars will be printed out The length of both types of scale bar need not be the same The scale bar within the image could be e g 20 um and the conventional scale bar below the image 50 um 167 The Image menu Properties Scale Bar Properties Format Defines the appearance of scale bars Scale Bar Properties x Display Format Size Scale bar styles Tope aa ie width Smal O Frame Transparent Tex
463. s position Define grid The whole stage area will be acquired You specify an interest area and the software calculates the number of images that will be necessary The sequence in which the images are acquired is always in the form of a horizontal meander 23 24 25 Create an overview image by defining a grid 1 If necessary switch to the Stage Area Mode since you can only acquire over view images in this mode see also The Modi of the Stage Navigator on page 336 2 Go to the area in the live image that you want to use as your starting position 3 Click the Scan m x n images button in the Stage Navigator gt The Create Overview dialog box will open 4 Here you specify the number of images you want to acquire gt The images that are to be created will be acquired symmetrically around this position i e if the preference is for 5 images two will be acquired to the left and two to the right of the current camera position gt In the Stage Navigator window the arrangement of the images will be graphically displayed 332 The Stage Navigator How to acquire an overview image x E Stage Navigator Unnamed q D S HE Aeaee ome P E Count Y E B Whole Stage Magnification Create one image from thumbnails Get seperate images of high quality Cancel 5 Select one of two options Create one image from thumbnails or Get separate images of high quality see page 331 6 I
464. s with the Smooth and Fine Coarse slide controls To ensure correct setting of the filter parameters the preview function should be used to continuously check the quality of separation Start with low values for the Smooth and Fine Coarse slide controls With this setting too many separation lines are generally drawn in Now increase the Fine Coarse parameter until you are close to the correct setting If some outlines can no longer be recognized then increase the Smooth parameter and then reduce the Fine Coarse parameter You will quickly achieve the best results with a few iterative changes to these parameters The parameters for this filter are defined in the Define Separator dialog box 239 The Process menu Separator Define Separator Boundary shape Ap Dark Jl C Bright f i Step Cancel Sigma 0 Execute Smooth ont l File 2 E rT Preview Fine Coarse C Bum black Window 10 qd l 2 Bum white Edit yt Help Classify the objects to be separated in the Boundary shape group The separator affects light or dark lines dividing objects of the same color or affects objects distin guishable from the background due to their intensity value Select the Dark option if light objects have a dark outline Select the Bright option if dark objects have a light outline Select the Step option if the individual objects are distinguishable by their varying intensity values Select this
465. scence in situ hybridization 11 Click on the Add button to take over the entry from the Value field into the pick list e Check the entries carefully Typing errors can only be corrected with great difficulty at a later date 12 Confirm the picklist by clicking OK to return to the Define Fields dialog box gt The Picklist group in the Define Fields dialog box can now be accessed 13 Select the Restrict input to picklist entries option so that only the given experi ment description can be accepted when you set up a new experiment 14 Repeat the procedure from step 2 to define further organizational fields such as for example approved by or estimated completion time 15 In the Define Fields dialog box click the Close button to complete the definition of database fields 67 Archiving Images Define organizational fields How to define organiza tional fields Organizational ID Define Fields All predefined figrvec database pe Time 5 Audio File fields Bits per Pixel 2B Calibration Unit amp Channel EE Document Creation Date amp Document Creation Time a Document Files Size E Document Insertion Date Document Insertion Time Document Modification Date Ey Document Modification Time Ey Document Replacement Date Document Replacement Time New organizational field is created Define Fields F Experiment No E Calibration Unit
466. se parameters are variable which means that filtering strength is adjustable Anytime you adjust a parameter the filter will immediately be applied to the original image and the resulting image shown in the preview window After you ve obtained the optimal filter setting you can apply the next filter to the image To do this select the next function from the list and then proceed to optimize its filter parameter s 258 The Process menu RGB Studio Loading filter sequences The Parameter group is context sensitive and is thus dependent upon which filter you select from the filter list The Value field will appear for most filters This is where you adjust the specific parameters of the selected filter using the arrow buttons next to the field You can also enter the desired parameter value directly in the field and then confirm with the Enter key or by clicking the Execute button The value range of the parameter will be indicated below the field You determine filtering strength by adjusting filter parameter s Any change you make to a filter s parameters will be immediately shown in the preview window Every new parameter value will be applied to the original image in its state before the currently selected filter was applied not to the image as currently shown The parameter value of the current filter of the filter sequence will be overwritten by any new value Using the arrow keys you can thus increase a parameter increme
467. se detection utilizing equidensities You can set the filter s sensitivity i e the gray value distances using the Define Filter gt Reimer command see page 231 245 User Filter NxN Lowpass The Process menu User Filter Performs the user defined filter The filter matrix is defined using the Define Filter gt User Filter command see page 228 Performs an averaging filter with a huge sized N x N matrix The properties of the NxN filter are defined using the Define Filter gt NxN command see page 230 Performs a lowpass frequency analyzing filter for noise reduction Noise will be filtered out and strong image contrast will be smoothed The filter matrix is 15x15 A frequency filter with sharp edges will be generated Original ee FFT FFT of the of the original filtered image image The original image has periodical interference superimposed on it which vanishes after lowpass filtering The FFT of the original image indicates the periodical interference via two pronounced maxima arrows After lowpass filtering these maxima have vanished from the FFT image Edge Enhance Performs a log filter that uses local neighborhood to enhance edges Use this filter for enhancing image edges and as a preparatory step toward particle detection The parameters of this parameter can be defined using the Define Filter gt Edge Enhance command see page 234 246 The Process menu Image Calcul
468. se dialog box 2 If the database is protected by a password select the Database gt Administra tion gt Logon command and enter the session password gt The Database gt Administration gt Change Database Password command is available 3 Select the Database gt Administration gt Change Database Password command gt You will receive a warning that the database is lost if you forget the pass word gt The Change Database Password dialog box opens 4 Enter your password in the New Password field e You can enter up to 14 characters 100 Archiving Images Protect with a password Use the Database gt Administration gt Change Database Password command to protect your database with a password It can have up to 14 charac ters For security reasons you have to enter the password twice consecutively a special character appears for each char acter The command is only available when the data base is opened exclu sively e The system is upper and lower case sensitive 5 Retype your password in the Retype password field gt The OK button is activated 6 Confirm the password by clicking OK e In future you can only open the database by using the correct password Change Database Password x Old password TA pa _ New password E Help Cancel ddi Retype password E 101 Acquiring images Acquiring images Acquiring images Acquire Snapshot Your image an
469. sed on the Normal template 2 Open an image database of your choice 3 Arrange your report and database windows such that they re next to each other but not overlapping e To do so use the Window gt Documeni Manager command Keep the Ctrl key depressed and select the Database and Report documents Click the Tile Vertical button and close the document manager Object templates 59 43 This is how to insert records from a database directly into a report se lect all database records you wish to include in the report Drag the im ages selected keeping the left mouse button depressed onto the first record object within the report window The records in the illustration have been selected as thumbnails Report Generator Record objects 4 Insert at least one record object To do so click the Record button located in the Report Objects button bar gt The mouse cursor will change its shape Click on the approximate spot where you want to have the record object placed in the report The position you click on represents the center of the record object You can position the record object after editing gt The Select Object Template list will be opened This is where you find all the record objects currently defined in the current report template If the report template only has one or no format at all for record objects the record object will automatically be inserted in a standard format gt The predefin
470. sert documents gt Documents are saved in the file format in which they are normally stored in your image analysis program This ensures that all documents can be reloaded into the image analysis software If you want to save a document in another format enter the required file name extension in the File name field e g Image bmp to save an image as a bitmap or Tab xls to save a sheet as an Excel file gt The current document title will be suggested as the file name together with the standard file format which would mean for example Tab2 for a sheet 8 Click on the Insert button gt The document will be saved and the insertion mask opened for the next document repeatedly until all documents have been filed 9 If necessary expand the database folder in the tree structure in which you have inserted the documents To do this doubleclick for example on the name of the database folder or click on the plus character in the tree structure nodes gt All inserted documents are listed under the record in the tree structure gt Each type of document is identified by its own icon gt The gallery view shows all records which are below the marked tree entry Your image analysis program creates a thumbnail preview for diagrams The other types of documents are represented by icons ew gt The record name is used to label the thumbnails in the gallery view To change the record name for documents select the Database gt Edit
471. sheet You will find a description of all the measurement parameters in the Select Measure ments dialog box In the measurement parameter list click on the name of a param eter to show the description and a schematic drawing in the dialog box You can print a list of all the measurement parameters from the online help The Radius Mean parameter is explained here as an example The radius mean of an object with any form e g rectangle is calculated in the following way the program calculates the center of the object and lays many straight lines through this point Two intersections of one line with the actual perimeter deliver the values for a radius The mean radius is the mean value of all radii determined this way The mean radius is the circle s radius when dealing with a circle 289 The Select Measure ments dialog box is divided into several sections 1 You will find various types of measurement objects in the tree view 2 The list contains all of the measurement parameters which are at your disposal The measurement parame ters are shown for the types which are marked in the tree view 3 The Selected Measurement list shows which measurement parameters are to be shown in the measure ment display and in the results sheet Types of Measure ment Objects Sequence of active parameters gt otep by step Interactive Image Measurement Measuring Arbitrary Structures Select Measurements I All
472. sing check box in the Process Cycle and or Sequence groups to activate preprocessing for the respective unit Click the Define button to define the respective processing steps The Define Pre Postprocessing dialog box will open Enter the macro commands that you wish to have executed at this point in the process into the Preprocessing steps or Postprocessing steps fields Click the Execute button to have a test run of the steps shown Click the Delete button to delete all steps defined until now 138 Acquiring images Multiple Image Alignment Multiple Image Alignment What is MIA Definition Automatic image acquisition Manual image acqui sition Arranging images Recognizing patterns Correlation With the help of the Multiple Image Alignment MIA command group you can have a maximum of 100 single frames combined into a composite image You have the choice between combining the single frames either manually or automatically whereby the intensities of the single frames will be matched with one another In this way you will obtain a high resolution overview image that you could not create as a single acquisition When certain types of microscope are connected the acquisition of composite images can be automated When you are working without a remote controlled stage you will have to use the manual method Thereby the edges of images that have already been made will be superimposed over the live image to help w
473. single complete file that contains all data necessary to the report 1 Select the File gt Report gt Export RTF command 2 Click the Browse button next to the Destination file field 29 Report Generator Report objects 3 Select the directory for the RTF file in the Save RTF dialog box Enter the name of the RTF file into the File name field Click the Save button to return to the Export RTF dialog box gt The complete path and file name of the RTF file is now located in the Desti nation file field Note that the RTF file has not yet been saved 4 Determine the resolution of the images in the RTF file and thus the file size of the RTF file in the Reduce image data group If you are planning on sending someone the report by e mail then it makes sense to keep file size as small as possible Select the Use JPEG compression check box 5 Enter 60 into the quality field This quality value determines the degree to which images are compressed low percentages mean a correspondingly high degree of compression gt The JPEG compression reduces the file size of an image but also gener ates typical image artifacts The more you compress an image the greater the loss in image quality JPEG artifacts are generally not visible in a printout at 60 6 Initiate exporting by clicking on OK gt The resulting RTF file you can now e g load in MS Word or send to someone by e mail The layout of the report remains completel
474. sions Background Subtraction i Constant ROI e Image OF Cancel Help Select the method to be used for correction in the Background Subtraction group Select the Constant option to subtract a fixed value from all pixel intensities Enter the value for the constant in the edit field Select the RO option to have the value that is to be used for the subtraction calcu lated from a previously defined ROI The mean intensity value will then be used Select the image on which the relevant ROI has been defined from the Select ROI from Image list If more than one ROI has been defined in this image select the ROI to be used from the Select RO list The RO option is only available when at least one ROI has been defined with the Image gt Define ROIs command Select the Image option to correct the background by subtracting another image Select the correction image from the Select Background Image list After setting the method make sure to define the subset to be corrected using the Dimensions tab 223 The Process menu Dimensions Dimensions Selects the subset of the data set for the correction Background Background Settings Dimensions Color Channels Layers From DEE to 25 El step hi All Mime Brames Hon E e Else E All copy non processed data OF Cancel Help This tab shows three groups Color Channels Z Layers and Time Frames Depending on the
475. sitions Positioning aids Status bar Ruler Grid Align button bar The buttons of the but ton bar are only active if numerous objects have been selected Mirroring Aligning overlap ping objects Report Generator Report objects e Press the Shift key when you move the corner point of an object via mouse if you want to keep the length width ratio of the object frame the same e Press the Ctrl key to keep the center point constant when altering size of the object frame e Select the object Move the object with the mouse by keeping the left mouse button depressed e You can also select and move more than one object at the same time This means that the objects positions relative to one another remain the same e To move a copy ofthe object and not the object itself press the Ctrl key while moving the object Use the mouse for an initial and approximate positioning of objects Select one or more objects You can pick up the selected object s by leftclicking and then keeping the left mouse button depressed you move the mouse to move the object s to where you wish to have it them Use the grid alignment to be able to position objects fast e Select one or more objects and use the arrow keys on the keyboard to move the object in the direction desired Press the Shift key to increase the distance moved each time you press a key from 0 1 mm to 0 5 mm e Select one or more objects and use one of the bu
476. slide control on the right border of the dialog box or the buttons shown here on the left When you use the buttons the thicker arrows will move the stage Upwards or downwards in steps of 100 um and the thinner arrows in steps of 10 um The current Z position will be displayed on the bottom left in the Stage Navigator win dow 330 The Stage Navigator How to acquire an overview image Note GJ The buttons in the dialog box Send Overview Image Zoom In Zoom Out Scan m x n images Scan Interest Area Properties E Help E k e 9 Aerea D ga g Add Overview Image Select Image Mode Save Folder Open Folder New Folder The Send Overview Image and Add Overview Image buttons are only of importance for users of the telePresence add in or the Intrascope software package For this reason they will not be explained further in this documentation How to acquire an overview image When working with the Stage Navigator the first step you take is to acquire or load an overview image When you acquire an overview image you first have to decide if it is going to be compiled as one image or made up of separate images In doing so take the following advantages and disadvantages into consideration Should the overview image be made up of one image or of separate images Option Create one image from thumbnails All the images will be saved with a reduced resolution in a single TIF file You can use the ove
477. sponding curve will be instantly displayed in the graph window 2 Position the red line where you wish to measure gray value intensity and then leftclick The line will be drawn into the overlay 3 If you drag the mouse another red line will appear and can be placed as wanted 4 Terminate the measurement with a rightclick The line profiles of all color channels will be drawn into a separate graph for each of the lines drawn into the image overlay Intensity Profile gt Arbitrary Creates a graph of the gray values along an arbitrary line How to measure an arbitrary intensity profile 1 Select the Measure gt Intensity Profile gt Arbitrary command 2 Leftclick on the starting position of the line to be drawn The corresponding curve is instantly displayed in the graph window 3 Move the mouse toward the end position A blue line marks the current length and orientation of the line being drawn At each end an orthogonal red line is displayed 4 Leftclick on the end position 5 Continue with drawing another line or terminate the measurement with a right click Intensity Profile gt Average Creates a graph of the gray values along a horizontal line How to measure an arbitrary intensity profile 1 Select the Measure gt Intensity Profile gt Average command 2 Leftclick on the starting position of the line to be drawn A rectangle with a central horizontal arrow is displayed 3 Move the mouse to position
478. standard wavelength of 540 nm will be entered Multiple Fluorescences 114 127 Acquiring images Extended Focal Imaging 8 Close the Color Channel Properties dialog box with OK to return to the Acquire Z stack dialog box 9 Click the Z Distance button to have an optimal number of acquisitions calcu lated for the Z stack taking the Z range that has been set and the objective in use into consideration Note Please note that you can only use image stacks with an optimal Z Spacing if you want to make a 3D reconstruction e g with the VoxelViewer add in or if you want to employ deconvolution 10 Click the Acquire button to start the acquisition Options for Z stacks of multi channel fluorescent images In the Multi Channel Stack group select the Acquire check box Only then will the defined fluorescences be offered in the Color Channel picklist in the Color Channel Properties dialog box Select the Z first option to bring a mirror cube into the light path and take all the images of this fluorescence at the different Z positions Then the next mirror cube will be brought into the light path and the corresponding fluorescence images will again be acquired at the different Z positions With this option every mirror cube will be loaded once only This option is preset The acquisition of a multi channel Z stack takes place much more quickly when you use the Z first option than when you use the Channel first option Select
479. structure of the multi channel data set one or more of these groups will be active Select the check box prior to the name of a color channel in the Color Channels group to select this channel for background subtraction Clear the check box to have the color channel exempted from the correction In the Z Layers and or Time Frames group use the fields From to and Step to define the frames to be considered Click on the All button to undo any constraint and select all frames of the appropriate group General Information on Filter Operations A Filter modifies the intensity of each pixel according to the intensity of neighboring pixels Why filter images Filters can be used to prepare for the actual image analysis for example for the correction of image errors resulting from an acquisition Filter operations have the following possible applications e correction of image interference due to statistical noise e reduction of acquisition errors e g CCD cameras sometimes provide errone ous gray values for individual pixels e restoration of poorly focussed images e suppression or accentuation of minute image detail e accentuation of basic structures such as gray value edges Many filters are convolution filters A convolution filter is defined using a filter matrix A matrix can be described as a geometrical array of positive or negative whole numbers that serve as a mask placed over individual pixels Any pixel that is to be 224
480. sword for the software version The commands are deactivated for all other users The insertion and loading of data and all queries for a database with session password is also permitted for every user who can open the database The following commands are deactivated when the database is opened without the session password e Export record files e Database gt View gt Arrange Fields e All commands from the Database gt Administration menu apart from the Logon command Warning amp Make a note of your database password You can limit the igiete jeeerds number of records Na limit which may be deleted in any one process in the Absolute Database gt Administra i percent tion gt Database Settings dialog box Step by step How to define a session password 1 Select the Database gt Administration gt Change Session Password com mand 2 Enter a password in the Password field Remember which characters are upper lower case 3 Retype the password in the Confirm Password field 4 Confirm the session password by clicking OK 5 Select the Database gt Administration gt Logoff command gt The commands for changing the database structure are now gray 6 Select the Database gt Administration gt Logon command if you want to release disabled commands How to assign a password to the database 1 Open the database exclusively e Todo so mark the Exclusive check box in the Open Databa
481. symbols 9 Copy the image object by pressing the Ctrl key each time you move the new image object to its intended position 10 Click the Save Page Template button and enter a new name e g ImageGal lery in the field 11 Confirm via OK to have the new template page added to the report template gt The new template page called ImageGallery will now also be available to you when you create a new report based on the current report template Saving report templates 1 Press F8 to open the Preferences dialog box and select the Report tab gt You ll find the standard path name for saving reports and report templates in the Directories group gt Your image analysis program will propose the Report directory for saving report templates 2 Enter the path name you want to be using for saving your templates into the User templates field e Ifthe report directory is not yet existent click the button next to the User templates field Click the Create New Folder button in the Select Directory dialog box to set up the directory 3 Confirm the new template path via OK 58 Report Generator Report templates 4 Click the Save button in the Standard or Report button bars gt When you save a report template for the first time the Save Report Template dialog box will be opened automatically The preset path is the path you entered into the preferences in the Report tab for saving reports 5 Enter a name for your
482. t C iH P Shadow Unit conversion M Adjust unit rit Auto Cancel Apply Help Determine the appearance of the horizontal and vertical scale bars in the Scale bar styles group The scale bar appearance selected applies to all three application areas i e viewport clipboard and printer Select one of three scale bar types from the Type list for the horizontal and vertical scale bars Select a small medium or large scale bar width from the Width list This width value is not an absolute length value rather it indicates the maximum permissible scale bar width a small scale bar select smal cannot be wider than 1 4 of the image width a medium scale bar select medium not greater than 1 3 and a large select large cannot be longer than 1 2 x image width The calibration length of the scale bar is computed based on the current image cali bration possible values are 10 um 20 um 50 um 100 um 200 um 500 um 1 um etc The height of the scale bar frame and lettering size are not affected by the Width selected here The size of the scale bar with regard to viewport display will be in rela tion to the size of the viewport Select a white yellow or a clear frame transparent from the Frame list Select the Transparent entry to ensure that the image information covered up by the scale bar is kept to a minimum The legibility of the scale bar may however suffer Set the color in which scale bar label is shown in the Te
483. t Select and Move tab has not been object selected Field object On the General tab the text object re only available in edit ob ceives the type AutoText ject mode 48 Report Generator Report objects otep by step Inserting text objects 1 Enlarge the display of a report page so that the text is legible Select an appropriate zoom factor from the Set Zoom list in the button bar of the report window e g 100 or enter the desired zoom factor directly 2 Click the Text button in the Report Objects button bar to insert a new text object into the report gt The mouse cursor will change shape and appear as a cross shaped symbol 3 Now define a rectangular area where you plan on inserting the text The text has to appear within the area you have defined to be displayed and printed out gt The new text object has now been selected and the word Text will appear within it 4 Click the Properties button in the Report Objects button bar to format the text within the text object 5 Select the font and font size in the Font tab Select the Italic format for the text You can also decide on what color you want the text to be 6 Click the Apply button to try out the text formats selected in the report 7 Decide on what kind of text to use in the Text tab e Select the Multiple Lines check box if the image comment is to have more than one line The Word Break check box will now become available e Select t
484. t the more noticeably will the morphology of the object within the image be altered Shot Noise will not be removed rather these noise pixels will simply be broadened and their intensity will be adapted to the image s average intensity The extent to which the NxN filter broadens edges will depend largely on the parameters set The Median Rank and Sigma filters reduce noise without broadening the rectangle The effect of both the Rank and Sigma filters is however highly dependent on the parameters set The Median and Rank filters eliminate Shot Noise completely Filter parameters There is a distinction between predefined and user definable filters Parameters of definable filters can be set thus allowing you to adjust the filter s effect Definable filters are located in the Define Filter submenu as well as in the Filter submenu The command in Define Filter will open a dialog box for the setting of filter parameters When you select a filter from the Filter submenu the filter will be applied using the parameters as they were last set How to filter an image 1 Load the image desired into the source image buffer 2 Select the destination image buffer 3 Open the Process gt Define Filter menu 4 Select the filter type desired A dialog box for setting filter parameters will be opened 5 Set the requisite parameters 6 Click the Execute button The dialog box will be closed The filtered image will be written into the d
485. t Derivation and 2nd Derivation The 1st Derivation displays the local maximums and minimums through the zero crossing Use the Smooth list to define to what degree the function displayed in your diagram is to be smoothed The higher the value you enter here the smoother the curve will be displayed Smoothing is useful with regard to derivatives because a derivative curve often has irregularities that make it difficult to make out the main path of the curve Smoothing will affect the display of the following functions Smoothing 1st Derivation and 2nd Derivation A Histogram is always displayed without smoothing Select a color for your active phase from the Color list This color will be displayed in the diagram s color bar indicating where this phase is The gray values belonging to this phase can then be displayed in this color in your image The color you select here will also be used for phase color coding Measure gt Phase Color Coding command Use the options located in the Preview group to keep an eye on the effect the thresh olds you select have on the image In preview whether you have set a frame or not your selections will be applied to the whole image Select the None option to turn off the preview None of the phases defined will be displayed in color within the image Select Current to have the phase s currently active gray values displayed in color in your image The preview display will be continuously up
486. t are defined in the active image database 4 Select any of the database fields and confirm via OK e When defining an object template you don t need to worry about selecting any particular database field What you are doing now is simply defining the layout You can then later on apply this layout to any database field you like gt No object templates have been defined for the Normal template which means that the standard field object will be automatically inserted If you have already defined object templates for fields the Select Field dialog box will be opened Ely 5 Click the Edit Object Template button in the report window button bar to switch Be over to the object template editing mode in this case for the field object e Please note that you are already in the object template editing mode You can edit object templates in several multi level steps Now define a field ob ject template gt You can now select the separate elements of the field object and then edit them gt All other objects even those of the active record object will only be shown for reference purposes and thus appear crosshatched These can no longer be selected 6 Now you edit the field object e The field name is an essential part of the field object You can omit the field name when defining an object template for a field object but you cannot replace it with a fixed text 60 Report Generator Planning report templates RE 7 C
487. t button located in the Report Objects button bar and pull open the sheet object 52 Report Generator Report objects 4 Load the sheet you want to insert into a report 5 Select the sheet object on the report page and click the right mouse button 6 Select the Select Sheet command from the context menu to open a list of all the sheets loaded and to insert the desired sheet Using sheets in a record object 1 Open an image database and insert a sheet 2 Generate a new report based on the Normal template 3 Use the Record button in the Report Objects button bar to insert an existing record object 4 Click the Edit Object Template in the report window button bar to switch over to ale the edit object mode 5 Select the image object and click the Sheet button located in the Report Objects im button bar and pull open the sheet object 6 Click the Properties button in the Report Objects button bar 7 On the Document Link tab enter in the File name field to have the first attached sheet inserted together with the record Determine the number of rows and columns which are to be inserted into the report The heading of a measurement sheet usually has the sheet cell number O Please note If you use 0 as the amount of rows or columns your image analysis program inserts exactly that part of the sheet which was active during the inser tion of the sheets into the database 8 Click OK to close the dialog box
488. t hand corner in order to invert the text or image Objects that overlap each other may partially or completely cover each other up The object that was inserted last is generally in the foreground You can however later alter the order of the objects Individual objects can then be partially or completely placed in the background 32 Report Generator Report objects You will find the Order and Group button bar in the Button Bars menu located in the report window s context menu otep by step Insert report object 1 Load an already existing report or create a new one 2 Click on any position on the report page to activate the report window gt The Report Objects button bar is only displayed if the report window is active You will find the button bar on the right edge of the user interface by default e The first button Select Objects of the Report Objects button bar is engaged by default In this mode you can select and edit as many objects on the report page as you like 3 To draw a new graphic object click the corresponding button in the Report E Object button bar e g the Rectangle button gt Within the report window the mouse cursor will now change shape into a cross shaped symbol indicating that you may now define the object Please note that no other action is allowed in the report as long as your image analysis program is waiting for the definition of an object You will notice this mode by the fact t
489. tains all user defined image formats as well The Clipping border Pixel group is for defining your own image formats You can tell an image format is a user defined one because free will appear next to that entry e g free 1000x1000 pixels Select either a predefined or user defined image format from this list The format you select will be at the top of the list the next time you access this tab Select the Multi Channel check box to automatically acquire images in a multi dimen sional image format You will recognize the file format by the image icon in the image manager For multi dimensional image formats an additional Dimensions tab will appear in the Image Information dialog box A graphic of the clipped borders is displayed within the Active area field and the dimensions of these clipped borders are displayed in the Clipping border Pixel group fields Binning The CCD chip is composed of many light sensitive units pixels These pixels can be read out individually binning 1x1 or the signal of neighboring pixels can be combined electronically on the CCD chip binning gt 1x1 Binning reduces the spatial resolution but increases the sensitivity and thus reduces the exposure time required for a good signal to noise ratio It further reduces the amount of data and 110 Acquiring images Set Input Set Input consequently increases the readout speed Therefore binning is recommended if weak signals have
490. te and VVindow buttons as well as the Preview check box are only avail able as long as the source image buffer contains an image As far as false color images are concerned the shading correction is only available if you have selected the Special gt Preferences gt Image gt False Color Images Click the File button to open the standard dialog box for the opening and loading of files Files will be saved using the SHC format Shading Correction Corrects inhomogeneous lighting conditions Note When working with multiple images with varying shading you must disable the Define Shading Correction gt Save shading image check box This means that a separate shading image will be calculated for each image If however you are working with image series with images that have the same shading you may select this check box The same shading image will then be used for all the images This makes shading correction much quicker When the Shading Correction command is used on a multi channel data set the Shading Correction gt Dimensions dialog box will be opened Use this dialog box to select on which parts of the data set the shading correction shall be performed 222 The Process menu Background Subtraction Background Subtraction Calculates the background subtraction This dialog box offers two tabs Background Settings Defines a method for background subtraction Background Ed Background Settings Dimen
491. template Your own report templates will now be included in the User Templates tab in the New Report dialog box 14 Now insert an image from the database into the adapted record object gt The image and the desired field entries as well will be included in the report Text objects Background Information Text objects You cannot enter text into a report directly Before you can insert text you have to reserve space for it The area reserved for text is defined by a text object Text formatting is determined by the text object s properties If you e g use different font sizes in your report you have to define a separate text object for each different text format Text entry mode When in text entry mode the pointer is positioned within a text object allowing you to insert text The text entry mode is active directly after a text object has been inserted If you want to edit texts in already existing text objects doubleclick the text object using the left mouse key Doubleclicking does not put you in the text entry mode but instead opens the dialog box for text properties The Text tab also allows you to enter or edit text Left click on an arbitrary position outside of the text object in order to end the text entry mode and to switch to layout mode Warning The text may have a somewhat different format in the text entry mode than in the lt P actual report Layout mode You can only edit the text object in the layout mode n
492. tents of this object will field be automatically replaced with the field name selected You can replace the field name by a definite text To do so dou bleclick the field object in the edit object mode The field name can be edited in the Record Fields tab Text vari Placeholder for the The contents of the database field is different for each image and able contents of the respec is automatically filled out anytime a record object is inserted into a tive database field i e report from an image database The lt Calibration Unit gt field entry the field entry will be replaced by e g mm if the image has been calibrated in This text variable ap mm pixel seat eee ast You can format this text object just like any other Of especial note ets When the field ob is that you can permit multiline text thus allowing space for data ject is inserted the field base fields such as the Memo type name appears here Be genic layout function such as frames and rectangles for use as colored highlighting for text object image ob layout function e g An image object defined in a field object cannot be used later on ject pictograms represent asa placeholder for images You cannot insert a new image into ing the various data this image object in this report base fields To save space be sure that you always insert images as links If you don t images will be re copied each time you insert a field ob ject into a report
493. than one page template You ll find all template pages that are defined in the current report template listed within the dialog box Depending on the report template you ll have very different page layouts available 8 Select the desired page template and confirm via OK gt You ll now see the newly added page within the report window The selected report template page influences the appearance of the page gt The status bar shows the current page and the total number of pages the report has gt The buttons for paging backwards or forwards are now available 28 Report Generator Saving Exporting report Saving a report 1 Press F8 to open the Preferences dialog box and select the Report tab gt You ll find the standard path for saving reports and templates in the Direc tories group gt Your image analysis program will propose a standard directory for saving reports the Report directory is a subdirectory of the root directory 2 Enter the path name where you want to save your future reports into the Reports field e g C Reports ProjectXY Z e If the report directory does not yet exist click the button next to the Re ports field Click the Create New Folder button in the Select Directory dia log box to set up the directory 3 Confirm the new report path via OK 4 Click the Save button in the Standard or Report button bars gt If you are saving the report for the first time the Save Report Document dial
494. the clipboard into the active image Inserted objects can be repositioned right away however because when they are inserted they appear selected Note The Paste Object function do not make use of the usual Windows clipboard It has its own memory for data you copy Please keep in mind that you cannot copy overlay objects via Ctrl V Bring to Front Brings the selected objects to the front Click the Bring to Front button to move the selected objects to the topmost position When several objects overlap only the topmost one is shown in the overlapping region Send to Back Sends the selected objects to the back Click the Send to Back button to move the selected objects to the bottom position When several objects overlap only the topmost one is shown in the overlapping region Bring Forward Brings the selected objects forward Click the Bring Forward button to move the selected overlay objects one level in the top direction Send Backward Sends the selected objects backward Click the Send Backward button to move the selected objects one level in the bottom direction Text Creates a text object in the overlay Move the mouse to position the text object on the image Move the mouse with the left key depressed to change the size of the text box Rightclick to fix size and posi tion of the box The Object Properties dialog box with the tabs Text and Font will be opened Press Shift and click the Text but
495. the rectangle Move the mouse with the left key depressed to change the size of the rectangle 4 Rightclick to confirm the measurement 5 Continue with drawing another rectangle or terminate the command by pressing Esc 296 The Measure menu ROI Area Measures the selected ROI areas 1 If you have not defined any ROI on the active image the Define ROIs dialog box will open 2 Close the Define ROIs dialog box when the definition of the ROIs has been finished If you have already plotted ROIs with the Image gt Define ROIs command The image with the defined ROI is brought to the foreground A small white square is attached to the mouse pointer 3 Successively leftclick the ROIs you want to measure 4 Click the right mouse button when you have finished selecting ROIs The last two steps are only necessary if the ROIs were defined prior to the use of the Measure gt ROI gt Area command by using the Image gt Define ROIs command The program calculates the area of the selected ROIs A measurement sheet displaying the results opens ROI number and area are displayed for each selected ROI The area unit depends on the image calibration The outline of a ROI is counted as part of its area 5 Click the x button in the title bar to close the measurement sheet You can save the measurement sheet as a SFS file on your hard disk Perimeter Measures the selected ROI perimeters 1 If you have not defined any ROI on the ac
496. the results opens ROI number and average intensity are displayed for each selected ROI 3 Click the x button in the title bar to close the measurement sheet You can save the measurement sheet as a SFS file on your hard disk Average Gray Value Measures the average gray value of all pixels within the selected ROI 1 If you have not defined any ROI on the active image the Define ROIs dialog box opens 2 Close the Define ROIs dialog box when the definition of the ROIs has been finished The image with the defined ROI is brought to the foreground A small white square is attached to the mouse pointer The program calculates the average intensity of the defined ROIs The pixels of the ROI outline are taken into account thereby A measurement sheet displaying the results opens ROI number and average gray value are displayed for each selected ROI Click the x button in the title bar to close the measurement sheet You can save the measurement sheet as a SFS file on your hard disk 3 w Kinetic Calculates the kinetics Use this command to quantify the changes of fluorescence intensity within ROIs over time or within a Z stack and to display the results graphically This command is available for the following types of multi dimensional images Z stacks multi channel Z stacks single color time lapse images multi channel time lapse images single color Z stack in time lapse images and multi channel Z stack in time lapse imag
497. the same Where the definition of the maximum size is concerned only one value will be deci sive the other value will be scaled down to keep the width height ratio the same If the Interest area has been interactively set in height and width at more than the max imum allowed dimensions the value that exceeds these dimensions by the most will be taken as the maximum value The other value will be proportionally reduced to keep the width height ratio the same Select this check box to immediately load the currently selected image segment in the Interest area into the active image buffer and not after you have doubleclicked it This check box applies only to the Interest Area Mode This option is only of importance when you have set an image overlap In this case the transition will be optimally displayed if you select this check box The separate images that make up the overview image are set out seamlessly Every inexactness of your stage will inevitably lead to a misalignment of the separate images i e the images look as if they have been tiled In this case enter an image overlap in percent and repeat the acquisition For the display of the overlapping area proportional image information from both images will be used and merged An alignment of the images by means of a correlation function as for instance with the MIA Multiple Image Alignment add In will not be employed Double lines in the grid indicate that an overlappin
498. ther applications The Red Green and Blue scrollbars will be available if one or more rows have been selected Use these scrollbars to define specific value s for all selected rows Please note that only the fields corresponding to the relevant color are changed All changes will be displayed on the monitor immediately Click the File button to open the standard dialog box for the opening and loading of files Files will be saved using the LUT format The LUT format is the only format Imaging C functions can use for altering an image s lookup table As becomes neces sary you really should save LUT s originally saved in the polygon or formula format as LUT files as well When you click the Linear button the values of the linear standard LUT will be inserted into the sheet If you click the Interval button the Set Thresholds dialog box will be opened Here is where you can define a gray value interval for the definition of LUT values When you exit the Set Threshold dialog box via OK the corresponding interval in the sheet will be selected When you click the Fill down button all values of the first selected row will be trans ferred to the other selected rows Using this command you can enter new values into one row and then have them copied to successive rows Click the Sheet button to export the active LUT to a sheet document The columns of the sheet are Index Red Green and Blue Edit LUT Polygon Defines the LUT
499. time gt The overview image will be displayed Deleting images Deleting a newly created overview image You can delete a newly created i e not yet saved overview image by clicking the New Folder button Then a corresponding warning message will appear Answer it with Yes and the temporary file in the lt Installation folder gt StageNavigator folder will be deleted and you will see the Stage Navigator s start screen display again Deleting complete folders Use the Windows Explorer to delete saved folders Deleting individual images from an overview image If you chose the Get separate images of high quality option when making the acqui sition you can delete individual images from an overview image In this way you can reduce the amount of saved data 1 Make sure that you are in the Single Image Mode The Modi of the Stage Nav igator on page 336 2 Click the image you want to delete then click the right mouse button 3 In the context menu select the Remove image option gt In this way the selected image will be deleted and you will see a gray area in the overview image gt The image is then irrecoverably lost The tif file will also be deleted from the hard disk The Modi of the Stage Navigator The Stage Navigator comes with four different modi that are tailored to support the basic operations Two modi are designed for image acquisition Therefore in these modi the yellow camera area is always visible
500. ting Method Formula Description Linear Ip A X r The background is approximated by a straight line Power Ip A X A power law is used Exponential Ip A e An exponential law is used 2 point linear Ip A X r The line is defined by the intensity values at two X positions Use the Reference group to define the X range that is to be used for the background fit The X range will be defined by a lower and an upper limit Click the Set Lower button to change the position of the first limit Move the line cursor to the X value you want and click the left mouse button to have the back ground recalculated and to return to the dialog box Click on the Set Upper button to change the position of the second limit The upper limit should be set directly before the interesting structure Move the line cursor to the X value you want and click the left mouse button to have the background recalcu lated and to return to the dialog box Calculates the inverse graph Each channel of the graph is replaced by its inverted value The new graph is written to the Destination buffer Calculates the absolute graph Each channel with a negative entry is replaced by the absolute of this entry The new graph is written to the Destination buffer Performs a FFT Performs an envelope smoothing Use the Envelope filter to remove high frequency noise from the graph The Envelope filter searches for the local maximum and minimum and calculates the average S
501. ting the derivation in the Smoothing width field The Smoothing width can be a number from 1 to 5 inclu sive Select the Absolute derivation check box to use the absolute value of the derivation as the result The new graph will be written into the destination buffer 2 Derivation Calculates the second derivation 2nd Difference Smoothing width Eo B 3 0 Absolute derivation Cancel Help 321 Graph Background Subtraction Enter the desired width of the range you want to use for calculating the second deri vation in the Smoothing width field The Smoothing width may be a number from 1 to 5 including Select the Absolute derivation check box to use the absolute value of the derivative as result The new graph is written to the Destination buffer Background Subtraction Performs a background subtraction Select the Graph gt Calculation gt Background Subtraction command to remove one of the graph s specific structures from the background The background intensity in this case will be calculated from the course of the curve for this specific structure Before you use this command change the display of the graph so that the interesting structure is visible For calculation the background a suitable X range before the specific structure must also be displayed Adjust the display so that the specific struc ture is approximately located in the middle of the graph document Make sure that
502. tion X2 will not be horizontally aligned but rather diagonally displaced When acquiring an image series for an automatic image alignment the stage has to be moved in both directions X and Y directions to guar antee that the images are positioned side by side e Calibration has to be performed only once but for each magnification and each logical input channel It is necessary to calibrate every magnification be cause changing the magnification may cause a different orientation of the im age e You have to recalibrate the system every time you change the position of the camera with respect to the microscope stage e There are some electron microscopes where you can realize a magnification us ing different imaging modes e g a low magnification can also be reached by switching to an overview mode Note that one calibration cannot be valid for the two magnifications because the image rotation will differ according to the imag ing mode For calibration the application acquires an image at the current stage position XO then moves the stage in the X direction and acquires a new image at the new stage position X1 The images at the positions X1 and XO are compared in order to get the displacement of the images The same measurement is performed for moving the stage in the Y direction The result of the calibration is saved for the present magni fication and the active logical input channel When acquiring the image series for the automatic i
503. tion currently set Each magnification requires its own calibration Using the Preprocessing list you determine whether the preprocessing steps should be performed for every image of the series Always only for the first image Only first image or for no image of the series Never The pre and postprocessing steps refer to the macro commands defined on the Macro tab for the current logical input channel Using the Postprocessing list you determine whether the postprocessing steps should be performed for every image of the series Always only for the last image Only last image or for no image of the series Never The fields X and Y display the present stage position in nanometers In the Shift type group you specify the shift device used for acquiring different object regions If you select the Specimen shift option the object will be moved while the camera stays fixed This shift type requires a microscope stage with a remote control The Image option is not relevant for your image analysis program During the calibration routine the transformation matrix is entered into the sheet displayed in the Calibration data group The calibration data deliver the actual stage movement to acquire a horizontal image shift In the Movements column you can follow the movements of the stage during calibra tion After starting calibration the stage is shifted step by step in the X then in the Y direction When calibration has been successfu
504. titled Current You can always alter this selection without having to repeat a measurement 4 Let s assume the Minimum and Maximum statistical values are not to be shown In this case in the Define Statistics dialog box select the Minimum and Maximum parameters and click the lt lt Remove button Click OK to close the dialog box gt The minimal and maximum values have disappeared from the measure ment display 5 Click on the Create Measurement Sheet button located in the measurement button bar 6 Mark the Generate statistics of the sheet s check box located in the Create Measurement Sheet dialog box and confirm with OK gt A sheet with the statistic parameters is created and shown in addition to the measurement sheet All of the measured values are acquired to the 287 Interactive Image Measurement Measuring Arbitrary Structures sheet A separate column is created for each value The statistic parame ters shown correspond to the parameters which you selected in the Define Statistics dialog box In addition to the i measurement sheets i Measurements of Image Pel Fa you can also export a p 20 Object 2D Object sheet with measure Area Perimeter EE a rm ment statistics Sa tarien 2D Object 2D Object E eee Base Unit pm pm 7983 72 4482 64 3718 14 1AA Ad 3677 28 Standard Deviation he ga 01 09 A Measuring Arbitrary Structures Background Information ably select an object
505. tive image the Define ROIs dialog box opens 2 Close the Define ROIs dialog box when the definition of the ROIs has been finished If you have already plotted ROIs with the Image gt Define ROIs command The image with the defined ROI is brought to the foreground A small white square is attached to the mouse pointer 3 Successively leftclick the ROIs you want to measure 4 Click the right mouse button when finishing selecting ROIs The last two steps are only necessary if the ROIs were defined prior to the use of the Measure gt ROI gt Perimeter command by using the Image gt Define ROIs command The program calculates the perimeter of the selected ROIs A measurement sheet displaying the results opens ROI number and perimeter are displayed for each selected ROI The perimeter unit depends on the image calibration Click the x button in the title bar to close the measurement sheet You can save the measurement sheet as a SFS file on your hard disk 5 w Average Intensity Measures the average intensity of all pixels within the selected ROI 1 If you have not defined any ROI on the active image the Define ROIs dialog box opens 2 Close the Define ROIs dialog box when the definition of the ROIs has been finished The program calculates the average intensity of the defined ROIs The pixels of 297 The Measure menu Average Gray Value the ROI outline are taken into account thereby A measurement sheet displaying
506. tive zero level Frecute Source for shading image Polynomial fit f Source 1 Source 2 T Preview Save shading image Window IY Automatic scaling Overflow 0 1 z Lower limit fo Upper limit E10 z i File Nell average filter iF Size ler Iterations Help PERCE In the Assumed Deterioration group you can define the appropriate correction method for the assumed cause of shading With the Multiplicative option shading correction will divide the current image by a correction image A new group will appear in the lower center of the dialog box Select the Automatic scaling check box to have an image resulting from shading correction automatically scaled Use the Overflow field to determine what percentage of all gray values are to be ignored in histogram calculation If the Automatic scaling check box is clear minimum and maximum gray values of the resulting image can be entered manually into the Lower and Upper limit fields If the option Additive has been selected shading correction will subtract the correc tion image from the current image A new group will appear in the lower center of the dialog box Select the Invert check box to have the resulting image inverted after filtering The value in the Offset field determines the gray value to be added to the resulting image In the Use for Correction group you determine which image is to be used for correc tion of the original ima
507. to accept the new scale As long as the button remains depressed the line cursors will remain visible and can be moved again by using the mouse cursor Y Scale The second group sets the scale of the Y axis of the graph Zoom Out gt lt Click this button to increase the displayed X range i e to compress the graph in the 308 Graph Graph Zoom Up Zoom Down Scale Y Max Y Auto Max Y Define Display Area Default size Log X Log Y Delete All Labels Window Mode ZIKA E hy a ee x Z E Se Overlay Functions Overlay Mode Click this button to decrease the displayed Y range i e to stretch the graph in the Y direction Click this button to increase the displayed Y range i e to compress the graph in the Y direction To scale the Y range up or down keep the corresponding button pressed The command will be repeated automatically at short intervals While working with these two scaling operations the initial Y value remains unchanged Click this button to enlarge any Y segment of the displayed graph to full axis size In the displayed graph click on the borders of the desired Y value segment Y range The first clicking produces a blue line cursor the second a green one Both diagram cursors can be moved with the depressed mouse button The current Y position is continuously shown in the status bar To accept the new scale press the right mouse button As long as the button
508. to set the starting point of the first segment 3 Move the mouse to draw the segment and click its endpoint This endpoint will be interpreted as the starting point of the next segment 4 Repeat this for any further segment of your polygon line 5 Click the right mouse button when the definition of the polygon line has been finished If the polyline is made of more than two segments your image analysis program will close it automatically A closed region has been defined on the image You return to the dialog box The new ROI is immediately added to the Active ROIs list Interpolating Polygon A ROI defined by the Interpolating Polygon drawing method is an interpolating Spline curve This is how you define a ROI with the Interpolating Polygon method 1 Click the Interpolating Polygon button located in the Tools group The mouse pointer switches to the image 2 Click the left mouse button to set points You need at least four points to define an interpolating polyline 3 Click the right mouse button when the definition of the polygon line has been finished Your image analysis program calculates and draws the best fit line through the points you have set A closed region has been defined on the image You return to the dialog box The new ROI is immediately added to the Active ROIs list Freehand polygon A ROI defined by the Freehand Polygon drawing method is defined by a freely drawn line This is how you define a ROI with the
509. to use it for the image acquisition and confirm with OK Click the Remove fluorescence button to delete the marked fluorescence from the picklist By clicking the Move fluorescence up or Move fluorescence down button you move the selected fluorescence one position upwards or downwards in the picklist The order of the acquisition of the individual images reflects the order of the fluores cences in the picklist You can influence this order as described above or sort the acquired images for example according to the exposure time To do so click on the header of the Exposure time column in the list Using these two buttons you can acquire an image from a single fluorescence 121 Acquiring images Define Fluorescence Acquisition Preview DE Snapshot kd Preview By clicking the Preview button you will begin the preview of the fluorescence that has been selected in the Fluorescence list Use this opportunity to adjust the acquisition position and to define the focus level Your image analysis program asks you to carry out the settings which have been defined for this fluorescence Confirm each time with OK or click on Cancel so as to stop the image acquisition The live acquisition subsequently appears in the image window Now you can set the exposure time in the Camera settings group in the Exposure time gt Preview field Note When you have marked the Link exposure times check box in the Camera settings group the exposure tim
510. tomer is displayed in the form view for all types of data When an image is inserted the customer should be displayed as information in the insertion mask 1 Select the Database gt View gt Arrange Fields command gt You will find in the View Type list in the Arrange Fields dialog box all the contexts in which database fields can be displayed e Remember that user defined database fields are not automatically visible in every view but must be explicitly arranged One exception is the inser tion mask User defined database fields are adopted automatically into the insertion mask Remember that organizational fields only appear automatically in the in sertion mask when a new database folder is created If the information from the organizational fields is also to be displayed when inserting imag es graphs and documents into a database folder then you must arrange the organizational fields explicitly for the insertion mask 2 Extend the display of Form View To do this doubleclick for example on the Form View entry or click on the plus character in front of it 82 Archiving Images Work in the database window e You can adopt different database fields into the form view for the lt Folder names gt Image Graph Document and Data data types The lt Folder name gt is different for each database and reflects the organi zational ID of your database 3 Mark the database folder In a project database the database folder
511. ton at the same time to insert a single line text object Single line text objects can be rotated at any angle Use Object Properties gt Font tab to define the angle 176 The Image menu Overlay Bar Aligning text Autosize bound Word Wrap Zoom with image Select the Use as default check box on the Font tab to save the current settings as your default setting The next text object you wish to insert will be inserted according to these settings Text tab Text Font Colors and Lines lt New text gt Text alignment Left D Center vertically P Autosize bound Word wrap I Zoom with image oro ao Use the text entry field The text you enter will always be automatically wordwrapped independent of how it is displayed in the overlay Please note that the text will gener ally be wordwrapped in the overlay somewhat differently as in the text entry field If the text you enter does not fit into the text object some of the text will not be shown within the image overlay You can then simply adjust the size of the text object accordingly You decide how you want the text to be aligned within the text object left center or right This check box can only be selected for single line texts When you select the check box an automatic frame the exact size of the text will appear around the text object The size of the text object will automatically be adjusted if you subsequently add or
512. try may only be an entry from the picklist a new entry may be made in the picklist and or the most frequent entry should be suggested as default value Organizational An organizational field belongs to the higher ranking database folder The organiza fields tional field contains information which can be related to all the documents within the database folder This may be for example customer and profit center All other database fields can be different for every single record However the orga nizational fields have the same value for each record stored in a database folder Icons for database The table shows icons used to identify database fields in the Define Fields dialog fields box Predefined organizational field E Predefined database field na User defined organizational field User defined database field Step by step How to define organizational fields You can only define database fields if the database is opened exclusively Following the setting up of a new database the database is opened exclusively 1 Select the Database gt Administration gt Define Fields command gt The Available Fields list contains the organizational ID e g Experiment number and the predefined database fields Available Fields list gt The Add Field dialog box will be opened Here you define the new data base field s properties gt The name ofthe new database field will be entered in the Field Name field User field cons
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514. ts The grouping that had kept the individual cells together will now be taken apart You will thus now see the marking symbols around each separate text object gt You can now e g rename the sheet or the columns add a comment on individual measurement values or have certain cells of the sheet high lighted in color 6 As needed you can edit the sheet Doubleclick e g on the sheet header to rename the sheet 7 Once editing is completed you select all the sheet elements by drawing a frame around the whole sheet keeping the left mouse button depressed 8 Click the Group button in the Order and Group button bar to have the separate sheet cells reassembled into a single object 9 Position the sheet keeping the left mouse button depressed and if necessary adjust sheet size Adjust sheet size by moving one of the sheet edges with the mouse keeping the left mouse button depressed 51 You can automatically fill an entire report with database information m Report Generator Inserting sheets 10 You now change the size of the sheet To do this you keep the Ctrl key depressed so that the sheet remains in the same position gt When adjusting the size of a sheet the font and font size remain unchanged The separate values will thus be pushed closer together and in extreme cases overlapping 11 Click the Properties button in the Report Objects button bar to have the sheet formatted At the same time you
515. ts of the image buffer cannot be changed as long as the image is protected and can be used only as a source buffer but never as a destination buffer All commands affecting the image buffer s contents will be disabled if the active image buffer is a write protected image buffer This is also the case for all commands that write into the overlay of a protected image buffer It does not affect files stored on hard disks Protected image buffers are indicated by the lock icon in the image buffer box If the active document is protected the lock button in the Standard button bar will appear to have been pressed down Drag amp drop commands acknowledge the locked status by showing the prohibited cursor symbol This command works as a toggle switch Applying this command to an unprotected image buffer or clicking on the corresponding button in the button bar will result in locking the image buffer and applying it to a previously locked image will release write protection If you wish to protect multiple images simultaneously select the images in the image buffer box Press Shift or Ctrl while selecting the images with the mouse Then select the Protect Image command or click on the button for this command Inversely you can select several images that are write protected the command will in this case delete the write protected status for all f the selected images You should only select either images that are all write protected or ima
516. ts that can be copied faster than they can be created from scratch e g graphic elements you have yourself created 62 Archiving Images Archiving Images Archiving Images Define a database Set up a new database Term definition STAR STructured The STAR database is integrated into the image analysis program It allows the ARchive structured storage of all of your images graphs and documents such as sheets otep by step Term definition diagrams and text At all times you have fast and easy access to even very large amounts of data The database has full network capability and can be accessed by several users simultaneously How to define the directories for data storage 1 Select the Special gt Preferences command 2 Click on the Database tab 3 In the Locations group select a directory for the storage of all Database files If several users are to access the database select a network drive which can be accessed by all users 4 In the Locations group select a Temporary storage directory This directory is used as temporary storage for files during data saving and archive work in the database 5 Check the preset backup volume capacity for the backing up of your database You can change this setting later when making the backup copy e Ifyou store your database on CDs enter the volume of the data carrier into the Backup volume capacity field in MB e g 600 e If your network database is backed up by the s
517. tten into the destination image buffer and the subsequent image buffers Z Layer Extracts the stacks This command decomposes a data set into its Z layers The resulting Z layers will be written into the destination image buffer and the subsequent image buffers Time Extracts the time series This command decomposes a data set into its time points The individual images of the time stack will be written into the destination image buffer and the subsequent image buffers Red Converts the red part of a true color image to a gray value image Green Converts the green part of a true color image to a gray value image Blue Converts the blue part of a true color image to a gray value image Hue Converts the hue values of a true color image to a gray value image The values of the color component H 0 0 359 5 will be projected onto the gray values 0 255 These H values will be linearly compressed and also rounded to whole numbers This is why this color extract will not contain all information of the H component The result of the compression of the value range is that varied shades of gray values will be more readily visible than in the other color extracts Saturation Converts the saturation values of a true color image to a gray value image The values of the saturation component S 0 255 will be assigned to the gray values 0 255 186 The Image menu Extract Intensity Extract Converts the
518. ttons of the Position button bar to move the object selected in the direction desired Each click represents 0 1 mm e Select one or more objects and use the Position and Size tab if you wish to enter the position of an object as an absolute numeric value When you select an object its exact size and position will be shown in the status bar When positioning or altering the size of objects its dimensions and current position are shown in relation to the rulers of the report window You can turn rulers on or off in the Report Properties dialog box The unit of the rulers is also determined along with the report properties The following units are available cm inch mm and pt Use the grid as a positioning aid while inserting objects You can display the grid in the report properties and alter its size When inserting an object it is automatically aligned on the grid Use the buttons on the Align button bar to arrange numerous objects relatively to one another The position of the reference object is crucial for alignment The reference object is the object you last selected It s easy to recognize with its gray selection markers any other objects have white selection markers Position and size of the reference object is unaffected by operations conducted Alion S m o Ue s s HE pa You can mirror an object by altering the size of the object Drag e g the lower right hand corner of a text or image object over the upper lef
519. two possibilities for using the LUT as a mapping function to be applied directly to an image s gray values These two possibilities are image operations that do in fact alter the original image s gray values 159 The Image menu Edit LUT e Process gt Intensity gt To Gray Scale Image The resulting image is a gray value image that will be written into the destination image buffer Its gray values are determined from an average of its three color components R G B 3 The lin ear standard LUT is then placed over the resulting image it will thus be dis played onscreen as a gray value image e mage gt Convert gt To RGB The resulting image will be a 24 bit true color im age and will be written into the destination image buffer The true color image s color components contain the LUT values of the false color image The true col or image s colors will thus be the same as the colors of the false color image e The following commands are not well suited to situations where you wish to ap ply an image s LUT to its gray values Process gt Intensity gt Modify Gray Val ues and Image gt Edit Image Image operations on These image operations alter the gray values of the original image beneath the LUT false color images The LUT will remain unchanged and then transferred to the resulting image The altered gray values are then displayed onscreen in altered colors Depending on the LUT even minimally altered gray v
520. u will still be able to unam biguously recognize the target structure at a later date even when you have set one fluorochrome for several target structures e Select Fluorochrome to name the images based on the fluorochromes e g Cy3 FITC DAPI e Select Target to name the images based on the given targets e g Centrin Nucleus Tubulin e Select Fluorochrome Target to combine the names in the form lt Fluorochrome target gt e g Cy3 Centrin FITC Tubulin DAPI Nucleus e Select Target Fluorochrome to combine the names in the form lt Target Flu orochrome gt e g Centrin Cy3 Tubulin V Nucleus DAPI Define Fluorescence Acquisition Acquires the fluorescence images Select an empty image buffer After accessing the command the Acquire Fluores cence Images dialog box will appear Here you define e with which fluorescences you want to acquire images e with which exposure time and which binning this is to take place each time e whether the image acquisition over several frames should be integrated e How long the pauses between acquisitions should be e g to enable you to carry out manual filter changes on your microscope e whether the acquisition is to be steered manually or automatically e whether a Z correction should be carried out e The information regarding binning and the integration over frames are camera specific and are therefore not av
521. ue images the linear mapping function will automat ically be preset and displayed in the diagram For this function y x or G r G o i e Original and resulting image do not differ when using the linear display Only the gray value distribution of an image area within a frame will be shown if you have set a frame Gray values are plotted on the X axis 256 values are displayed for 8 bit gray value images and for binary image When working with 16 bit gray value images only those gray values actually included in the image will be displayed A 16 bit image can include up to 65535 gray values Say a given 16 bit image has for example 2783 gray values then only those 2783 values will be shown The Y axis has different scales for each of the two function curves The red histogram curve plots the number of pixels per gray value For the blue mapping function the X and Y axis are the same The axes show the gray values of the image The mapping function plots the gray values of the X axis against the gray values of the Y axis Defining a mapping The blue mapping curve can be defined interactively right in the diagram This lets function right in the you adjust an image s intensity and contrast You can retain some of the curve as diagram desired and just change parts of it e g if you want to continue using parts of the predefined linear curve Or you can define a totally new mapping function First of all the mouse cursor will tu
522. uire a multi channel fluorescence image 114 Acquire whole stage area 332 333 Acquisition button bar 17 Activate Stage Navigator add In 343 Add Graphs 324 Shape 268 Adjust Colors 208 Display 152 Align Graphs 325 Multiple Images 146 Sources 258 Alignment of Camera and Stage 329 Animate Image Stack 22 Apply Filters 259 Archive Database files 98 Area ROI 297 Arithmetic 324 Arithmetic Operations 250 Arrange Fields 82 Auto preview 343 Automatic Rotation Correction 329 Automatically update active image buffer 339 Average Gray Value ROI 298 Average Intensity ROI 297 Background color 338 Background Subtraction 223 322 Backup 96 Backup Database 96 Binarize image 219 Binary image 7 Blend images or image series 267 Brightness 203 208 Burkes 263 Burn into Image 171 Button Bar Acquisition 17 Image Analysis 24 Image Display 24 Image Stack 24 Overlay 172 Stack Navigator 18 Standard 17 Button bar Stage Navigator 330 Buttons 331 C Calculator 247 Calibrated input channel 329 Calibration 104 113 147 165 317 Camera 113 Camera Area 330 Camera area 338 Camera Control 102 Cancel the acquisition 333 334 Change Color Depth 262 Zoom factor 3 Change Z position 330 Channel Delete 112 Duplicate 112 New 112 Choose View 89 Clipping 150 Clipping border 110 Color of frames 338 Configuration Software 10 Configure Input Display 110 Format 110 Image intensity 108 Info 103 Input 109 Macro 109 Magnificati
523. ull the ellipse to a desired shape and size by keeping the left mouse button depressed Position the rectangle by moving the mouse 3 Click the right mouse button to confirm the size and position of the ellipse Creates a line object in the overlay When you insert lines they appear selected Lines color is predefined red The preset line weight is defined in the Pen size field in the Special gt Preferences gt View tab After insertion click the Object Properties button to define color and line weight Length orientation and position of lines can be altered subsequently as you like To insert a line 1 Click the Line button The mouse pointer jumps into the image window 2 Define the starting and ending point of the line by clicking the left mouse button 178 The Image menu Overlay Bar Arrow Polyline Polygon Creates an arrow object in the overlay When you insert arrows they appear selected Lines color is predefined red The preset line weight is defined in the Pen size field in the Special gt Preferences gt View tab After insertion click the Object Properties button to define color type of arrow and line weight Length orientation arrow type and position of arrows can be altered subsequently as you like To insert a line 1 Click the Arrow button The mouse pointer jumps into the image window 2 Define the starting and ending point of the line by clicking the left mouse button 3 Clic
524. ulting image will have the same bit depth as the original image The image is assigned the same size that all the pixels of the shapes are displayed at Fill Shape Generates a new image in which the shapes are filled with a color of your choice If you wish to move the pixels before they are filled then press Shift wnen selecting this command The Select Fill Color dialog box will be opened This is where you select the color or gray value for filling the shapes in the new image This dialog box varies depending on the image type Select the desired color or gray value Click on OK A new image will be created in the destination image buffer All the pixels inside the shapes will appear in the color selected Crop Shape Generates a new image that only contains the shapes Background and shapes are assigned a constant color The Select Background Color dialog box will be opened This is where you select the color or gray value for the image background This dialog box varies depending on the image type Select the desired color for the image background and click OK The Select Fill Color dialog box will be opened This is where you select the color or gray value with which the new image s shapes are to be filled Select the color desired and click OK The resulting image will have the same bit depth as the original image The image is assigned the same size that all the pixels of the shapes are displayed at 272 The Process
525. umn Select the statistical parameters you wish to have computed with the Define Statis tics command Select the sheet needed by clicking onto the sheet document If you wish to have the Statistical parameters computed from only one sheet column and not all columns select that sheet column Click on the sheet column header to select the column 305 The Measure menu Statistics The Statistics command generates a sheet which is called Statistics of which refers to the original measurement sheet The sheet contains a column called Statistical Function which contains the Base Unit and the names of the statistical parameters The second column contains the column selected from the original measurement sheet and values calculated This provides a clear correspondence between values in the measurement sheet and the statistical sheet If you have statistics calculated for all columns of the measurement sheet each column as named will appear in the statistical sheet as well 306 Graph Graph Graph Graph window For displaying and processing of graphs i e one dimensional data your image Graph Buffer Box analysis program offers an additional document window The graph windows always available It is located in the document area and contains special buttons for setting the graph display q in the Image Manager there is an additional toolbar referring only to graphs Which type the active document is determines which tab
526. ur image analysis program suggests three ways to create a database Express To store your documents use the predefined Folder 1 database folder You can set up further database folders for document storage at any time Template You adopt the structure from a template model or an existing database You define the organizational ID and the required database fields in the data defined base The organizational ID is the central field of your database and may not be altered later for existing databases In an experiment database for exam ple the organizational ID is the experiment number All documents that are entered into the database must be allocated to the relevant database folder with the experiment number Step by step How to set up a new database A wizard guides you step by step through the setting up of the database 1 Select the Database gt Administration gt New Database command 2 Enter the name of your database into the Database name field gt The database wizard suggests an identically named subfolder in the data base directory for the storage of the database files Even if you change the suggested directory you should still set up a separate directory for each database 3 Click on the Next gt button 4 Confirm the question as to whether the database directory should be set up 5 Select the User defined option Click on the Next gt button 6 Enter for example Project as the des
527. ur measuring processes from acquiring the images to measuring the results of the analysis The functional scope includes routines for image processing that can be handled completely automatically Save the automation profiles for different analytical tasks to measure similar sample classes at the push of a button In conjunction with motorized stages the automation environment supports the auto matic analysis of several single samples Each work step can be checked with a test sequence for a selected sample position or for an example image An automatic process is divided up by the automator into the following partial tasks e Image input Image Input tab e Image processing Processing tab e Measurement operations Measurement tab Each of these partial tasks is specified on a separate tab in the Define Processing dialog box and processed consecutively after starting the application When processing several images image acquisition processing and measurement are executed in a cyclic course The results are entered in measuring tables and saved In contrast to the processing of individual images there is no difference between the source and the destination image buffer for an automatic analysis The processed images are overwritten again during the analysis and are lost The results of the fully automated analysis are measuring tables containing the data from many images The Image Input Tab Use this tab to define the source which supplies
528. use within your image analysis program and you are still able to insert the image and its calibration data into MS Word You should however avoid burning scale bars if possible simply because you thus reduce your chances of scale bars overlapping one another and you also avoid having image information overwritten 170 The Image menu Overlay Bar Overlay Bar Multi dimensional image Overlays are vector graphics Shows hides the Overlay button bar The Overlay button bar is used to access functions for working on the overlay Most of these functions are not available via the menu Edit Overlay Select All Select None Object Properties Layer Burn Overlay Delete Layers Load Objects Save Objects ie Objects Objects p Pasie Object fal a e talta on A Oyelay gt Bring to Front Sent to Back Bring Forward Send Backward Text Rectangle Ellipse Line Arrow Polyline Polygon Highlighter The Overlay is used to add information in the form of texts markers or other graph ical elements to an image Although image and overlay form a unit the data are stored independently on the computer s hard disk Imagine the overlay as a trans parent foil covering the image Drawing and changing the overlay does not affect the image data Moreover you can fade out or delete the overlay at any time One advantage of using the overlay is that image editing operations are not affected by labeling and adding markers
529. use key depressed and moving the mouse Rightclick to confirm position and size of the ROI Multiple Fluorescences The mFIP acquisition process is a basic function of your image analysis program It supports the acquisition of a multi channel fluorescence image You can carry out this acquisition process either with a manual or with an automated microscope When you use an automated microscope you can acquire this multi channel fluores cence image with a single command as soon as you have defined the fluorescence acquisition one time With a manual microscope your image analysis program will support you with messages that will conduct you through the acquisition process The result of a mFIP acquisition is in every case a multi channel image in which the individual fluorescence acquisitions have been combined You can view the indi vidual fluorescence acquisitions and naturally also superimpose them Use the Adjust Display button on the Image Display toolbar to have the appearance of the multi channel image matched to your monitor This is how you acquire a multi channel fluorescence image Prerequisite e You are using a fluorescence microscope to examine a sample with multiple flu orescence markings Example Your cellular tissue sample has been marked with the following fluorochromes MitoTracker Red Bodipy FL and DAPI e You have set up and calibrated an input channel for the light microscope and the camera e When you work with a
530. user programs these are for example the text files with the doc file name extension in Word and the sheets with the xls file name extension in Excel 94 Archiving Images Archive data There are four images in the marked database folder Select the Load with Documents command to load all the images into the image manager lel ES 0 OAE l l yegl as amp ExampleD atabase apl stereo images Perspective 24 ExampleDatabase apl E3 Water 3 Extended Acquisiti 3 Multiple Image Alig CI Phase analysis CJ TEM acquisition Choose iew Set Default User Field Values ial naet r eal oto Record J Extende Load Document 0 Filtering Load with Documents E3 Particle 39 Calibratic Arrange Fields E Fluoresc Copy lable aio Sethings ExampleLi atabase view Etuthon Eber Define Contest Menu Archive data Background Information Archiving e Temporary storage directory Storage Archiving allows you to archive older files from the current database on a CD or other data carriers The original data are subsequently deleted from the database You can naturally continue to work with the entire database i e formulate queries or insert and load data The thumbnails of the stored records are still available If you load a stored record you will simply be asked to insert the appropriate storage medium Complete database folders only are sto
531. usly into a report Create a new report page that has at least one image object Select the desired images in the image data base or in the image manager and drag the selected images onto the first image object If the number of images selected is greater than the number of image objects repre sented on the current report page your image analysis program will automatically add any pages needed The added pages use the page template of the active page Note Please note that you cannot reload an image already inserted into a report into your image analysis program Sequence when The sequence of the images inserted into the report is determined by the sequence inserting in which you select them from an image database Mark the images in the order in which you want them to be inserted into a report To do this depress the Ctrl key and mark the images with the left mouse button The numbers in the top right corner of each thumbnail represent the order of the selection The images from the image manager are always inserted in the sequence of the image buffers The order in which they were selected is of no significance Image types You can insert all image types which can be loaded by your image analysis program into a report Generally the images are inserted into a report in the way they are shown in the image window For example this means that a 16 bit image is inserted into a report with the current display LUT If your image analysis pro
532. usting pixel intensity often remains unused This means that images will appear weak in contrast or an image will seem too bright or too dark The Maximize Contrast command will effect a linear spreading of the available value range up to the maximum value range possible However an image lacking in contrast will scarcely be improved if it contains single pixels that are either very bright or dark Gray value images 8 bit gray value images have a maximum value range of 0 255 An image s value range is spread by having the darkest pixels set at a gray value of 0 the brightest at 255 16 bit gray value images are spread in the same way but with a value range of 0 65535 False color images 8 bit false color image are processed just like 8 bit gray value images False color images are in fact gray value images that are displayed on the monitor in color via a look up table LUT To look up the LUT used for the display of the active image use e g the Image gt Edit Image command The Maximize Contrast command alters the gray values behind the image s active look up table LUT The LUT has no effect on how gray values are adjusted The LUT itself remains totally unchanged The coloring of the resulting image may differ from the original image This is because the active LUT may assign different colors to the adjusted gray values which means that the coloring of the image onscreen will look different True color images 24 b
533. ve been already defined when the database folder was set up and cannot be altered when data is inserted into a database folder Arrange Fields Views ype Available mE Info Window Client et Form View LES Table View a Query by E sample El Insert Insert Image Fields 1 3 Record Name Comment 71 Experiment Desciption Current tjel Record Hame ok Comment Expenment Desciption Cancel Help Serto allah thie tipe Get Default Set Default Insert HER2 Fluorescence In Situ Hybridisation Human Breast Cancer Tissue labelled with a probe against the HER2 gene Texas Red and Chromosome 17 FITC patients with amplification of the HER2 gene are treated Skip Cancel A EE Help Archiving Images Insert data Insert data Create a new database folder Background Information Insert database There are several options for creating a new database folder folder 1 Menu command open the Database gt Insert submenu To set up a new data base folder select the first command in the submenu This command is different for each database and reflects the organizational ID of your database s 2 Button in the database window In the database window s button bar click the lt Organizational ID gt insert 3 Context menu activate the database window and click the right mouse button to open a context menu Open the Insert submenu Se
534. ve search filter For queries by free filter and queries with SQL you have the possibility of saving your search filter so that it may be reloaded later if required Representation of The data found is indicated in the Query results database folder directly under the query results the database symbol in the tree structure e The Query results database folder is updated with the new query results for each search e Query results are no longer present after a new start of the program 90 Archiving Images Work in the database window otep by step How to select the database fields which you need in the query mask In your database there is the user defined User field You want to be able to find all the images the user Mr Smith inserted into the database in 2003 1 Select the Database gt View gt Arrange Fields command 2 Mark Query by Example in the View Type list 3 Select the fields in which you want to search for images e g the user defined User field and the predefined Document Insertion Date field 4 Click OK to close the dialog box How to search for images in your database 1 Select the Database gt Query by Example command gt The Query by Example dialog box contains the selected database fields 2 Enter the required entries into one or more fields For example enter Mr Smith into the User field and 31 12 2002 into the Document Insertion Date field e During the query by exampl
535. ven if there would be enough space on the CD The reason for this is these database files must once again be modified after executing a successful burning procedure e Due to file security your image analysis program does not support the Multi session functionality from the burner software You cannot write anymore ad ditional files to a CD or DVD after a successful burning otep by step This is how you create a backup copy of your database The backup is to be written directly to the CD DVD System Require You have access to a CD DVD burner You have Nero Burning ROM software ments 1 Check the settings for the temporary backup directory Use the Special gt Pref erences command Select the path to which the data is going to be temporarily stored to from the Temporary storage directory field located in the Database tab e Please note There must be sufficient space for burning in the directory to D gt which the temporary backup directory has been stored 2 Check the directory name under which the database files of the database are stored To do this select the upper most entry in the database s tree structure located in the database window The database path is shown in the form in the lower right 3 Place an empty CD or DVD in the burner 4 Use the Database gt Administration gt File Storage command to call up the file storage wizard 96 Archiving Images Archive data 5 Select Backup Click o
536. vidual sections as required to do so move the mouse pointer to the section edge and move it with the left mouse button depressed On the lower left you will find the tree structure in which the data is arranged The upper level is the name of the database Below this are the main entries which are characterized by the organizational ID of the database A maximum of three sub levels are allowed under a main entry Each type of document is identified by its own icon Doubleclicking on an entry extends or reduces the active level The record which is marked in the tree structure is displayed on the upper left Doubleclicking on the icon in the preview window jumps up to the next higher level in the tree structure On the lower right you will find a sheet with the columns Field name and Field value All the field information which you have selected with the Arrange Fields command for Form View is displayed here You will find the gallery view on the upper right of the database window It shows all records which are below the marked record A thumbnail preview is displayed for images diagrams and graphs The other document types are represented by icons Doubleclick on a record to load the record Doubleclick on a folder symbol to open the database folder Records which are linked to other data are represented in the gallery by a document icon and a plus character Click on the plus character to display the linked data in the gallery view
537. ween maximum and minimum values of the neigh boring pixels Percent Z gt Imax 100 a max _ min Z max 234 The Process menu Define Filter Rank Percent lz lt Imax 100 Cmax Imin gt L Imin where lz intensity of the central pixel in the original image lnax maximum intensity within neighboring pixels lain minimum intensity within neighboring pixels Original Edge Enhance Filter SA Percent 50 Left In the unfiltered image you can see four intensity levels Their edges are blurry Right After being filtered with the Edge Enhance filter the edges are sharp The parameters for this filter are defined in the Define Edge Enhance dialog box FE Percent nS Cancel T Preview Execute Var diy Help Enter the dimensions of the neighboring pixels area N 3 9 into the Size field The gray value of the central pixel is compared with those of the neighboring pixels The larger this area is the less detail the resulting image will contain The preset size is 5 The Percent 1 99 field defines the threshold for assigning the central pixel either the minimum or maximum value of surrounding pixels When you select a lower percentage darker gray values of the original image will predominate a higher percentage will result in the lighter gray values predominating When you select a percentage of 50 the mean gray value of the original image will not be altered The preset p
538. will be indicated by selection markers the last one you select has gray markers and the rest white Or keep the left mouse button depressed move the mouse to draw up a frame that encompasses all the objects you wish to select You have numerous possibilities to alter the properties of an object e Doubleclick on the object e Select the object and then click the Properties button in the Report Objects but ton bar e Select the object then rightclick and choose the Properties context menu com mand Select Objects Move Image Properties Save Properties As Default Line Polyline Polygon Rectangle Sie Closed Curve m Ellipse Report 0 Tee SSS OS SSS Se SS SSeS Graph Diagram Sheet Field Record Clear Object Fit Frame to Image Detail Zoom Image AutoText Text You can alter the size and position of all report objects either in the object properties by entering absolute numeric values or alter them directly with the mouse You can use the ruled borders of the report window as positioning aids e Select the object keeping the left mouse button depressed and drag one of the selection markers in the direction desired When simply altering size the po sition of the selection marker opposite the one you have selected will remain un changed 31 Altering object position via mouse R Positioning objects approximately Fine tuned posi tioning Use the buttons of the Position button bar to fine tune your po
539. window 11 In the Finish Object Editing dialog box click the Exit without Save button 12 Pull a record with a sheet from the database onto the altered record object Inserting diagrams Background Information Diagrams can only be internal graphics Some commands e g Histogram auto matically generate diagrams Otherwise you use the Edit gt Diagram gt New command for a graphical display of values from a measurement sheet Diagram properties e Diagrams like sheets can also be used as placeholders e When enlarging diagrams the font size is correspondingly enlarged as well This is not the case with text objects or sheets Keep the Shift key depressed while adjusting diagram size This ensures that the length width ratio of the page re mains the same and the lettering will not be distorted e Diagrams cannot be rotated The buttons in the Rotate button bar are not avail able e To alter the background color of the diagram go to the Fill tab Text labels have nothing to do with this They remain black lettering on white e Determine the color and the width of the frame outline in the Line tab The line width of the axes is preset and cannot be altered Metafile format WMF stands for Windows Metafile Format a format used for data exchange between Windows applications Diagrams are inserted as Windows Metafiles After the RTF Export has been completed you can edit them in MS Word You can also e g alter font and
540. ximum value is 256 In the Frames field you enter the number of frames if you are working with a Sony DXC 950 video camera The maximum value is 255 frames In the Acquisition options group you define the waiting times the acquisition proce dure and the Z correction In the Delay field you enter how many seconds will elapse before the image acquisi tion for a specific fluorescence takes place This may be useful for example if manual adjustments to the microscope e g shutter or filter need to be carried out for this particular acquisition Activate the Manual check box if you want to acquire the fluorescence image manu ally The application then asks you step by step to carry out the relevant adjustments In the case of an automatic microscope you will be asked to confirm the next step each time Select the with preview check box in order to have a live acquisition displayed before you acquire the single image You should use this possibility because it will enable you to set the exposure time for the individual fluorescences optimally Select the no messages check box in order to deactivate all input messages You should only clear this check box for your first fluorescence acquisition Your image analysis program will then describe every single step that you need to take to make the acquisition This will no longer be necessary when you make acquisitions at a later date and will only tend to impede the acquisition Select the Aut
541. xt list This option is only avail able when you have no frame showing i e Transparent frame Select the Shadow check box to have a shadow displayed behind either the white or yellow frame This check box is only available if a frame is being shown i e you have not selected Transparent Determine how the unit selected is to be treated in the Unit conversion group you can have the scale bar unit automatically adjusted or use a constant unit The unit selected here will be used in all application areas for monitor display printing and for the clipboard The unit selected will also be used for the scale bar you can have appear below the image when you print out an image To have this scale bar printed out select the Print scalebar check box located in the File gt Define Page Layout gt Header Footer tab 168 The Image menu Scale Bar Please note that although the unit in use will correspond e g um the length of the scale bar on a printout and in the clipboard may be different than the length of the scale bar on the monitor Select the Auto entry from the Unit list to have the scale bar unit automatically adjusted to onscreen display or to image size The unit will be adjusted such that the numerical values of the unit are always between 1 and 500 1000 um would thus automatically become 1 mm The largest and smallest units available via this auto matic unit adjustment are km and pm respectively The automatic
542. y Click the List button to open a list containing the last calibrations completed successfully The Calibration List dialog box The Channel column indicates the name of the logical input channel used for the corresponding calibration When renaming the input channel the channel name in the calibration list is automatically updated The Shift Type column indicates the shift method for acquiring the image series The Magnification column indicates the magnification used for the corresponding calibration The Last Calibration column indicates the date the system was calibrated Click the Close button to return to the Calibrate dialog box Click the Delete button to remove the marked entry from the calibration list This option is necessary because the list cannot be updated automatically when an entry becomes invalid because e g the position of the camera was changed Click the Edit button to have a look at the calibration data of the selected entry Clipping Cuts off same colored image edges of the assembled MIA image or cuts off the edges of the individual component images with single pixel precision Lett lo B Right jo B Cancel Top jo l Bottorn lo B Test Interactive Help After being assembled the MIA image often has a narrow one color edge This command is for clipping off that undesired edge Some cameras produce artifacts when acquiring the image These artifacts can be dark image borders containing
543. y to the Graph Informa tion dialog box In this tab the modifications made to the active graph are recorded according to the previously defined command groups Convert to Sheet Creates a spread sheet from current graph Use the Graph gt Convert to gt Sheet command to convert the coordinates of the displayed graph into a new data sheet When you convert a multi graph one sheet column will be generated for each graph contained in it Series of Graphs Converts a multi graph to a series of single graphs Some of the graph analysis commands are only available when only one curve is on display in the graph document Select the Graph gt Convert to gt Series of Graphs command to create a series of single curve graphs from a graph document containing several data sets All newly created graph documents will have the same X range but different Y ranges 327 The Stage Navigator The Stage Navigator The Stage Navigator Summary of Features With the Stage Navigator you can acquire overview images of a sample and use them for a precise navigation This assures that you always know your exact location on the sample You can recognize interesting areas in the sample move to them in the live image and acquire images with a higher magnification In the overview image a grid within a grid enables you to recognize the areas you have acquired with a higher magnification You can deactivate this display if you want to BEI 25
544. y unchanged in MS Word e The file size of RTF files can be very large You can reduce the file size by saving the report in MS Word as a Word document in DOC format Printing the report 1 Select the File gt Print command to print out the finished report gt The Print dialog box is context sensitive This simply means that the func tions being offered by the dialog box depend on what document is active Before you print a report you have to activate this dialog box 2 Select the Full image option from the Images group within the Print dialog box 3 Select the All option from the Print Range group to have the report printed out in its entirety Start printing by clicking on OK Report objects Background Information Report objects A report page usually includes various kinds of objects These may involve image and text objects as well as graphic objects For each object there are individual char acteristics which can be defined which are different for each object type A certain object type the record object can consist of numerous other objects You can define individual object templates for record objects In doing so you not only create a wealth of different record objects but also guarantee a uniform appearance of the record objects in different reports Placeholder Several objects serve as placeholders Image objects and record objects are typical placeholders These objects are usually defined in a report template I
545. y behind the channel name The camera must be aligned parallel to stage movement direction Otherwise the individual images in the overview image will appear twisted in relation to each other Small rotation angles less than 3 can be compensated by the software Should the camera position be changed e g because you have meanwhile attached another camera you will have to repeat the alignment procedure How to adjust the automatic rotation correction 1 In the live image go to a distinctive area of the sample and adjust the settings focus exposure time etc to provide an optimal image 2 Start the Stage Navigator and click the Properties button 3 Click the Preferences button gt The Stage Preferences dialog box will open Stage Preferences Slignment Adjust Rotation i edust View list Horizontal scale Vertical scale V Fine adjust 4 Select the Fine adjust check box to enlarge the stage movement during the acquisition of the two images that are to be used for the calculation of the rota tion angle In this way the search for the same image structure will be carried out in a far smaller image section gt To use a larger image section for the pattern recognition you can clear the Fine adjust check box When there are larger rotation angles this can lead to better results 5 Click the Adjust button to let the rotation angle be automatically determined 329 The Stage Navigator Sta
546. y containing the image This number will of course change when you for example put the image into another image buffer The Frame 6 10 fielddisplays the number of the current image and the total number of images in a data set The Created field displays the time and date at which the image was created The date and time an image file was last altered will be displayed for images not acquired via your image analysis software 194 The Image menu Image Information The Channel field displays the camera used to acquire an image This field will be blank for images not acquired via your image analysis software The Magnification field displays the magnification used when the image was acquired via your image analysis software For some microscopes your image anal ysis software can directly read out the magnification from the microscopes remote control Magnifications of images acquired in other application programs will always be defined as 1 Use the Image gt Calibrate Image command to adjust the magni fication of an image acquired in another application program The Resolution field displays image size in pixels and information depth bits pixel For example an entry of 2080 x 1544 x 16 would mean that the image width is 2080 pixels The image height is 1544 pixels The image can have up to 65536 different color values per channel The Width and Height fields display absolute dimensions of images These values are det
547. y either depressing the middle mouse button or the Esc key 281 Interactive Image Measurement Save Load and Edit Measurement Results gt The pointer is released 8 Select another measurement from the Measurements button bar Select the Closed Polygon button for example to measure any area with the help of the polyline 9 Set the individual points by depressing the left mouse button Close the area by depressing the right mouse button gt Inthe measurement display you will now find in addition to the measured area the perimeter of the measured object 10 End the area measurement by either depressing the middle mouse button or the Esc key e You can insert additional measurement values onto an image or respec tively delete single measurement values and measure them anew Save Load and Edit Measurement Results Background Information Measurement Display These images which have been loaded contain measurement values Select the name of an image in the tree view to switch to that image You can add new measurement values to an already existing measurement or delete single or entire measurements The measurement display is a tab in the image manager and contains all of the measured values The measurement display is empty as long as no measurement has been undertaken As soon as the first measurement has been started the measurement display will be filled The values are organized in a tree view The u
548. y the mean value of the four directly adjacent pixels Histogram Number of Pixels 80 Grayvalues 175 The image shows only statistical noise The gray values are distributed in a Gaussian shape around the mean gray value of the image The parameters of the Sigma filter are defined in the Define Sigma Filter dialog box Define Sigma Filter Cancel Execute e Help Enter the diameter of the surrounding area in pixels into the Size field You can select diameters ranging from 1 to 50 at intervals of 1 The preset value is 3 All pixels within the surrounding area defined in this way will be taken into consideration when the mean is determined In the Sigma field you enter the half width value s of the Gaussian distribution used The Sigma value defines the gray value interval in which the surrounding area pixels are to be located The Sigma parameter influences Gaussian distribution A small Sigma value gener ates a narrow distribution with a large maximum value A large Sigma value gener ates a flat wide distribution 95 5 of all curve values are within the double Sigma width 237 The Process menu DCE DCE Defines the DCE Filter The DCE filter enables you to selectively enhance weak differences in contrast The DCE filter renders image structures visible that are barely distinguishable from one another in the original image Resulting images are thus more detailed and appear more focuss
549. y thus disappear from the image This suppresses noise points and non extreme unevenness will be smoothed out Other available smoothing filters are the Mean NxN Rank and Sigma filters The Median and Mean filters are directly comparable When neighboring pixels gray values are distributed symmetrically with respect to the average value both filters will yield the same results asymmetrical gray value distribution will yield varying results with the two filters The Median filter is more stable i e it reacts less sensitively to individual greatly deviating values 244 The Process menu Filter Pseudo Sobel Roberts Reimer Performs a gradient filter for creating synthetic topographical effects The extent of the three dimensional effect can be determined using the Define Filter gt Pseudo command see page 231 Performs a gradient filter for edge detection with a noise insensitive Sobel filter Filter Matrix 101 A 2 4 2 02 gt a o0 00 7 101 1 24 This filter employs two matrices based upon which the values a and b are computed The resulting pixel value is thus D The Sobel filter is a genuine edge detector and does not react so sensitively to noise effects This is because differentiation is conducted using the lines and columns beyond the next immediate ones Thus any interference in lines and columns directly next to your central pixel cannot influence the results The Sobel filter is of the c
550. y used in record objects to sup along with every image plement the standard autotexts e g the se quential number is supplemented with the word image graphic lines object autotext sequential image numera This type of autotext is only available within a tion record object field objects automatic printing of sin Field objects are composite objects that are gen gle line contents of data erally at least comprised of the name of the base fields field and the field s entry automatic printing of Memo database fields multi line sheet cell placeholder for a special Many special Add Ins deliver standardized mea value from a sheet cell surement sheets which are always constructed the same way Insert the sheet below an image in the database Use this field function to acquire individual measurement results i e the G value of a grain size analysis into the record object sheet placeholder for a sheet a Sheets diagrams or graphs which are stored diagram or diagram or a graph below an image in a database can automatically graph be inserted into a report To do so create record objects with placeholders i e for a sheet Field objects Field objects are fields of text linked to the content of a specific database field They refer to a particular image database which must be open when creating a report Field objects are usually comprised of the name of the database field the field name and of the fi
551. ystem administrator enter the value 0 into the Backup volume capacity field 6 Close the Preferences dialog box by clicking OK Organizational ID All data is entered into a database folder within the STAR database The name of the Record folder is defined by the central field of the database the organizational ID You define the organizational ID when creating the database and cannot change it for databases that already exist When the database contains data on individual experiments the organizational ID is for example Experiment Number In an express database the Folder Name is the preset organizational ID A record is set up for every document that you enter into a database Images or other documents such as for example sheets texts diagrams graphs and reports can thus be stored in the database The records can be arranged hierarchically in the database 63 Archiving Images Set up a new database Database folder The term database folder describes a folder within the tree structure of a STAR database The database folder is not a file folder at operating system level You will find a database folder only in the database window but not in MS Windows Explorer Database fields Database fields contain all the information that you want to store together with a document The entries in database fields are linked to the relevant document and enable you to easily find any document in the database Database types Yo

User Manual - CALM - Confocal and Advanced Light Microscopy

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