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1. emitter observed emission YFP ente sion 450 500 550 nm Figure 4 Left Spectral match between eYFP fluorescence and optical filters HQ480 40x HQ500 20x Q515LP and HQ535m right reference images of an eYFP sample 10 9 5 Spectral Unmixing with cell R cell M cell4R cell M provides a software module named Unmixing that gives you a powerful tool to separate information from strongly overlapping emission spectra of two or three fluorochromes Spectral unmixing is composed of two steps Calibration and Unmixing It is currently possible to separate two or three different color channels cell s cell software Manual Chapter 10 Intensity Analyses 217 10 9 6 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 Ui Calibration 1 Acquire multi color re
2. GFP_YFP_Orig Ei 74757688 P_YFP_unm 768 x 6 3 Image pn OBS Megaview gt 768 576 cell s cell software Manual Chapter 3 Brief Introduction and First Steps To start the cell4M cell4R software double click the cell4M cell4R icon on your desktop or open it via Start gt Programs gt cell R M The screenshot below shows the program s graphical user interface GUI It is composed of a b Menu bar access to pull down menus with assorted commands Tool bar direct access to the most important commands for image acquisition processing and analysis Status bar shows the connected CCD camera and displays information depending on the ac tive functions Viewport Manager top left below button bars shows a thumbnail of the active image a red rectangle indicates the current zoomed in sector of the image in the Viewport The rectangle can be mouse dragged across the thumbnail to bring a different area into display Image Manager underneath the Viewport Manager consists of two parts the Operand Box an operational area to determine source and destination buffers for image processing the Image Buffer Box listing the currently loaded images or graphs Four tabs are avai
3. Filter Cube This column is active only if the microscope features a motorized fluorescence filter turret and if it has been configured on page Microscope gt Filter Cubes The fluorescence filter cube that matches the excitation filter of the image type has to be selected here so that it will be moved automatically into position for the image acquisition Observation Emission Filter This column is active only if the microscope features a motorized observation filter wheel and if it has been configured on page Microscope gt Filters The emission filter that matches the excitation filter and filter cube of the image type has to be selected here so that it will be moved automatically into position for the image acquisition Fluorescence color The Fluorescence Color which can be defined in the exact same way as described for Excitation filters see Chapter 15 2 1 Configuring the Excitation Filters determines the color of the image acquisition command icon in the Experiment Manager and more important the color with which here TxRed images or color channels will be displayed onscreen In the example a black to dark orange color palette will be used If no color is selected a black amp white gray scale display will be used 342 Chapter 15 cell M cell R Configuration OLYMPUS 15 4 Configuration of Additional Shutters Additional shutters which are not integral parts of the 1X81 or BX61 frames those are configu
4. 20 x 30 00 5 5 3 3 6 Experiments with online analyses The cell R cell4M software allows to calculate a ratio image and an intensity kinetic online during the execution of an experiment The Ratio commands can only be placed after a Multi Color Frame containing exactly two Image Acquisition commands In other cases a corresponding error message will be generated upon verification of the Experiment Plan A Kinetics command can be placed either behind an Image Acquisition or a Ratio command the calculation will be done for this very command The example below shows an Experiment Plan for a Fura 2 calcium ratio time series with online display of the images acquired with 340 nm excitation online calculation display and storage of the 340 nm 380 nm ratio images and online calculation display by default and storage of the intensity kinetics of the ratio image 65 66 Chapter 5 Experiment Manager OLYMPUS alte k F Furas4o Furazs0 EJ 50 x 500 00 ms 5 3 3 7 Complex experiments with trigger pulses and several time loops The Experiment Manager is powerful and flexible enough to set up much more complex experi ments than in the examples above As an example let s imagine the following ratiometric calcium experiment 1 First a series of Fura images at the two ratio wavelengths 340 nm and 380 nm is to be acquired for a certain time at moderate speed In the Experiment Plan this will be a Time Loop Frame cont
5. 5 The Background Subtraction Thresholds and Output Scaling functions are described in Chapter 10 8 2 Generating a Ratio Sequence 6 Confirmation with OK creates an Ffret Fdon ratio image O Note that such a ratio image itself has little meaning Only an increase of the ratio by a decrease of the Fdon signal along with an increase of the Ffret signal may hint at the occurrence of FRET processes 10 12 3 2 MicroFRET Quantification According to Youvan FRET Analysis Settings Dimensions Info Method Ratio I FRETn Gordon 1998 D M fret sia 2001 Correction Factors Ratio A Fdon 0 697060 CFRNFP ow Ratio B Facchi 0 031351 Input Inages Channels Selection 4 FRET cfp exc FDon cf 5 FRET yfp exc Face yf w i4 FRET cfp exc FFRET Background Subtraction None Select ROIs From Image O Constant 4 FRET cfp exc 11 ROI Select ROI O Image ROLL ROT 1 Thresholds Threshold 1 Threshold 2 e Ife e Ife Scale Factor ooo The method described by Youvan is a subtractive method The correction factors generated as described in Chapter 10 10 4 FRET Image Correction Factors are used to estimate the contribu tion of the channel bleed through to the signal in the FRET channel acceptor emission caused by cell a cell software Manual Chapter 10 Intensity Analyses 231 donor excitation The bleed though contribution is then subtracted
6. 7 configuration Use standard configuration and include functions defined in selected configuration O No update ignore new functions of this program version No update If the configuration you wish to load is however a reduced version of an older standard configuration you would do better to use the No update ignore new functions of this program version option In this case the configuration will be loaded precisely as it is O Any commands that are exclusive to the current program version are not part of the older configuration Thus these commands will not be available If you want to use them it will be necessary to add them manually to the configuration using update commands e g the Specials Define Menu Bar command This option should be used in cases where you simply do not know in what way the older configuration differs from the current configuration If necessary repeat loading of a configuration to achieve the result desired using an other option If you re happy with the results of your loading of an older configuration then save this now current configuration using the Configuration Save command in the Special menu The next time you load this configuration you ll be able to load it without any further queries 13 4 3 Save Use this command to save the current configuration Application Select the Save Configuration command to save your individual configuration You can recall t
7. Language version When programming C modules function names will be shown in either English or German depending on which language you ve selected in the Language list This setting also determines what language is to be used by the macro recorder for recording menu functions Command window Activate on output C modules and macros are able to write results into the command window Select this option to have the command window opened automatically or placed in the foreground if it was already open When working with macros keep in mind that due to an output for exam ple using the operator or the printf function the active document will lose its active status the command window will be activated in correlation with this check box A macro that refers to an active sheet document for example will locate the command window instead and thus can no longer continue working correctly Display string pointer values Tick mark this option to have not only strings displayed at output in the command window but the corresponding address as well Example Select the check box Enter the following two lines into a text document run the sample macro by pressing lt F5 gt char szInfo 20 Example szinto The following line will be output in the command window 0x59b7 0x000c gt Example This check box is not selected in the standard setting of the program strings are used frequently and having addresses included al
8. 30 Blind Deconvolution Filter Parameters Iterations ho E Sub olume Overlap ho E Microscope Widetield Fluorescence w Channel Settings Execute _ This is a true intensity restoring deconvolution method The algorithm removes out of focus inten sity from each Z stack layer and adds it to the layer of origin Thus the image resolution is strongly enhanced and the overall fluorescence intensity within the z stack conserved At the same time the signal to background intensity is improved Because the global intensity is conserved the deconvolved images are especially well suitable for quantitative intensity analyses Available Z stacks either monochromatic or multi colored Requirements The images need to be calibrated in the X Y and Z dimensions see Chapter 7 1 Calibrate Image otherwise an error message will be generated For meaningful results a back 174 Chapter 8 Image Processing OLYMPUS ground subtraction has to be carried out before the calculation see Chapter 10 5 Background Subtraction Iterations The higher the number of iterations the better will be the result Mind however that this method is time consuming in general 10 iterations are a good starting point that usually renders considerably improved data After about 50 or 100 iterations improvements will hardly be visible with further iterations Sub Volume Overlap See Chapter 8 4 5 Inverse Filter cell amp cell Software Manual
9. aasasasesenenenunnnnnnnnunnnnnnnnnnnnrnnnnne 194 Chapter 10 10 INTENSITY ANAIVSES wisivcsiseccdsvenietiwintstawdacctaeunvisceonssectedevevssaieniias 195 Sophisticated analyses of 10 1 Pixel Value o oo cece ec ec eeececececececeeeceeeeeeeaeaeaeaeeseceseseseneeeaeaeaeanenenets 196 fluorescence intensities 10 2 Histogram cccccccccecccccseeeeesseeeeeceeceeeeeeeeseeeeuaaasasseeeeeeeeeeeeesessaaaaes 197 mostly for time se 10 3 Line Profiles WMLGMSILY xxsictecccte ets tclteesnecsleateseleec tes csanecedenecendndt 198 quences and Z stackS 49 3 4 Horizontal Line Profile cccccccscceseccecececceseccecesceceseeceeescasereaees 198 10 3 2 Vertical Line Profile oo cece ceeeceseeeeeeseeeeeeeeeeueeeeeeueeeeeeneenenens 199 10 3 3 Arbitrary Line ProflE ssn eitceiuans cetnuiiwcaeenaueer E a 199 10 3 4 Average Intensity of Neighboring Pixels ccccceeseeeeeeeeeeeeees 200 10 4 Regions of Interest ROIS cccccccesssseeeeesseeeseeseeessaseeensaserensaes 201 cell amp cell Software Manual Contents 1041 GOMGral ve tepies ash eE E ctieierl aude TRN 201 10 42 Drawing ROIS sirere dearcdeeietxiedeldneneieenss 201 10 4 3 ROI Measurements 2 D ccccccsseeeceeeeeeeeeceesecseeeseeeesseeeesees 204 10 5 BackoQround SuUDWaCHON tari it a 205 10ST General desrena tart rn meres ameter ener erence a a 205 10 5 2 Subtracting the Image Background ccceececseeeeeeeeeeeeeneeeeeens 205 10 6 Intensity K
10. group will appear in the lower left corner of the dialog box in the Polynomial fit group This is where you determine whether the two dimensional fit is Linear Square or Cubic Polynomial fit Linear O Square Cubic 8 1 3 Executing a Shading Correction The Shading Correction is executed via Process gt Shading Correction The shading correction as defined under Define Shading correction will be applied In case of multi dimensional images the Dimensions dialog box opens Here you can choose the range of images in the different dimensions Color Channels Z Layers and Time Frames for which the analysis shall be performed in case only a subset of the data is of interest By default the entire data set is selected With the option Step in Z Layers and Time Frames you can select every second third fourth image and so on to be analyzed Shading Correction EEEEE Dimensions Color Channels Z Layers bL els E IE se IE Time Frames rom 1 Ele or Es E C cell s cell software Manual Chapter 8 Image Processing 131 8 2 Filters 8 2 1 General A Filter modifies the intensity of each pixel according to the intensity of neighboring pixels Why filter images Filters can be used to prepare the actual image analysis for example by cor rection of image errors resulting from acquisition Filter operations have the following possible ap plications e correction of image interference due to sta
11. switch filter cube filter cube contrast insert Selection displayed in icon open or close shutter switch transmission contrast insert switch transmission lamp on off switch camera port for IX81 status only switch between camera and status ocular for 1X81 only _ absolute intensity voltage displayed in icon move DSU in or out open or close shutter switch nosepiece position relative movement or position move Z drive cell s cell software Manual Chapter 5 Experiment Manager 5 3 2 13 Additional atomic commands the MT20 and CCD Cameras Commands tool bars CCD Cameras EJ CCD Cameras Additional atomic command icons change attenuation light intensity ea switch excitation filter filter cube Ee DAPI open or close shutter of status MT20 MT10 4 fo ty 7 open or close shutter trig status as 2 ope Min dae gered via TTL port acquire image binning ROI exposure time syn Acquisition chronization display Acquisition Not synchronous This option causes the camera to operate without each image being triggered The advantage of this mode is that camera exposure occurs while the data of the previous image are being read out The cycle time can thus be reduced to the read out time O This mode is only supported by certain cameras and available only for monochromatic experiments It does not make sense to combine this command with a
12. 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 the ordered sequence is to replace the central pixel The adjacent area 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 The Sigma filter is for filtering out statistical noise Individual pixels of gray values which deviate greatly from their surrounding area will however not be affected by the Sigma filter cell s cell software Manual Chapter 8 Image Processing 133 Gray values of Sorted sequence Gray values of the original image Matrix d the filtered image Lx x 84 85 99 80 109 120 80 85 104 109 120 J 72 404 155 Smoothing filters The Smoothing filters class comprises filters for averaging gray values sur rounding 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 flattened out The resulting image will thus seem somewhat blurry due to this type of filtering Applications of various smoothing filters The diagram below comparatively shows the effects of various filters used f
13. 281 282 Chapter 13 The Special Menu and the Window Menu OLYMPUS None A newly taken snapshot will always be placed into the active buffer in the Image Manager overwriting the any image currently being place there Image buffers All A newly taken snapshot will always be placed into the next buffer in the Image Manager overwriting the any image currently being place there Image buffers Circular Switch A newly taken snapshot will always be placed into the next free buffer in the Image Manager unless all buffers are occupied In that case the last one to filed will overwritten again and again Prefix for images This setting applies to the standard names cell4R cell4M provides when you acquire an image using the Acquire Snapshot command Enter the prefix of an image name into the Prefix for images field A prefix consists of up to 8 characters of upper or lowercase letters numbers or special symbols Each new image you acquire will then be given automatically this prefix The default is Tv Incremental number This setting applies to the standard names cell4R cell4M provides when you acquire an image using the Acquire Snapshot command In the Incremental number list select the number to be given to the next snapshot to be acquired Each successive image s num ber is raised by 1 This value is a whole number with a maximum of eight digits The lowest value available is 1 This number will automatically be reset to 1 every t
14. Available ROIs ROL1 ROIL ROIZ ROS Ril 3 ROS Sheet Highlight selected ROIs Reference Image Start frame i E Number of Frames ho E Thresholds Threshold ose Scaling Offset 1000 El Scale Factor 1000 g Kinetic field This field is only active if the Output option Kinetics has been marked Here you have to select from which data set the ROIs shall be taken and for which ones the analysis shall be per formed If the option Highlight selected ROIs is activated those ROIs are shown with bold lines If Sheet is activated in addition to the graph also the corresponding spreadsheet is generated Reference image field Here you determine the range of images that will be averaged and taken as the reference for the analysis For example if the Start frame is set to 1 and Number of frames to 5 the first 5 images of the sequence are intensity averaged The resulting average image is the F in AF F ratios 209 210 Chapter 10 Intensity Analyses OLYMPUS Thresholds field The thresholding is performed to prevent low signal areas and background areas from resulting in areas of pronounced noise in the AF F sequence the reason being the division of small values by other small values The threshold is set in the respective box For any pixel with an intensity value below the threshold the ratio value will be set to 1 the lowest possible value so that it appears black in any standard false color display Output Scaling fi
15. Description Uninstall Transmission Control Protocol lnternet Protocol The default Wide area network protocol that provides communication across diverse interconnected networks Show icon in notification area when connected Notify me when this connection has limited or no connectivity Realtek RTLB139 810x Family Fast Ethernet NIC 2 P FPIK General Advanced Driver Resources Power Management The following properties are available for this network adapter Click the property You want to change on the left and then select its value on the right Value 10Mbps Full Duplex r 100MbpsFull Duplex 100Mbps Half Duplex 10MbpsFull Duplex 10MbpsHalf Duplex Auto Negotiation Property Link Down Power Saving Link Speed Duple Network Address Optimal Performance Receive Buffer Size WakeLUp on SAPYPING WakeUp on Link Change WakeUp using 4PM Mode 6 Return to the CTR Properties window with OK cell cell software Manual Chapter 16 Installing the cell M cell R Software 361 7 Click on the Configure button and go to the Advanced tab of the Realtek window that opens 8 Select the Property Link Speed Duplex Mode and then the Value 70Mbps Full Duplex from the shortlist Confirm with OK Additionally it is important the PC to controller network connection is NOT protected by a firewall 1 To control this go to the Advanced tab of the CTR Properties window and click on the Settings button 2
16. Iterations h e Size H Lower lirit Upper lirit Overflovy Assumed Deterioration In the Assumed Deterioration field you can define the appropriate correction method for the as sumed cause of shading Multiplicative Check here if it is assumed that the shading results from a multiplicative effect The shading correction will divide the current image by a correction image the Source for shading image Additive Check here if it is assumed that the shading results from an additive effect The shading correction will subtract the correction image from the current image A new field will appear in the lower center of the dialog box Select the Invert check box to have the resulting image intensity inverted after filtering i e to get a negative image Enter a gray value to be added to each pixel in the resulting image in the Offset field This may help to adjust the display nicely afterwards espe cially in case of transmission images Define Shading Correction Assumed detenoration Preparation of shading image OF C Multiplicative NeW average filter Cancel Interactive zero level Execute Source for shading image Polynomial fit Preview Source 1 Save shading image UIE Source 2 indow C Invert File Iterations NaH average filter Offset Em h JE Size E 128 Chapter 8 Image Processing OLYMPUS Source for shading image In this field you determine which image is to
17. a Open Settings gt Control Panel System from the Start menu b Select the Advanced tab c Check that the User variables Temp and tmp are set to D Temp Otherwise change the variables to D Temp by selecting Edit How to run cell R cell M in a normal user account under MSWindows XP 1 Log on as Administrator 2 To create a new user using the Computer Management click Run in the MSWindows Start menu In the command line of the window that appears type mmc c windows system32 compmgmt msc and click OK 3 Select Local Users and Groups gt Users and Action gt New User and fill the required fields The new user is member of the group Users only 4 Check that the newly created user has permission to write into the Archive directory where the databases are created when working with cell4R cell4M To check the secu rity of a directory file the Security tab in the Properties dialog to be opened via right click has to be made visible a Start the Control Panel b Open Folder Options c Select the View tab and make sure that the option Use simple filesharing is NOT selected 5 To check the security settings of the Archive use the Windows Explorer and do the fol lowing Select the Archive directory Right click and select File gt Properties Select the Security tab Click Add write the name of the new user under Enter the object names to select and exit with OK The user is added to the list Group or
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19. 9 For length and area measurements the cell R cell M software provides a designated environment Measurements Chapter 9 Measurements for length and area measurements with its own button bar and the Measurement View in the Image Manager All measurements become linked with the parent image and are automatically stored as part of it and can be displayed and modified at will MGEASUFEMENTS TOODA wacesscdsecsvandncevisiccdeveventevinsriceneserecaveaserdeeesae 176 Drawing Tools for Length and Area Measurements 2 00008 177 PON ome reese mere eI tase Dee ire rote MD MOEN Ney Oey OAT RE DEINE REN Ae Cater ras wer 177 CTU e ai Oxo 0 a astern anne tema ramnnend E e tom eters ners eee eerie sere 177 T le jdm EA arte em reat meron ae et te iene ie eae at nee A atin ene ee ee ere 178 EE yh preteens ee aces aati erat AE te eee E E A T Ane eetece 180 PANS E cee E E E E anced 181 Mag VV ANO dn a a 186 Magic Wand Options cccccccccsseeeeeeeeeeeeeeeeeeeeeeeeeeseseeeseeeesseaes 187 PRC SUING eea OE E Ge E E E REER 188 WOVE O MOI eoe e r Ea E 188 Create Measurement Sheet cccccsseccseeecsseeceeseeseeseesaeeeesaees 189 Deleting Measurement Results cc ccceceeceeeeeeeeeeeeeeeeeeeneeeeaeeeees 190 image HINK ossai anra a aa T AEE EAA a ANa ATAN 191 Show Hide StatiStiC iscisenisriitaniiniiani kaniin ininda nnani na iniaa anran 191 Select Measurements cccsseccceeeeceeeecceeeceeseesaeeeseueessae
20. C Dialog box function What will happen The New Module command lightens your workload when you wish to gener ate a new directory then set up a source file SFM there and then have this file integrated into a make file MKU The text of the source file SFM will be opened in its own text window within the Graphical User Interface GUI The new module will automatically be compiled into an executable file SXU then loaded and made into an active module What s it for The New Module dialog box is where you determine which key segments of the new module source text are to be givens e the Main function e the Exit function e one of various MAPI Sample functions with varying properties MAPI functions are the only ones that can be used as menu commands O The various settings available in the New Module dialog box generate different source texts We recommend that you should try using these source texts when you first try programming a C module these can serve as examples 304 Chapter 14 Imaging C OLYMPUS Adding source files to a new module If the Create sub directory check box is the only check box that has been selected the Edit Mod ule dialog box will be opened Here is where you can have files added to a C module This allows you to integrate existing source and definition files into a new C module Create sub directory Current directory When you have selected the Create sub directory
21. Clicking on the Delete All button deletes all expressions from this list Command Clicking on the Command button will give you access to any expressions you might need The Command dialog box will be opened 14 16 Toggle Breakpoint Use this command to set or delete a breakpoint in the active text document You may also use the lt F9 gt key Available This command is available as long as a text document is opened and active 2 Breakpoint 325 326 Chapter 14 Imaging C OLYMPUS In a text window the symbol of a hand at the beginning of a line indicates a breakpoint Definition When a command for which a breakpoint has been defined is executed the execution of the Imaging C program or module will be interrupted The definition at the location of the break point will be displayed and the Debug button bar will appear The source text will appear in gray The mouse cursor will be transformed into the shape of a hand The breakpoint icon will be trans formed into footprints a In the Debug mode this footprint icon will always appear in the line to be executed next This en ables you to either view the value of a variable or to continue execution in single steps Possible breakpoint positions Breakpoints can be set in any line A breakpoint can however be invalid In this case the breakpoint will be skipped and ignored To locate invalid breakpoints have a look in the Edit Breakpoints dialog box a minus sign denotes an
22. Image Display gt Edit LUT to edit an existing color palette or to cre ate a new one The library of palette files provided together with cell4R cell4M Imaging Software is stored in the folder Cell R M LUT on the hard disk Available This command is available for all image types with the exception of 8x8 bit true color images False color palettes or lookup tables LUTs make it possible to display gray value images as well as individual color channels of nx16 bit images in color This is done by the palette assigning a certain color to each gray value of the image For details see previous section 6 7 5 Different palette types There are three different ways to define palettes these includes e Type all palette values directly into a sheet 88 Chapter 6 Image Display and Navigation OLYMPUS e Generate palettes interactively by defining polygons e Use formulae to have the palette computed The Edit False Color command opens a dialog box which provides tabs for each of the above mentioned methods Each of the three methods generates a different palette file format LUT LUP and LUF respectively In effect you are editing a separate palette in each of the three tabs see below in the Edit LUT dialog box i e each time you go to another tab you will not only switch to a different editing method but also to an altogether different palette of the image itself In general it is not feasible to automatically c
23. Resulting images are thus more detailed and appear more focused 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 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 filter Bandwidth defines the range of gray values belonging to the lesser gray value modulations 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 intensity the lesser gray value modula tions are comprised of When the bandwidth is narrow gray value modulations of a minimal dy namic range will be strongly enhanced This means that low bandwidth values result in greater cell cell software Manual Chapter 8 Image Processing image detail via a suppression of high amplitude contrast modulations Broad bandwidths result in greater contrast but also contain le
24. Tel 49 89 89 55 805 660 Fax 49 89 89 55 805 6606 Email info olympus sis com www olympus sis com OLYMPUS LICENSE AGREEMENT between END USER and OLYMPUS SOFT IMAGING SOLUTIONS regarding the OSIS cellAM cell4AR SOFTWARE PRODUCT IMPORTANT READ CAREFULLY Below you will find the contractual agreements governing the use of the OSIS cell4M cell4R SOFTWARE PRODUCT These conditions apply to you the user and to OLYMPUS SOFT IMAGING SOLUTIONS With any of the following actions you explicitly agree to be bound by the conditions of this contract purchasing the software opening the package breaking of one of the seals or using the software In case you do not agree with any of the conditions of this contract please return all parts of the product including manuals and the software protection key before using and without delay Remove all software installations of the product from any computer you might have installed it on Return all electronic media of the product or completely destroy all electronic media of the product and send proof that this has been accomplished For a refund please return every thing to where you purchased the product 1 Scope 1 This License agreement explicitly covers only the software disk ettes or other media you received with the purchase and the software stored on these media the manuals as far as they were developed and produced by OLYMPUS SOFT IMAGING SOLUTIONS 2 User rights
25. Tw Source 2 2 Tye Source 2 2 Tye Operatian Operation Subtraction Ww Subtraction ka Source 2 Constant Value Single Image 2 Tye Subtraction AOR 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 addition subtraction multiplica tion or division the gray value range of the result s may be greater than that of the original im age 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 re duce 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 bi nary representation of 8 or 16 bit numbers must be taken into consideration Arithmetic Operations 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 152 Chapter 8 Image Processing OLYMPUS AND Pixel values in binary form are compared bit by bit If the bit is 1 for both
26. Which gray values become white and which black To transform a gray value image into a binary image it has to be known which pixels of the original image are to be displayed in black and which ones in white This is 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 intervals Use the Process gt Set Thresholds command to define these gray value intervals which is done by defining one or more phases 167 168 Chapter 8 Image Processing OLYMPUS Binarize If only one phase has been defined currently the Binarize command will automatically 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 Binarize Phase Phase 1 OK Cancel Phase 2 Select all 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 that are not next to one another displayed in white Binarize Color Image If only one ph
27. Zoom 76 EFI Extended Focal Imaging 98 Experiment 70 Marker 73 Experiment Manager 14 38 Digital port TTL 54 66 Execution Center 41 Experiment Plan 42 Microscope 58 Wait 55 Filter 131 DCE 146 Differentiate 135 Edge enhance 143 Laplace 136 Lowpass 143 Mean 137 Median 137 NxN 142 Pseudo 138 Rank 144 Reimer 140 Roberts 139 Separator 148 Sharpen 134 135 Sigma 145 Sobel 139 User 141 FRET 224 Graph 236 Preferences 293 Illumination system 25 59 Configuration 330 Image Align 156 Animate 17 Calibration 102 Combine 117 Convert 119 Extract 116 Information 121 Markers 123 Mirror 155 Navigation 17 93 Overlay 99 Parallel Navigation 97 Resize 152 Rotate 154 Separate 116 Image Manager 9 297 Imaging C 302 Intensity DeltaF F 208 Histogram 197 Kinetics 207 Line profile 198 Pixel value 196 Live View 12 Load 14 Macros 266 Magic wand 186 Measurements 176 Angle 180 Area 181 Length 178 Preferences 289 Results 188 Select 191 Statistics 193 Menu Bar Define 274 Microscope 26 Configuration 333 Module 303 Motorized stage 29 50 67 Calibration 32 Configuration 344 Network connection 359 Parfocality correction 346 Phase Analysis 222 Color coding 221 Preferences Database 294 File 284 Graph 293 Image 280 cell amp cell Software Manual Measure 289 Module 291 View 282 Projection 97 Ratio Analysis 210 Regions of Interest ROIs 201 Save 13 Sca
28. add up to the color that all pixels of that particular intensity value are displayed in on the monitor If all three color components have the same intensity R G B the pixels are displayed in a shade of gray Possible Colors If all intensity values and combinations thereof are used each pixel can be dis played on the monitor in one of 256 256 256 16 777 216 colors Pixels of false color images 8 bit or 16 bit in contrast to 3x8 bit true color images are restricted to one of 2 256 different colors this is a limitation of the false color palettes These 256 can however be selected from all 16 777 216 colors Using palettes to alter gray value images An image s palette only defines how gray values of an image are displayed onscreen The original gray values under the palette remain unchanged The collection of palettes The palettes Blue Cyan Green Magenta Red and Yellow are sim ple linear monochrome black to color palettes Polaris Solaris Thermal Gamma3 and Dither are kind of standards that are probably of limited use in fluorescence imaging 17 ase az 192 176 The Rainbow1 2 3 palettes first second and third from left respectively shown above are espe cially useful for contrast enhancement The most appealing one might be Rainbows that begins with a black to blue gradient SemRainbow1 and 2 fourth and fifth from left respectively with their inherent dark to light gradient
29. 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 165 166 Chapter 8 Image Processing OLYMPUS 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 The HIS tab sets thresholds based on the HSI Hue Saturation Intensity model The RGB and HSI tabs both have the same structure Below is a description of only those controls that have a function differing from that in the RGB tab Set Color Thresholds Phase MEREM Hue 211 3 B 206 3 ae one pat og 124 Current m 88 8 On Background _ Include pixel Include Exclude Curve Color used in diagram Color value magenta Saturation light blue Intensity black 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 intensity For Hue color tone you can enter values between 0 and 360 steps of 0 5 are acceptable The Hue Sat Satura tion and Int intensity fields indicate the upper and lower limits for the respective hue saturation and intensity channels The easiest way to define color ranges is to go right into the image and select a portion o
30. it only alters the on screen display The execution of the command Image gt Image Display gt Black Balance is done in the very same way as the white balance described above 6 2 6 Gray Scale This command turns a false color image display back into the original gray value image display The image s false color palette will be deleted from the Viewport It will be lost if the palette itself is not part of the provided library and has not been saved as a separate file Available This command is only available for single channel 8 bit or 16 bit false color images This command does not affect the image data it only alters the on screen display 83 84 Chapter 6 Image Display and Navigation OLYMPUS 6 2 7 Fluorescence Color This command is used for displaying single channel images or individual channels of a multi channel image with the monochrome palette defined in the cell R cell M Configuration Soft ware see Chapter 15 cell R cell M Configuration for the corresponding filter of the Illumination System MT20 MT10 used for acquisition for more details see Part B Hardware Manual Available This command is only available for single channel 8 bit or 16 bit images and for n 16 bit images in the single channel display mode via the Select Color Channel button O This command does not affect the image data it only alters the on screen display 6 2 8 Edit Fluorescence Color Images acquired via the Experime
31. s cell software Manual Chapter 15 cell4M cell4R Configuration 343 15 5 Configuration of the PIFOC Use the PIFOC dialog for the configuration of an optional piezo electric objective drive or nose piece drive called PIFOC piezo focus OBS System Configuration Illumination System General General Pifoc Available Excitation Filters Burner Full Control E Inverted Microscope Upright Microscope Errors ECTOSEO De Maximum Limit 100 00 General 2 Drive Objectives Filter Cubes Contrast Inserts Filters Current Position Step width 0 50 Image Types Shutter Image Splitter ap Stage oo pm a counts General First you have to select if a PIFOC is available and whether an upright microscope or an inverted microscope is used The selection determines the convention of the objective movement This means for an inverted microscope that an upward movement of the objective is displayed as a positive movement Range The Maximum limit defines the maximum traverse path and the Step width of the PIFOC A standard objective drive has a range of 100 nm while a nosepiece drive has 80 nm Step width This parameter defines the step size when the respective slider is moved in the Illumi nation Control window upon mouse click see Chapter 4 3 Move The scroll bar represents the traverse path of the PIFOC You can move the PIFOC within these limits either stepwise by clicking on the arrows or via m
32. C A Bisector Outer Max Distance Help rat Bisector Outer Max Angle C Bisector Outer Mean Distance Get Default Selected Measurements Object Type Port r Point Length Line Value Angle i 2D Object Perimeter 20 Object The area of a measured object is number of pels of the measured object times calibration factors int and direction Tree View It is located in the upper left hand part of the dialog box and lists the different types of measurement objects Click on any type to list all measurements possible for this type in the Measurement Parameters list Measurement Parameters list Add new measurements to the Selected Measurements list by marking the respective check box or remove measurements by unmarking the check box Selected Measurements list Click on a measurement function in the list to show the description and a schematic drawing in the dialog box Move the selection up or down with help of the Arrow Up or Arrow Down buttons on the right or deselect the measurement by clicking the Delete but ton It can be added anew to the list any time x Delete If changes are made the Measurement Display becomes updated automatically upon closing the dialog with OK cell amp cell Software Manual 9 4 7 Define Statistics fia Define Statistics This button opens the Define Statistics window Define Statistics Available Current Average Deviation Delta Max Mean Delta Min Minimu Kurtosis Masimum M
33. Correction Focus List Correction If you observe a focus drift do the following 1 Go to any list position and correct the focus Right click the position in the list to open the context menu and click Set Current Posi tion As Reference A green letter R will appear to the left of the position number in the list and the command Focus List Correction in the context menu will become enabled 3 45075 37290 6264 51 56640 5 47974 35775 5729 58 Upon executing the command List Correction a message like the following will appear Olympus 0Y100 4 Click Yes and the Z coordinates of all positions in the list will be adjusted by the amount given in the message Correction of a tilt of the sample If the sample is tilt relative to when the positions list was defined a simple focus correction will not help In that case setting three reference points defines a reference plane With the help of this plane the software determines individual focus corrections for each position in the list 1 Go to any list position and correct the focus 32 Chapter 4 Image Acquisition and Hardware Control OLYMPUS 2 Right click the position in the list to open the context menu and click Set Current Posi tion As Reference A green letter R will appear to the left of the position number in the list 3 Define two more reference positions by repeating steps 1 and 2 at two other positions in the list E 2 44517 3945
34. Illumination System MT20 MT10 anew 6 Next you have to fill in the customer information and confirm it 7 Inthe Display Device Selection window you have two options a Single Screen Windows Display Use this option if the system features only one monitor You can also use it in case of two monitors The cell4R cell4M interface window can simply be dragged open to cover both screens Together they behave as if they were one big screen It is convenient in this case to place the Viewport in the second monitor while all control windows and the database remain in the first b Dual Screen Dual Screen Win If the system has two monitors you may choose this option The second monitor will then be filled out entirely and unchangeable with a second Viewport The Viewport Manager allows switching focus from one to the other Viewport via mouse click 8 Click Next gt and decide if you want the Life Science Demo database from the second CD to be installed Click Next gt This database has not changed in the new version and there is no need to install it anew 9 In the following three windows choose the Destination Folders where the program files are to be written image files are to be stored and image databases are to be saved The setup will suggest the existing folders probably D cell R M D cell R M Images and D Archive It is most convenient to accept each time and simply continue with Next gt 354 Chapter 16 Installing
35. Images cccccccecssseeeeeeeeeeeeeeeeeenaes 220 10 10 Phase Color Coding and AnalySiS ccccccssseeeeeseeeesseeeeeeeeeeesaees 221 10 10 1 Phase Color Coding cccccsssccceeseeecseeeeeesaseeeeeeseeesaeeeeseaeeeesaees 221 10 10 2 Phase AnallySIS ccccscccsseecescccseeseeeeceeeceeeesaeeseeeesaeeeeseeseeeenaeees 222 WU Coed O ee E E E 222 10 12 The FRET Software Module ccccccccssseecseeeeeeeeeeeseeeeeeenaeeeesaees 224 10 121 mags ACQUISINION wessentsoieinetsus cacessnrcaaweaaestanpoundaccansteanceseneadeasncioees 224 10 12 2 FRET Image Correction Factors cccccccecseseeeseeeeeseeeeeseeeeeeeenees 226 Wg 2 FREF ANAN SiS pereen e e E EE Eaa 228 Image Analysis Image Analysis The analysis features explained in this chapter are very important for cell biological applications many can be called with the buttons in the Image Analysis toolbar 10 1 Pixel Value Use the command Measure gt Pixel Value to determine coordinates and measure gray or color value s of a single pixel Shortcut Right click an image and select Pixel Value from the context menu that pops up Checking the values on the flight Right click on the image and click on Pixel Value in the ap pearing Context Menu The status bar in the lower left corner of the cell4R cell4M window dis plays the gray value for each color channel at the current mouse cursor position Stop You can terminate the action by opening the re
36. In case of aCC12 camera select OBS CC12 4 Exit with OK 5 Calibrate the camera pixel size as described in Chapter 7 1 Calibrate Images 16 4 Single User Systems Administrator Users If the system is used exclusively with Administrator rights by one or several users the following has to be observed By default the hard disk of the cell R cell M computer has two partitions e Partition C the smaller one contains the operating system standard software and enough space to install additional programs e Partition D the large one contains the cell R cell4M software and the folder Archive where the databases are usually created The archival path can be changed in cell4R cell4M via Special Preferences Database in the field Database files The path should not be set to C otherwise memory problems will occur soon while using the system The corresponding error message would be Could not reserve enough memory Make sure that the Temporary storage directory to be set in the same dialog box is on the same disk partition as the database directory otherwise problems might arise at least in case of fast data acquisition cell cell software Manual Chapter 16 Installing the cell M cell R Software 357 16 5 Multi User Systems Note the following if user accounts in addition to the Administrator are to be installed on the cell4R cell4M computer There are two main differences between operati
37. Installing the cell M cell R Software ccsscssseesseeneees 351 The details to take care 16 1 Updating the cell4M cell R Software cceeeeeeeeeeeeeeeeeeeeeeees 352 of when installinganew 16 2 Updating the cell M cellAR Hardware Control 00sseeee 354 software version 16 3 Selecting the Camera c ccccccccscscsssccssscsssseseescseeesseeesseeesseaeeseeeeses 355 16 4 Single User Systems Administrator USersS ccceseeeeeeeeeeees 356 16 5 Multi User SySteMms cccccseccceseeceeeeeceeeeceeeecaeeeseeeeeseueesseeeesees 357 16 6 PC to Controller Network COnnectiOn cccccccececececececececececeees 359 Contents OLYMPUS cell amp cell Software Manual Contents cell a cell software Manual Chapter 1 Introduction 1 1 Introduction Thank you very much for purchasing Olympus state of art cell M or cell R Imaging Station and for your confidence in our products and service It is Olympus main objective to provide you with solu tions able to meet your experimental demands and thus pave the way to your scientific success 2 Chapter 1 Introduction OLYMPUS Introduction The concept and technology of cell4M cell R introduces the next generation of imaging worksta tions cell4M cell4R is designed as a modular imaging system for a broad range of life science experiments that supports the Olympus microscopes of the IX and BX series Hallmarks of the systems are e
38. Make sure that Off not recommended is activated in the Windows Firewall window O The PC to controller network connection is not a computer safety risk and there is no reason for a firewall protection This internal network connection is used only for the system commands traffic and not a gateway to the internet or intranet Windows Firewall General Exceptions Advanced pis CTR_R Pro pe rties alta x our PC is not protected turm on Windows Firewall ication Ad d General Authentication Avance Windows Firewall helps protect your computer by preventing unauthorized users from gaining access to your computer through the Internet or a network Windows Firewall Protect my computer and network by limiting amp C etti 1 or preventing access to this computer from aSa iian O Dn recommended the Internet This etting blocks all outside sources from connecting to this computer with the exception of those selected on the Exceptions tab Internet Connection Sharing Allow other network users to connect through this computer s Internet connection f Select this when you connect to public networks in less secure locations such as airports ou will not be notified when Windows Firewall blocks programs Selections on the Exceptions tab will be ignored Leam more about Internet Connection Sharing Avoid using this setting Turning off Windows Firewall may make this computer more vulnerable to viruses
39. Memory size 26094 KB File size Created 05 06 2003 11 30 Color Channels Channel Name Color Attenuator Exp me Bin Magnification B Te HA 1 Furaz40 CL 100 00 130 0 Resolution 353 310x 16 Width 38 12 pm Height 33 46 pm Image intensity Gray Value Comment How to open the Image Information dialog box Double clicking on the image manager Double click on any image buffer within the image man ager to view information on that image you can also alter this information Double clicking on an image buffer will automatically activate that image buffer e lt Alt Enter gt key Use these keys to view information on the image within the active image buffer 122 Chapter 7 Image Data Handling OLYMPUS Image Manager context menu Right click on any image buffer to open the corresponding con text menu and then click on Image Information The information then displayed refers to the image you right clicked on it doesn t have to be the active one Viewport Manager context menu Once an image is being displayed on the monitor simply right click on the relevant Viewport within the Viewport Manager to open the corresponding context menu and then click on Image Information The information then displayed refers to the image you right clicked on it doesn t have to be the active one Image Document context menu Once an image is being displayed on the Windows monitor simply right click on the
40. Menu and the Window Menu Macros are little programs that allow repeating series of processing and analysis steps upon a click of a button Here we explain how such macros can be generated Also you will find in detail how personal preferences for the software user interface can be set Moe MATOS ois cc uxc8icias sass tase untae ntnaunadecedenaduasaunncncadecad uteduantamaduaauaientncueaicea 266 eS Wel E EE Nl PE AE A A N E E E E E TE 266 t12 RECO MaC Osad a a i 266 ASS EXSCGUHINO MACIOS sarriro a E a aa e a EAA REE Da ANRA 267 13 14 Stop Macro RECOrdE aessa E 269 Toal RUNM IIA ChO se cies e a aa a Os chinie tae aticseesedeceesaies 269 1S TOs Ogie SIC scr ssetaceia tar aticeiu gots lulestratiedanacs A 269 ISET TACSCUIMNIGKOFCISl erone a e 270 13 1 8 Set as Default Macro vaceciccccesccaccasedencassececcasixcerecesiiecdesiactexsadiaccst 271 T919 Dee MO Oa ics seca telah ee a ant a a N 271 toe AJOIN Manage s eraa di 2 3 t39 Dene VICI Ga E a 274 13 4 GUI Configuration ccccccccseccesesseeeseseeecseaseeeseseeeseaseeeneneeesneaees 277 toak RES 21 BRA ee a ni ee eR oe ee Pee ae eR eee a 2 8 ee WO ACR chen cas cemoncede E sata aecsie E E saat tesebeetensaaine 278 BS 4G OWO A E E OP OER E E E REE OOT 279 TOs Prelog Se E E die 280 13 5 1 The Preferences gt Image Tab o nssnnonnnesnsnnnnrnrnnnrnnnrenrrerrrnrrnene 280 13 5 2 The Preferences gt View Tab snsnsensesnnnnnnnnnnnnnnnnnnnnrnnnrnnrnrnrnnnne 282 13 5 3 The Pref
41. OLYMPUS Graphs 1 Histogram of Dapi Fitc TxRed ens Fase Qe v greer 101 Histogram o v Fi Histogram of Dapi Fite TxRed 13000 Gray vale 10 3 Line Profiles Intensity 10 3 1 Horizontal Line Profile Use the Measure gt Intensity Profile gt Horizontal command to determine the gray value intensity along a horizontal line How to measure a horizontal intensity profile 1 Select the Horizontal command A horizontal red line will appear in the overlay 2 Position the red line where you wish to measure gray value intensity and then left click 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 command with a right click and confirm yes cell a cell software Manual Chapter 10 Intensity Analyses The line profiles of all color channels will be drawn into a separate graph for each of the lines drawn into the image overlay O This command works currently only on the active frame not on an entire multi dimensional image set 10 3 2 Vertical Line Profile Use the Measure gt Intensity Profile gt Vertical command to determine the gray value intensity along a vertical line Conduct the measurement as with the Horizontal Intensity command O This command works currently only on the active frame not on an entire multi dimensional image set 10 3 3 Arbitrary Line Profile Use the Mea
42. Resonance Energy Transfer studies are based on the observation at different wavelengths which can be performed conveniently with the fast switching filter wheel U FFWO see the U_FFWO Instruction Manual for installation and configuration A second option is the Dual View Micro Ilmager beam splitting device that allows the simultane ous acquisition of two chromatically separated fluorescence images of half frame size The Micro Imager splits the camera field of view into two halves They cover identical specimen areas but show different spectral contents Depending on the alignment the split may be horizontal or verti cal The cell4M cell4R software is able to overlay the two image halves into one dual color image To do so certain settings in the ObsConfig software are necessary see Chapter 15 9 The Configu ration of the Dual View Micro lmager The FRET software add on to the cell4M and cell4R imaging software provides different analysis algorithms Ratio MicroFRET FRETN N See also the original papers MicroFRET D C Youvan et al 1997 Biotechnology 3 1 18 FRETN G W Gordon et al 1998 Biophysical Journal 74 2702 2713 Neer Z Xia Y Liu 2001 Biophysical Journal 81 2395 2402 10 12 1 Image Acquisition 10 12 1 1 Snapshots and Live View with the Dual View Micro Imager 1 0 314 600 Image split cell a cell software Manual Chapter 10 Intensity Analyses The Camera Control window features the
43. Table Select the String table check box if you wish the new C module to support different languages In this case the source text will contain the Operator instead of strings and followed by a con stant e g IDM_SAMPLE in place of Info in the status bar Example Imaging C Code export UINT SampleFunction lt MAPI IDM SAMPLE IDM SAMPLE IDM SAMPLE 1 gt dlgOutput IDS MESSAGE return IDOK Dialog box Function Screen shot Sample Dialog Select the Dialog box function check box if you wish to have a look at sample programming of a dialog box using Imaging C What will happen is that a sample dialog box will be created when you activate SampleFunction Example Imaging C Code define IDD BEEP 100 define IDD CHECKBOX 101 define IDD EDIT 102 MYDIALOG DIALOG 130 72 186 76 STYLE DS MODALFRAME WS POPUP WS VISIBLE WS CAPTION WS SYSMENU CAPTION Sample Dialog FONT 8 MS Sans Serif BEGIN DEFPUSHBUTTON OK IDOK 133 12 48 14 PUSHBUTTON Cancel IDCANCEL 133 30 48 14 PUSHBUTTON amp Beep IDD BEEP 133 50 48 14 cell cell software Manual Chapter 14 Imaging C 307 LTEXT amp Edit Controls 1 6 20 51 8 EDITTEXT IDD EDIT 60 18 54 12 ES AUTOHSCROLL CONTROL eCheckbox Control IDD CHECKBOX Button BS AUTOCHECKBOX WS TABSTOP 10 41 71 10 END Callback function of the dialog box BOOL SampleDlgProc HWND hDlg UINT uMsg WPARAM wParam LPARAM lParam
44. This method performs the same pixel by pixel intensity division to create a ratio image used for ion ratio imaging see Chapter 10 8 Ratio Analysis No channel bleed through correction is being ap plied Dual emission images have to be acquired using Experiment Plans as described in Chapter 10 10 1 2 Dual Emission Experiments with the Dual View Micro lmager or Chapter 10 10 1 3 Dual Emission Experiments with an Observation Filter Wheel cell a cell software Manual Chapter 10 Intensity Analyses 229 The following filters are needed Donor exciter Dual band dichroic mirror Donor emitter Fdon channel Acceptor emitter Ffret channel Create a ratio image as follows 1 Select the dual emission image Fa 2 Click on the FRET Analysis button to open the dialog window FRET Analysis settings Dimensions Info Method Ratio CONFRET Youvan 1997 FRETn fGordon 1998 CO NiFret Kia 2001 Correction Factors Input Images Channels Selection 4 FRET cfp exc FDon cf 4 FRET cfp exc FFRET Background Subtraction None Select ROIs From Image Constant 4 FRET cfp exc 11 4 ROI Select ROT Image ROLI ROIL v Thresholds Threshold 1 Threshold 2 w Em Qutpuk Scaling Scale Factor 1000 E 3 Select the Method Ratio 4 Make sure that the Fdon and Ffret channels are selected correctly 230 Chapter 10 Intensity Analyses OLYMPUS
45. adjust the size of the displayed images given as of the original image size If you select Auto the image size is automatically adjusted to fit into the size of the Viewport A Adjust Zoom This allows adjusting the zoom factor to the current Viewport size It has the same function as Auto in the Zoom Factor shortlist Adjust Window This function fits the Viewport size to the size of the displayed image This command is only avail able in the single view mode Some basic information is given here about the active data set From left to right it lists The Viewport number here 8 the name of the data set here 3 D Time Lapse the current point in time out of a total number of time points here T 5 20 in case of a time sequence the current layer out of a total number of layers here Z 1 11 and the magnification here 150 m Full Screen This function generates a full screen view of the active image Additionally the Menu bar the Full Screen button bar the Acquisition toolbar and the button bar of the active document Viewport database or graph remain available 78 Chapter 6 Image Display and Navigation OLYMPUS a E E E E close Full Screen returns to standard view HE waala oe Es Select previous buffer displays the image in the p Image Manager buffer atop the current image a ja pom Select next buffer displays the image in the Image Manager buffer below the current image E Select a docu
46. again As always a right mouse click termi nates the command im Rectangle Rectangle A rectangle opens the size and shape of which can be adjusted by dragging with the left mouse button cell a cell software Manual Chapter 10 Intensity Analyses o Rotated rectangle Rotated rectangle With this command rectangles of arbitrary orientation can be drawn The left mouse button toggles between the Move and the Rotate modes indicated by the crosshair and the circular arrow respectively in one corner Dragging without a mouse button clicked performs the moving and rotating In either mode a left button drag changes size and shape The best way to draw is to open a rectangle at any position and rotate it so that the edges are parallel to the desired position Then change to Move mode and position the corner opposite to the crosshair to its final position and finally adjust the size of the rectangle via left button mouse drag Virtual Virtual This button is currently without function TA nit ae Fro m Particle From Particle This button is currently without function Es Magic wand Magic wand See Chapter 9 3 Magic Wand for details m m amp Magic wand options Magic wand options See Chapter 9 3 Magic Wand Options for details Move Move This enables to move any ROI highlighted in the Active ROI list B Delete Delete This removes the ROI highlighted in the Active ROI list 203 204 C
47. analysis features explained in this chapter are very important for cell biological applications Line intensity profiles illustrate intensity changes along arbitrary lines within an image Commonly regions of interest are defined to determine average intensities and follow their changes through image series to quantify dynamic processes and display the results in graphs and sheets Meaning ful quantification usually requires background subtraction Special image ratio algorithms are used to quantify ion imaging experiments Spectral bleed through in multi labeled specimen skews quantita tive analyses but can be corrected with spectral unmixing The enhanced chromatic resolution pro vided by this technique also allows clearly distinguishing spectrally very similar fluorochromes TOS Pixel VNC acer e a a aa ENa eens 196 TO PSO OLIN oi a a 197 10 3 Line Profiles INteENSIty ccccceccceeeceeeeceeeeeeeeceeeeeseesaeeeseeesaeeenes 198 10 3 1 Horizontal Line Profile cccccscccsececeeeeeeeeseeeeeseeseeeeeeeseeeenes 198 10 32 VeRICal LING PrOMe ixcwshivestwodsioswelibastued end a e a ii de 199 10 3 3 Arbitrary Line Profile cccccceccseecceeeceeecceeeceeeeseesaeeeeseeseeeenes 199 10 3 4 Average Intensity of Neighboring Pixels ccccceeeeeeeeeeeeeeeees 200 10 4 Regions of Interest ROIS ccccecccseseceesesseeeseeeeeeseeeeeeseneeesnaeees 201 TOBA Generalne oieran fotsetin cree a E Sata ete
48. are particularly nice for displaying small features on a black background The ColdHot palettes below are designed to highlight extremes in intensity i e the dimmest and brightest features of the image They also allow judging optically the dynamic ranges of image ar eas ColdHot4 fourth from left with a rather narrow gray center was designed to easily distinguish if scaled accordingly areas of increasing and decreasing signal intensities in AF F plots The palette currently loaded is marked with a tag in the menu Image gt Image Display and in the shortlist of the Select Color Channel button cell s cell software Manual Chapter 6 Image Display and Navigation 87 6 2 9 1 The palette bar displaying the current LUT It is possible to have the current lookup table LUT of displayed in the Viewport To do so open the Scale Bar Properties window via Image gt Scale Bar gt Properties and select the Palette bar check box This function can be used for 8 bit and 16 bit gray value images and false color images In case of multi color images the palette bar will be displayed only if just one of the color channels is being displayed in the Viewport This can be done via selection from the pull down menu of the Select Color Channel button see Chapter 6 3 2 Multi Color Images As soon as the Viewport becomes smaller than a certain size the palette will disappear 6 2 10 Edit False Color Use the command Image gt
49. be a MKU make file for this module A text document has to be open and active in the Graphical User Interface GUI Module Info Click on the Module Info button to view information on the module selected The Module Info dialog box will be opened This button is only available if you ve selected a module in either the Loaded or the Other lists Activate Click on the Activate button to activate the module selected The previously active mod ule will be deactivated This button is only available if you ve selected a module in either the Loaded or the Other lists Deactivate Click on the Deactivate button to deactivate the active module The Active field indi cates which module is active This module is the one you had either recently activated or opened it does not necessarily have to have been selected in one of the two lists After you deactivate a module no C module will be currently active This button is only active when a C module is active Module The Module field indicates the path of the module currently selected in either the Loaded or the Other lists File The File field indicates the path of the active text document Using the Add button you can add this text document to the selected module Active The Active field indicates the path of the active module Only one module can be active at a time cell s cell software Manual Chapter 14 Imaging C 319 14 10 1 The Define Search Path for Modules Dialog
50. be displayed in the overlay and written into a sheet which will be generated automatically cell s cell software Manual Chapter 9 Measurements If you do not want the results to be shown in the overlay select Show labels gt None in Spe cial Preferences Measure or Measurement Properties button in the Measurement View or key F8 9 2 Drawing Tools for Length and Area Measure ments 9 2 1 Point A Point Use this command to measure parameters of single pixels With each left mouse click a colored cross is drawn in the overlay The XY coordinates of pixels are measured by default The X coordinate is then shown in the images overlay while the X and Y coordinates are displayed in the measurement display in the Image Manager To determine further positions click left A right click lets you exit the Point function The results are displayed in the overlay One can then start to count a second set of objects or terminate the command with another right click 9 2 2 Touch Count ae Touch Count Use this command to count objects in an image Several measurement series can be conducted successively to count objects of various classes With each left mouse click a colored cross is drawn in the overlay at the position of the tool tip and the count is increased by 1 A right click stops the counting The result is displayed in the over lay and written into a sheet generated automatically One can then start to count a second
51. be given to the multi color image set in the folder of the current experiment in the database If no name is given it will be cell s cell software Manual Chapter 5 Experiment Manager stored under a default name Each color band will carry the name set in the corresponding Image Properties page see the previous Chapter 5 3 2 1 Image Acquisition Properties Display If this option is selected an on line image with a color channels will be dis played and constantly updated on the Viewport during the experiment for example a time lapse or a Z stack acquisition In this case the Multicolor frame shows a monitor symbol For further de tails see previous Chapter 5 3 2 1 Image Acquisition 5 3 2 3 The Z Stack Frame E Z Stack frame Use for subsequent acquisition of images at different focus Z levels Execution of this command will cause the following e The commands within the 3 D box are repeated on subsequent focus Z levels e The images will be stored as Z stacks e Multi color Z stacks can be acquired if the Z Stack Frame contains a Multi Color Frame with more than one Image Acquisition icon O The command can only be executed when a PIFOC or a motorized Z Drive is avail able Properties Z Device If both a PIFOC and a motorized Z Drive are available you have to select the targeted device from the pick list Absolute Movement Current pos This read only information displays the current position of the
52. be used as correction image This can be either the image itself that is being corrected itself Source 1 or it can be a reference image from the Src 2 image buffer in the Image Manager Source 2 i T s D 1 gt 2 orc fe Dest Src Mask Preparation of shading image Define here how the how the correction image the Source for shading image is to be preproc essed before the correction O Use this option if a reference image for correction has been acquired and does not need further processing Such an image might either be taken without a sample or without illumination The Source 1 option is disabled in case of this option because otherwise the active image would either be divided by itself or subtracted from itself re sulting in empty images consisting entirely of pixels with the value 1 or 0 respectively NxN average filter The NxN filter is a smoothing filter to eliminate noise as explained in Chapter 8 2 15 It averages the pixel values in a square area defined by the value in the Size field around each pixel and assigns the result to it This process can be repeated several times depending on the number set in the Iterations box The resulting shading correction is also known as unsharp masking MaN average filter Iterations ae Size A Interactive zero level This is a useful option if obvious illumination gradient is visible in an image for example due to the illumination not being centered correctly
53. buffer will be raised successively to the 8th image buffer and then go back to image buffer 1 Multiple view In this case the circular switch range is meaningless If you re working with several Viewports the destination image buffer will be set to the next respective Viewport If for example you re working with four Viewports assigned respectively to image buffers 2 6 3 and 8 the des tination image buffer will be successively set to 6 3 8 and then 2 again If you re working with two Viewports for example which are currently assigned to image buffers 7 and 8 the destination image buffer will switch back and forth between image buffers 7 and 8 Write protected image buffers If the next image buffer is write protected the one after that one will become the destination image buffer Allow operations on false color images Select this check box to apply image operations that change the gray values of an image to the false color images as well Several image operations applied to the gray value image do not affect the displayed false color image i e the resulting image has altered gray values although the lookup table remains the same For this reason image operations applied to false color images may yield unexpected re sults Image acquisition Sequence The option chosen here determines where a newly acquired snapshot will be placed in the Image Manager and whether it will be stored in the active database or not
54. camera Of course focusing can be done via the ocular but this will not be explained here Click Shutter in the Illumination System MT20 MT10 control box to illuminate the specimen Click Acquire in the Acquisition toolbar or the Camera Control window The following happens cell cell software Manual Chapter 3 Brief Introduction and First Steps The camera starts acquiring images at maximal speed None is stored instead the newest image overwrites the previous one in the temporary buffer The current image is displayed in the active Viewport A single image icon appears in the Image Buffer Box 8 Focus your sample with the microscope Z drive Even if the optional PIFOC is available for focus change its total range may be too narrow to find the focus at the beginning For this reason it is usually better to start with the Z drive 9 If necessary change the Binning factor in the Camera Control window 1x1 in order to get highest spatial resolution larger factors increase the signal intensity at the cost of resolution 10 Adjust the Exposure time for a good signal to noise ratio Fa 11 Click Snapshot in the Acquisition toolbar or the Camera Control window to stop the Live View The very last image is being stored as a Snapshot in the Image Buffer Box and displayed in the Viewport y 12 Click Shutter in the MT20 MT10 control box to stop the illumination 13 Save your image as described in the n
55. cell amp cell Software Manual Chapter 3 Brief Introduction and First Steps 17 3 Displaying Sequences Image Navigator EJ E H M w Navigation Time Lapse experiments and Z stack acquisitions generate series of images that are all stored together within one file The individual images can be accessed with the navigation buttons First Previous Next Last in the Navigation toolbar The number in the Go To field represents the actual frame displayed in the Viewport An additional feature enables the user to animate image sequences and play it as a movie Pressing the Animate button opens the Animate Image Stack window Animate Image Stack Animate Here you find the buttons to start Play the animation to Stop it and to play it in the Reverse mode imi Reverse Stop and Play For a detailed description of the navigation tools see Chapter 6 3 Image Navigation 18 Chapter 3 Brief Introduction and First Steps OLYMPUS cell amp cell Software Manual 4 The following chapters explain how to take snapshots or live images what camera parameters have to be adjusted for good quality and how to use the illumination system with the excitation filters and Image Acquisition and Hardware Control the motorized parts of the microscope Simple Image Acquisition si rssececendaceeessaceeeeind caveiredeeneiecedeeeeeedeeneaneces 20 Snapshot and Live VICW ccccccsseeeceseecceeeeceeeecceeeceusessee
56. 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 have 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 progres sion 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 represent 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 left clicking 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 the color channels before you can adjust any thresholds Left click on one of the phases in the color bar Keeping the left mouse button pressed you can move both thresholds simultaneously Use th
57. default the first one to be acquired being displayed in the top position The arrangement can be changed as usual via the Arrange Viewport button 71 72 Chapter 5 Experiment Manager OLYMPUS 5 4 2 3 Experiment start IY Start Once the Start button is enabled the information Prepared is displayed in the status bar at the bottom of the Experiment Manager window Upon pressing the button the experiment starts im mediately In order not to slow down the data transfer only a limited number of user interactions which might be required for online data inspection are allowed during the course of the experi ment These include e Viewport settings all functions in the Viewport toolbar e Display settings all functions in the Image Analysis gt Image Display dialog box of cell4R cell4M Likewise the two buttons in the Image Display toolbar Display Intensity and False Color remain active All other functions as well as all other programs are blocked during the course of image acquisition The status bar of the Experiment Manager window displays information about the acquisition status After starting the experiment the second field from the left in the status bar shows the in formation Running indicating that images are acquired The field in the middle displays the experi ment time hh mm ss ms Information about the actual number of Frames Acquired and Stored and the Total number of frames of the experiment is shown
58. displayed in the Viewport if the image is zoomed to an extent that is larger than the Viewport The rectangle is interactive It can be freely moved within the Viewport Manager to display different areas in the Viewport It can also be resized by mouse drag to change the zoom factor in the Viewport display 3 1 3 The Viewport The Viewport window allows displaying one image or a number of images at the same time The number of Viewports to be displayed and their arrangement can be set using the Arrange View ports button in the toolbar of the Image window Just mark the columns and rows by moving the mouse cursor over the schematic Viewport which opens with 4x4 image icons symbolizing inde pendent image areas The maximum number of images that can be shown at one time is 16 4x4 by default This setting can be increased to 5x5 as maximum via the Display Properties Right click on the Viewport Manager to open the Display Properties window and change the Viewport limit entry accordingly cell s cell software Manual Chapter 3 Brief Introduction and First Steps 11 Pt mages TEjo Wwe ASE ddew 2x 2 Images M4 Fura 53 Display Properties Ed Display Properties settings Zoom Settings Zoom Image manager Viewport Zoom Magnifier Prefered monitor Windows Automatic Factor Locate image in viewports 10 C Show comments Ox Crosshair Windows 50 Viewport caption viewport limit 10 Background pa
59. gt Y io H 1038 al EJ Properties Image Frame Display Display Brightness Automap Map fixed from 0 to 4095 Coloring Gray Scale Image type color Pseudo color LUT Image Type Select the type of image to be acquired from a list of pre defined types specified in the Image type dialog box of the cell4R cell M Configuration Software see Chapter 15 cel AR cell M Configuration This selection determines the excitation filter and thus controls the filter wheel of the Illumination System MT20 MT10 used for acquisition Depending on the Image type chosen the icon is displayed in a predefined color the same color used afterwards to display the image on the screen Light Intensity Adjust the intensity of the incoming light by selecting the percentage of transmitted light from the list of 14 intensities This selection sets the attenuator wheel of the Illumination Sys tem MT20 MT10 cell s cell software Manual Chapter 5 Experiment Manager Objective This function appears only if a motorized microscope is configured Select an objective from the pick list Contrast Insert This function appears only if a motorized microscope is configured Select an insert from the pick list Exposure Time Control the image intensity range by selecting an appropriate exposure time Ex posure time adjustment can easily be done under visual control while acquiring images in Live mode see
60. here the smoother the curve will be displayed Smooth ing 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 thresholds 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 updated 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 acti
61. however you generally loose image information Convert To 6 Bik Separate Extract To RGB r 7 9 2 To 8 Bit Use this command to convert cell4R s cell4M s proprietary nx16 bit multi color images into nx8 bit multi color images with up to 256 colors per channel or 16 bit gray value images into 8 bit gray value images with up to 256 shades of gray 120 Chapter 7 Image Data Handling OLYMPUS 7 9 3 To 16 Bit Use this command to convert nx8 bit multi color images into nx16 bit multi color images or 8 bit gray value images into 16 bit gray value images This might be important if you want to carry out certain arithmetic operations O This conversion doubles the required storage space 7 9 4 To RGB 3x8 Bit This command converts all other image types into the standard 24 bit true color RGB format This might be necessary for the export of data because many other software programs can only read this format and 8 bit gray scale images O In certain cases there is a different result between a conversion into nx8 bit format where n 3 with the To 8 Bit command and a conversion into RGB 3x8 bit format For example consider a nx16 bit image with a cyan a yellow and a red channel With the To 8 Bit command the result is an nx8 bit here 3x8 bit image with a cyan a yel low and red channel With the To RGB 3x8 Bit command however the data of the three original channels are distributed after rescal
62. images the result will be 1 otherwise it will be 0 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 the source 2 image buffers will be compared and the greater pixel value selected from the respective image The resulting image will thus have maximum intensity Minimum The images in the source and the source 2 image buffers will be compared and the lesser pixel value selected from the respective image 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 Negatives 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 00
63. in all major aspects according to the descriptions in the manuals OLYMPUS SOFT IMAGING SOLUTIONS as the producer of the software provides this warranty It does not replace or restrict other warranties or liabilities provided to the User by local or other sales people or organizations OLYMPUS SOFT IMAGING SOLUTIONS does not guarantee that the software is defect free that the software fulfills the specific requirements of the User or that the OBS cell4M cell4R SOFTWARE PRODUCT works with other software provided by the User 2 OLYMPUS SOFT IMAGING SOLUTIONS further guarantees that the software storage devices floppy disks CD ROMs etc and the manuals are free of material defects Defective storage devices or manuals will be replace free of charge if they are returned to OLYMPUS SOFT IMAGING SOLUTIONS within 90 days of purchase and accompanied by a proof of purchase 6 Liability 1 OLYMPUS SOFT IMAGING SOLUTIONS or their sales organiza tions cannot be held liable for damages or injuries resulting from the use of the software or the lack of capabilities of the software unless the User can show gross negligence on the part of OLYMPUS SOFT IMAGING SOLUTIONS This applies without exceptions also to losses of productivity or profit interruptions in the flow of business or manufacture loss of information and other financial losses Without exceptions the possible liability of OLYMPUS SOFT IMAGING SOLUTIONS is limited to the amoun
64. into Overlay option in Image Analy sis gt Scale Bar the Scale Bar is shown only in the currently active image cell s cell software Manual Chapter 7 Image Data Handling 109 7 3 Show Markers Time and Z Information The Experiment Manager allows the user to set markers by hand mouse click during the execution of an experiment This is useful to mark certain actions that are carried out or events that happen during an experiment While the markers are always shown in a Kinetics graph they are displayed in the images only if the option Image gt Show Markers is activated by mouse click With this option activated the time and Z information of the displayed image will be shown at the bottom left corner as well 4 40um 00 12 351 7 4 Grid Use the command Measure gt Grid to place a measuring grid of arbitrary size over an image This command for determining the size of the grid frames and the color of the grid lines opens a dialog box see below Image size The Image size field contains the size of the active image in the calibration unit Grid size Determine the size of grid frames in the Grid size field 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 automatically if you type 0 The grid size
65. invalid breakpoint Valid breakpoints Breakpoints are valid where Imaging C statement are executed e g e initializations e if else do while for e closing brackets e lines ending in a semicolon if a statement comprises several lines Skipping breakpoints Here are the exceptions e pure definitions such as the definition of a variable without a simultaneous initialization e if an expression has not yet been translated by the Imaging C interpreter not applicable to loaded C modules e the closing bracket of functions e if lines are to be skipped upon execution of the program e if you ve selected a condition in the Condition dialog box whereby breakpoints are to be skipped Moving on to next breakpoint Using the Bookmark Next command in the Edit menu or pressing lt F2 gt you ll move on to the next breakpoint To move to the previous breakpoint use the Book mark Previous command or press lt Shift F2 gt Maintaining breakpoints after closing text s You do not lose breakpoints within a text as long as you don t close cell4R cell4M if there s a source file A source text document along with its breakpoints can be closed and then the source file can be reopened at a later time Breakpoints will continue to be displayed If you save the source text breakpoints will not be saved The next time you open cell4R cell M all breakpoints will have been removed cell cell software Manual Chapter 14
66. is moved over the diagram area it changes into a horizontal double headed arrow on top of a vertical line 11 2 1 The Cursor Changing the XY Scaling in the Diagram P Upon a left mouse click on the graph diagram the cursor changes its shape to a double arrow cross Moving the cursor up and down stretches or compresses the Y scaling Moving it right or left 237 238 Chapter 11 Graph Display and Graph Analysis OLYMPUS stretches or compresses the X scaling Alternatively the scaling buttons of the Graphs button bar can be used this is explained in detail in Chapter 11 2 3 The Graphs Button Bar Upon zooming in scroll bars appear 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 11 2 2 The Cursor Measuring Individual Graph Points If the mouse cursor is located over the diagram s area the X value of the current cursor position and the corresponding Y value where the black horizontal line crosses the curve are given in the status bar of the graph document at the bottom left corner l X 39 003 5 Y 1 01e 003 11 2 3 The Graphs Button Bar The button bar at the top of the graph document is used to change the displayed X and Y range and to edit graph overlays The different buttons are arranged in functional groups 11 2 3 1 Scaling the X axis Zoom In Click the Zoom In button to decrease the displayed X range i e to stretch the graph i
67. like An Add In is an additional program developed for special tasks that you can integrate into your program using the Add In Manager All add ins are optional i e cellSR cell4M does not require any add ins to be fully functional Add ins can be Imaging C modules SXU as well as program libraries DLX Available add ins The Available add ins list displays all add ins available in your program pack age These add ins are automatically a part of the program when you install it They are not how ever automatically activated Activating add ins Select the check box next to the name of the program to activate the add in A number of add ins can only be used after you re start cell4R cell4M If this is the case you ll re ceive a message to this effect Some of the available add ins will already have been activated as they make up essential compo nents of cell4R cell4M Take the image database for example it s an add in If you clear the check box next to the entry Graph Processing the whole Graph Analysis menu will be gone the next time you start up cell4R cell4M 274 Chapter 13 The Special Menu and the Window Menu OLYMPUS Add ins that have not yet been activated remain on offer We recommend however that you acti vate only those add ins that you really need Otherwise you ll have too many superfluous button bars or commands making the appearance of the Graphical User Interface unnecessarily complex Add i
68. 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 standard 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 pre sent 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 analysis 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 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 Click the Include Pixel butt
69. measurement series can be conducted successively As soon as the button is clicked a blue ellipse circumscribed by a red rectangle between four blue lines will appear in the image overlay The left mouse button toggles between the Move and the Rotate mode indicated by the crosshair and the circular arrow respectively in one corner Drag ging without a mouse button clicked performs the moving and rotating In either mode a left button drag changes size and shape The best way to draw is the following 1 Click the Ellipse button to open an ellipse and move it roughly to the desired position 2 Adjust the size by dragging with the left mouse button pressed 3 Switch to Rotate by a single left click 4 Rotate the ellipse to the desired position 5 Switch back to Move by another single left click 6 Adjust the size of the ellipse via left button mouse drag 7 Fix the ellipse with a right click A new ellipse with rectangle will appear 8 Proceed as before or exit with press lt ESC gt The result will be displayed in the overlay and listed in the measurement display 9 2 5 6 Closed Polygon B Closed Polygon Use this command to measure an irregular area with straight border segments Default measure ments are area and perimeter Several measurement series can be conducted successively 1 Left click on a point at the border of the area A red line will appear in the overlay be tween the starting point and the moving mouse cu
70. ment result and the relevant unit of measurement Include name Check here to have the name of the method displayed with each measurement Decimal places Here you determine how many decimal places will be displayed This field is only active if the Measurement results option is selected Scale Select the scale of the unit from the picklist Label font Zoom text with image Mark this option if the label text shall be zoomed together with the image Label style Use alternating colors Select this check box to have different colors automatically assigned to different measurement objects or series of measurements This color appears in visualization of interactive measurements and in the corresponding labeling and sheet entries Colors will be as signed cyclically to the various measurements their sequence is red green yellow blue ma genta cyan and then back to red This sequence is preset After finishing a measurement series and then beginning another color assignment will start with red again O These settings do not affect the colors of ROIs Inactive Color and Active Color If the Use alternating colors check box is deselected you can choose one of the 16 MSWindows colors to mark the currently active measurements objects in the overlays in one color of choice and the inactive measurement objects in another Color selection applies to Display of interactive measurement elements such as lines and X s The corr
71. of the circle of interest with a left click an x will appear 2 Select a second point at the arc of the circle of interest with a left click another x will mark this position 3 Move the mouse and a circle passing through the two marks will be displayed 4 To fix the circle select a third arc point with a left click 5 Continue by drawing a second circle or exit via right click or lt ESC gt The result will be displayed in the overlay and listed in the measurement display 9 2 5 4 Circle with Center and Radius Circle with Center and Radius Use this command to measure a circular object by selecting the circle s center and one point of the arc Default measurements are area and perimeter Several measurement series can be conducted successively 1 Select the center of the circle with a left click an x in the center and a circle will appear 2 To adjust the size move the mouse Fix the circle by clicking on a point at the arc of the circle of interest 3 To fix the circle right click when desired size has been achieved A new circle appears with the sizes of the previous one Proceed as before 4 Proceed as before or exit with lt ESC gt The result will be displayed in the overlay and listed in the measurement display 184 Chapter 9 Measurements OLYMPUS 9 2 5 5 Ellipse Ellipse Use this command to measure an elliptical region of arbitrary orientation Default measurements are area and perimeter Several
72. 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 im age 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 reas signed an intensity value of 254 so that only the separation lines have an 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 O After separating objects the resulting image is the ideal basis for automatic particle de tection As the separation lines are only one pixel wide you must set fourfold connec tivity in the Define Detection dialog box so that the separation lines will actually separate the particles For eight fold connectivity the particles will be interconnected via the pixels positioned diagonally to each other and as such will be detected as a single particle Black Select this option to have object outlines shown in black on a white background White Select this option to have object outlines shown in white on a black back ground 150 Chapter 8 Image Processing OLYMPUS Burn black Select
73. open the dialog box you just have to modify the entry as fol lows DefineNxN Now when you activate the macro the Define NxN dialog box will be opened and you can adjust the parameters Logging dialog box parameters When you call up a dialog box there are two ways to log the entered parameters during recording of the macro Command window output Anytime you like you can have values within a macro output in the command window Use the operator to do this Working with loops Quite often you want to apply a recorded function series to more than one image This can be easily achieved by supplementing the macro with a basic for loop Example Select the Horizontal Distance command in the Measurements toolbar The macro re corder will record the following entry HorizontalDistance Let s suppose you wish to conduct this analysis for 10 similar images The images are in image buffers 1 10 To solve this task set a for loop around the commands recorded int i Variable i is defined for 1 1 1 lt 11 1i Op Display i Image buffer is set HorizontalDistance Imaging C Online Help To get a precise description of the recorded commands use the Imaging C online help Select a command in the macro text and then press lt F1 gt The Imaging C online help for this command will automatically be opened cell s cell software Manual Chapter 13 The Special Menu and the Window Menu 269 13 1 4 Stop Macro R
74. option Enable image split as long as the Dual View Micro Imager is configured in the ObsConfig software as described in Chapter 15 9 The Configura tion of the Dual View Micro lmager When this option is enabled the software will automatically create a dual color overlay image out of each image captured by the camera in Snapshot or Live mode This is done by splitting the image into two halves and using one half as the first and the other one as the second channel of the overlay The color of the channels and whether the split is done vertically or horizontally depends on the settings in the ObsConfig software 10 12 1 2 Dual Emission Experiments with the Dual View Micro Imager The Experiment Manager treats the acquisition of images through the Dual View Micro Imager like any other image acquisition see Chapter 5 3 3 4 Time lapse experiments monochromatic or in multiple colors with the difference that it creates an automatic image split and overlay as de scribed above The resulting time lapse series contain dual color images of half frame size It is possible to calculate online a ratio image sequence out of the overlay as well as kinetics of intensity changes see Chapter 5 3 2 6 Online ratio image Chapter 5 3 3 6 Experiments with online analyses and Chapter 10 8 Ratio Analysis An Experiment Plan would look like this for example i gt Ha gt CAEN Ratio Kinetic 100 x 1 00 s 10 12 1 3 Dual Emissio
75. overlay and written into the measurement display 5 Draw a second line or click lt Esc gt to terminate the measurement 9 2 4 Angle 9 2 4 1 3 Points Angle Al 3 Points Angle Use this command to measure an arbitrary angle using two lines initializing from the same point It is called 3 Points Angle because each line has a separate end point but the same starting point The angle will be displayed in the overlay and listed in the measurement display cell4R cellAM measures angles clockwise between the angle s first and second arm Thus the order of drawing is decisive 1 Position the mouse cursor at the origin of the angle and left click 2 Position the mouse cursor onto a point of the angle s first arm A straight line will ap pear in the overlay 3 Left click to fix the first arm It will be drawn into the overlay 4 A second arm will appear starting from the initial point of the first arm upon movement of the mouse Draw the second arm in the same way 5 Right click to terminate the measurement O Note again that the angle is measured clockwise exclusively and does not depend on a clockwise or counter clockwise movement of the cursor cell s cell software Manual Chapter 9 Measurements 181 9 2 4 2 4 Points Angle A 4 Points Angle Use this command to measure the angle between two lines It is called 4 points because each line has a separate starting and end point The intersection divides eac
76. 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 pur pose 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 Background 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 de pressed you can adjust the frame s size and position as you wish To set the frame right click Click the File button to open the standard dialog box for the opening and 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
77. reduces the possibility that a wrong focal position is being detected Display If this option is checked online images of the Autofocus scan will be displayed Use ROI In case of thicker specimens it may be that structures are brought into focus that are not the ones of interest for the user In such a case an ROI can be defined that will be considered ex clusively in the focality analysis of the Autofocus scan If the option is activated and one clicks the Autofocus button to execute an Autofocus scan first a red rectangular ROI drawing tool will appear in the active image to enable the user to define an autofocus ROI via mouse drag and confirmation with a right click compare Chapter 10 4 2 Draw ing ROIs Once this is done the Autofocus scan will be carried out automatically Low Signal mode If this option is checked a Mean filter see Chapter 8 2 8 will be applied on each image before evaluation of the sharpness 4 6 2 Executing an Autofocus Scan First of all an Autofocus mode needs to be selected from the Autofocus button shortlist The selected mode will be executed upon clicking the Autofocus button If the Use ROI option in the Autofocus options is active an ROI has to be defined before the Autofocus scan is carried out see previous chapter Autofocus normal In this mode the Range and Step parameters set in the Autofocus options see previous chapter are used This is the default mode and mostly the one of choice if
78. relevant Viewport within the image document to open the corresponding context menu and then click on Image Information Besides basic image information i e gray or color values you will see other information having to do with the image as well This includes for example calibration data and or any comments you may have made concerning the image All this information is saved along with the image You can access it via the Image Information dialog box 7 10 1 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 Image name The name can be changed it can be up to 31 digits long File name When you load an image the File name field will contain that image file s complete path An image you ve processed in cell4R cell4M 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 cell4R cell4M this field will be blank until you have saved the image File names can be copied into the clipboard Select the complete file name using the mouse and use the lt Shift Delete gt keys Use the lt Shift Insert gt keys to get the file name from the clip board 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 Wh
79. set of objects or terminate the command with another right click 177 178 Chapter 9 Measurements OLYMPUS 9 2 3 Length 9 2 3 1 Vertical line E Vertical line Use this command to measure a vertical distance between two horizontal lines Measurement Results The distance to be measured will be denoted by a vertical line labeled with the length by default This vertical distance will also be listed in the measurement display How to define a vertical distance 1 Upon clicking the button a horizontal red line appears at the mouse cursor Position it at the horizontal distance s starting point 2 Left click and the red line will be drawn in the overlay 3 A second vertical line will appear at the mouse cursor Position it at the horizontal dis tance s end point 4 Left click and a vertical line representing the distance measured will be fixed in the over lay and labeled with the length The fixed horizontal line will vanish The remaining horizontal line at the mouse cursor can be used for a new measurement 5 Continue the measurement in the same way 6 Terminate the measurement with a right click 9 2 3 2 Horizontal line H Horizontal line Use this command to measure a horizontal distance between two vertical lines Measurement Results The distance to be measured will be denoted in the overlay by a horizontal line labeled with the length by default This horizontal distance will also be listed in the meas
80. the Breakpoint list will go from D to E Delete Click on the Delete button to delete the break point selected from both the list and the text document cell s cell software Manual Chapter 15 cell4M cell R Configuration 15 cell M cell4R Configuration A special cell M cell R Configuration Software ObsConfig is provided for the configuration of the cell4M cell4R Imaging Station components for the Illumination System MT20 MT10 the motorized microscope components and for the optional PIFOC z drives We recommend to use the cell4M cell4R Configuration Software also for the optical alignment of the arc burner and the illumination coupling to the microscope Optical alignment with the cell4M cell4R Configuration Software is described in detail in Part B Hardware Manual If your cell M cell R imaging station is equipped with a motorized Olympus BX or IX series micro scope please note that the configuration of the motorized microscope components is carried out within the cell M cell R imaging software as described in this chapter 15 1 The Illumination System MT20 M7T10 cccccsseeeeseeeeseeeeseeees 330 15 1 1 Configuring the Excitation Filters c cc eeeeeeeeeeeeeeeeeeeeeeeeeeaeees 330 15 1 2 Burner ConfiQuration cccccsccccssssceeeeceeseeseeeseeseeeseeeeeesseeesssaes 332 15 1 3 Using MT20 MT10 without the Imaging Software 008 333 15 2 Configuring the MiCrOSCO
81. the cell M cell R Software OLYMPUS 10 In the next window choose in which group program shortcuts for Start Programs are to be added The setup will suggest the folder cell R M It is most convenient to accept this and simply continue with Next gt The installation will be executed 16 2 Updating the cell M cell R Hardware Control ie SS obsUpdate exe The cell4M System Coordinator cell R Real Time Controller in the imaging PC and the proces sors of the MT20 MT10 electronics run their own software so called firmware which need to be updated for version 3 0 as well Before doing this switch on the MT20 MT10 and wait a few sec onds for it to initialize Then execute the obsUpdate exe that can be found in the cell4M cellAR folder or under Start Programs gt cell M cell R The software checks the current firmware ver sions and opens a dialog box listing the current status If the necessity of an update is indicated click the Start Update button A Make sure the MT10 MT20 illumination system is switched on a obsUpdate CTR_M Pci04 ZF Update required CTR_M PIC 1 22 Ok MT10 2 46 Ok A 4b least one connected device requires an update Start update Cancel A Carefully read the warning message that appears and follow its instructions Then click the Start firmware update now button Do not use the computer or any of the periph erals for other tasks during the update process and do not unpl
82. the ratio e g GFP in the respective box and then saved it using the Save button Depending on the number of different fluorochromes the ratios are respectively stored in the folder cell4R M Unmixing ratio2 or ratio3 This procedure has to be repeated for each of the fluorochromes used that is for all reference images 10 9 7 Unmixing 4 Unmixing Based on the calibration for the different fluorochromes the unmixing of a multi labeled sample can be performed Unmixing Unmixing Output Output One image per channel Single image One image per channel Background Subtraction Background Subtraction C Constant o C Constant o i Ono Ratio data Ratio data 1 Fluorochrome 9 GFP 1 Fluorochrome 2 Fluorachrome FP 2 Fluorochrome w w cell s cell software Manual Chapter 10 Intensity Analyses 1 Acquire a multi color image of a multi labeled sample using the same filters that were used for the calibration described above Load the multi color image of a multi labeled sample into the active image buffer Our example image is a two channel image of a GFP YFP labeled specimen taken with the GFP and the YFP excitation filters Press the Unmixing button to open the respective dialog The cell4R cell4M Software automatically recognizes the number of color channels and the dialog will have two or three fluorochromes to be selected Select the ratio data for the differ
83. this button to have all lines of separation erased from the overlay Set All Click this 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 inter actively will reappear in the overlay Erase Click this 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 right clicking all deletion of outlines can be reversed one step at a time To do so press Shift and right click simultaneously Trace Click this button to select specific outlines Before using this function we would recom mend you click the Erase All button Polygon Click this button to be able to add missing outlines freehand Draw the arbitrary polygo nal figures desired within the image Right click to return to the Edit Lines dialog box cell s cell software Manual Chapter 8 Image Processing 151 8 3 Arithmetic Operations This command opens a dialog box containing commands to execute various predefined arithmeti cal functions Arithmetic Operations Ed Arithmetic Operations Arithmetic Operations Dimensions Input Input Source 1 1 Tvl Source 1 1
84. this graph information can be changed by the user and will be stored along with the graph Graph Information Pr at General Title netics of Ratio Image Fura 2 Graph data Date Graph start Graph end Channel width User Graph range Components Time 13 58 Comment Data type Channels Version 4 15 1653 m anis Y axis Title Title Intensity Graph data field Here information about the active graph parameters is displayed Graph start displays the start point together with the unit of the abscissa Graph end displays the end point together with the unit of the abscissa Channel width displays the interval and unit between single data points on the X coordinate Graph range displays the total range and unit of the abscissa Components gives the number of graphs displayed in the graph document Channels displays the number of data points on the X coordinate 11 3 4 Sheet Image analysis done with the analysis tools from the Image Analysis menu automatically creates a graph but no data sheet To create data sheets for graphs use Graph gt Convert to gt Sheet Data sheets can be edited with the commands in the Sheet context menu to be opened via right click cell amp cell Software Manual Chapter 11 Graph Display and Graph Analysis 247 R ol Tut Cret Ratio Copy Ctrl C Delete Delete Edit Column Header Sort Ascending Sort Descending Create Copy Sutofilter Create
85. tion of experiment 5 1 5 2 5 2 1 9 2 2 5 3 5 3 1 5 3 2 5 3 3 5 4 5 4 1 5 4 2 5 4 3 A Graphical TOOlnrenr ana a E E O 38 Concept of USAGC is casectoceindnsscalntensntinstatesadincaraetiiaswedstuevancutiehexcuaeetadens 39 Experiment Manager Components cccsscccceseeeeseeceeeeesaeeesaaees 39 Arrangement and Customization cccccsssceesseseeeseeseeeeeeseeesaaeees 40 setting Up Experiment Plans tisrsccceideesiecsetedeccesteseredetecdeecesiezecetseetes 42 Types of Graphical Icons and the General Principles of Usage 42 The Command Symbols and Their Properties Pages 06 43 Types of EXDOMIMENIS sesio A 60 Conducting Experiments Data ACQUISItION sccccseeeeeeeeeeees 69 Opening a Database cccccssecccceeseeeceeseeeesaseeesseeeessseeeeeeeeneaes 69 EX CUTING AN ExpenmeN T einen ida 70 Data Stora JE soina e e E A EE E 74 37 38 Chapter 5 Experiment Manager OLYMPUS 5 1 A Graphical Tool The Experiment Manager is a universal and easy to use tool for planning preparing and executing experiments with the Imaging Station cell R cell M Planning an experiment Your experiment may for example require capturing fluorescence images of a multi labeled sam ple and to display and archive them as composite multi color images Or it may be more complex and involve acquisition of precisely timed image sequences in multiple fluorescence channels and on multiple focus levels W
86. to profit from C modules There are a large number of ready made C mod ules which can be integrated into cell4R cell4M without your having to know they are in fact C module functions and not internal functions A C module consists of one or more files The files are compiled which means that they are proc essed in such a way that an executable C module SXU is created Before generating this executa ble C module a Make File MKU is necessary A make file contains among other things a list of the files comprising the C module The following file types are part of a C module File types Fierameorensons mesno OO header definition of variables and constants resource definition of dialog boxes C module configuration Definition Imaging C expressions will always be compiled automatically in the Interpreter Mode when the expression is executed cell R cell4M makes use of this mode in the following situa tions e when an expression is executed from the command window e when you use the macro commands Define Macro and Run Macro in the Special menu cell s cell software Manual Chapter 14 Imaging C 303 14 2 New Module Use Special C Module gt New Module to create a new C module New Module Current directory D cellASModule Options Create sub director Main function Exit function MAFI Sample function C Onlnithenu function Auto configuration C String table
87. top position of the stack is acquired the PIFOC or motorized Z drive moves back to the position it was at before the experiment started with the first layer If it is desired that the stacks of different color are stored together within one four dimensional data set XYZ color so that the colors are displayed together as multi color images for each layer a Color Frame has to be drawn around the Image Acquisition icons The experiment will be the same as above but for the way of data storage 3 Multi color Z stack The Image Manager indicates a Z stack acquired with a multiple excitation wavelengths with a colored stack icon 21x 0 50 pm It is not possible to interchange the order of frames that is to have the Multi Color Frame as outer and the Z Stack Frame as inner frame Upon Plan Verification see 5 4 2 Executing an Experiment an error message will be generated A Time Lapse or Stack command must not be inside a Multi Color command 5 3 3 4 Time lapse experiments monochromatic or in multiple colors A time lapse Experiment Plan is similar to that of a Z Stack Plan here a Time Loop Frame has to be drawn around one or several Image Acquisition icons see the examples below The result will be data files one or several respectively that contain all the images taken at the different time points 1a Monochromatic time sequence The Image Manager a time sequences acquired with a single excitation
88. 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 ignore1 of the darkest pixels when cal culating 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 ROI button to define the portion of the image to be used for automatic threshold cal culation 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 pa rameters OK is not available when the Auto Settings tab is active O 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 cell cell software Manual Chapter 8 Image Processing 8 5 2 Set Color Thresholds Sets color thresholds using the RGB or HSI color space Thresholds cannot be set for a single image or image
89. within the menu bar a command will be moved up or down within its menu Delete Click on the Delete button to remove the menu element shown in the Menu field Individual commands can be removed from a menu or you can remove a whole menu O Deleting a command in this case does not mean that this command is no longer avail able to you Any command you ve deleted can be re inserted into a menu anytime from the list of commands 275 276 Chapter 13 The Special Menu and the Window Menu OLYMPUS Modify Add Click on the Modify button to alter the name of a menu or command When you ve selected an entry in the Menu list you can alter the name of the entry in the Menu field As soon as you ve entered the new name the Add button s function will be changed to Modify Insert sub menu Select the Insert sub menu check box to be able to use the Add button to insert a new menu Enter the name of this menu into the Menu field The new menu will be located at the same menu hierarchy level as current entries in the Menu list So if you wish e g to insert a new main menu do it when the list of main menus is being shown Add Click on the Add button to e Create anew menu 1 10 11 12 13 Select the Define Menu Bar command in the Special menu Enter the desired menu name into the Menu field e g amp Name At the same time you can also define a short cut for opening the menu more quickly en ter
90. 001111 Result 10100101 165 8 4 Image Geometry 8 4 1 Resize The function Process gt Image Geometry gt Resize creates a copy of different size of the active image without cutting of parts like the Extract ROI cropping function does see Chapter 7 7 Ex tract The original data remain untouched Factor Set here the resizing factor for the spatial dimensions X Y and in case of Z stacks Z can be set The corresponding Size will be set automatically cell cell software Manual Chapter 8 Image Processing Resize Properties New Size a Y Z on fe E gee 128 JE o00 vor Ratio Zoom axes independently wt Interpolation Trilinear x42 Arithmetic Operations Bicubic 73 Linear 7 Image Geometry Resize RGB Studio Rotate Mirror 3D Images F Align Size Alternatively set the resulting size in the spatial dimensions X Y and in case of Z stacks Z The corresponding Factor will be set automatically Ratio Maintain XY ratio If this option is selected a change of the X Factor or X Size causes automati cally a corresponding setting of the Y Factor or Y Size and vice versa Zoom axes independently If this option is selected it is possible to set for example different Fac tors for X and Y Doing so leads to a distortion of the resulting image Zoom axes homogeneously This option is available for Z stacks only Here all three Factors X Y
91. 1 OLYMPUS SOFT IMAGING SOLUTIONS permits the User for the duration of this contract to use the software on a single computer and a single terminal on that computer This license is explicitly non exclusive i e the User does not have an exclusive right to use the software As a licensed user you can copy the software from one computer to another by using a computer network or other storage devices as long as it is assured that the software can only be used on a single computer or terminal at any time and that the conditions set forth under 4 are observed 2 The User has the right to produce a copy of the software only for backup purposes 3 Additional user rights Only if OLYMPUS SOFT IMAGING SOLUTIONS provides the User with permission in written form the User can incorporate parts of the software into other software developed by the User A distribution of the software can only be made in compiled form as part of the soft ware developed by the User under strict observation of the conditions set forth in the written permission to the User The User must include the OSIS cell4M cell4R SOFTWARE PRODUCT copyright notification with the User s software The User has to make sure that OLYMPUS SOFT IMAGING SOLUTIONS cannot be held liable for any damages or injuries resulting from the use of the User s software that include parts of the OSIS cell4M cell4R SOFTWARE PRODUCT 4 Copyright 1 OLYMPUS SOFT IMAGING SOLUTIONS or its subs
92. 10 MT20 cell4M cell4R Imaging Software integrating the Experiment Manager for planning and execut ing experiments Viewport Viewport Manager and Image Manager for managing and displaying the images on the desktop Image and Graph Analysis tools to analyze the acquired data and a database for storing and archiving the images cell4M cell4R software updates only Update Software for the cell M System Coordinator cell4R Real time Controller and the MT10 MT20 electronics cell s cell software Manual Chapter 3 Brief Introduction and First Steps 3 Brief Introduction to the Software and First Steps The following sections explain the basic cell4M cell4R features and functions They also introduce the most important terms used in the software and in this manual and thus should help you to get started For you to follow the contents of this chapter it is necessary that the system hardware and software has been installed and properly configured For a detailed description of the system installation and configuration read Chapter 15 cell M cell R Configuration of this manual carefully as well as the Hardware Manual especially Chapter 7 System Assembly and Adjustment 3 1 The cellAM cell4R User Interface ccccccsseceesseeeeeeeeeeeeseeeeesseeeees 8 Slt WAC MMAGS NIAMNAGCK aces ies eetintctseed sca nenn alee aon aa 9 3 1 2 The Viewport Manager ccscccssccsseessseecseccseeseesecsesseeeessesseeeese
93. 145 2 Configuring the MicroSCOpe sssssssssescececeecereceeecerseseereeseereren 333 arp leeee 15 2 1 General Configuration cccccccccececscesscscssesseesesesesserseseeeeseneeeens 333 1522 Z Drive CONMIGUIATION ssncsscswerscentssuedccseectienterndetrshentiecdemdeetsime sat 335 15 2 3 Configuration of the ODJeCtIVES ceeceeeeeeeeeeeeeeeeeeeeeeeeeesaaeeeees 336 15 2 4 Configuration of the Fluorescence Filter Turret s 0000 337 15 2 5 Configuration of the Transmission Contrast Inserts 000 338 15 2 6 Configuration of the Filters of a Filter Wheel ccceseeeeeees 339 15 3 Definition of Image Types ccccccseccssecceseeceeeeeeeeeaeeeeeeeseeeseeesaees 340 15 4 Configuration of Additional Shutters cccccscceesseeeeeseeeeeeneeeeeens 342 15 5 Configuration of the PIFOC cccescccssseeeeseseeeeeeeseesseeeeessaeeeees 343 15 6 Configuration of the Motorized Stage ccccsesseesssseeeseeeeeeneeeeeens 344 15 7 The UCB Control Box Light Panel cccccsseceesseeeeeseeeeeeneeeeeens 345 15 8 Parfocality Correction Of Objectives ccccceccesseeeseeeeseeeeseeeeesees 346 15 9 Configuration of the Dual View Micro IMaget c ccccesseeeeeees 347 15 9 1 Configuring the Emission Filters cccccccssseceesseeeeeseeeeeeneeeeeens 347 15 9 2 Configuring the Image Types ccccceesseeeeseeeeeeseeeeeeeeeeeesneeeeeens 349 Chapter 16 16
94. 16 PX AC lis cnssiaiasaseineveragtebeeructameetectercaeel acatec hee eel aed Pe denl an oaiiley 116 COMDE sh aces ee Be ee i al at 117 aenor direa ne eet nee eer ee eae ier eee ene Re ea eee 117 COMBINING Data SOUS sax sca x Sela Saale Pico weal teats eal eget 118 Conven A ass casa le Sadgeahenc tadnaceadelee a aai 119 GONG Mall sizes couse oe ele eet eile E nl cee osteitis 119 Chapter 7 Image Data Handling 101 102 Chapter 7 Image Data Handling OLYMPUS F392 VO GHB rcieded cotereeseteled ceecnettiodidely suet ih led siieseh edad eul tele als 119 FOIS TOTO Bitarna a ee a 120 108 TORGB 3x8 Bili eE TEESE 120 TO O lV Speen es a te ore ort Oe ee ee aS 120 FAO MWAAGES normato sassa TEE E 121 FAO MAS General TaD asr E E A cease we 122 7 10 2 The Dimensions and Markers TabS ccccccececeseeeeeeeeeeeeeeeeeeeeees 123 7 1 Calibrate Images 7 1 1 Why Calibrate Images Without a proper image and input calibration you cannot expect to obtain correct distance measur ing results when using the commands of the Image Measurement menu Even drawing the scale bar into the image assumes correct image calibration Sometimes it is necessary to recalibrate an image e g after loading third party images or if the system calibration is no longer valid but the dimensions of specific structures in the image are known If the system does not have a communi cation link to the microscope it is the user s responsibility to adjust
95. 2 4 3 Adjusting the Database WINdKOW ccccceeeeeeeeeeeeeeeeeeeeeeeeneeeees 257 12 5 Working with the Database snesnesnenseenennnnnnnnnnrnernnnnnernnnnne nne 260 AZo EOAGING DOCUMENTS krsnera E A EEE 260 12 9 2 INSEMING Documents snis pna d 260 1209 QUEN a E aes 262 12 5 4 Administration Defining Organizational and Database Fields 263 Chapter 13 13 The Special Menu and the Window Meni c ceseeseee 265 Setting personal prefer 13 1 IW aCrOS ica casas aiata erat dear erence ten Gin cates eatuaa Seletae Satta eeu eae ait uch 266 SSS UAE SOUS BM SINS alls ctuatirerel deat sen natnds aceetathnamencseanietybvaratasncsabaictaeieneaees 266 Heel ntenace 13 1 2 Record MACO uoeesesssessesseesseeseessessesseesetssesseessesssesstssesesstssteateseen 266 too VEXECUUING MACIOS asvan aren ttieanthl leer a 267 13 1 4 Stop Macro Recorder c ccccccsececceeeeceeeecceseeceenecceneeceeeeceees 269 Woe RUM MACIO 3 x0 iren sdodeuns a hocaveautaradnds E EEG 269 T6 SNe Step erpa aE rea e EEE 269 13 1 7 Reset Interpreter n nnnnennennennnnnnnennnnnnnnnnnnnnnnnnnnrnnennrenenneennnnnnne 270 13 1 8 Set as Default Macro ccccccceccccssseccseecceeeeceesecsaseeceueesseeeessaees 271 131 9 Define WIACKOS octane a a E a 271 19 2 ACC IN Manag i seisena enerne a E anaE AOA EEK AAt 2 3 T39 Deine Menu Ol es adeatinore ccs aten coin een seieieu ste orca Omri tain Gal 274 ASA GUIS OMMGUrAUION asnan a 277 TFoA
96. 23 PRET Analy SiS es a a EE 228 Chapter 11 11 Graph Display and Graph AnalySIs c scssseceseenseensenseees 235 Analyses of intensity 11 1 Graph DOCUMENTS sactaactatatacctancsdetevanceteuaarnaneabescaaiweeieadaateaus luseasbieer 236 kinetics of fluorescence 11 2 The Graph WIA OW 2csnid u Soec tase cedcadeve cd Gsee sed esidscdituds ead eee totseondees 237 nage Ume Sees 11 2 1 The Cursor Changing the XY Scaling in the Diagram 237 11 2 2 The Cursor Measuring Individual Graph Points cccseeecees 238 T23 TheGraphs BUtON Barebone eaa a 238 Ge The Graph Meninis nh e a eee e ee enaa aeee 242 TST Markers and WADCIS iea a TE 242 11 3 2 Protecting and Deleting a Graph ccccccesseceeeseseeeeseeeeeeneeeeens 245 11 3 3 Graph INTOrMAalON ox 222 eevee ere eek ieee ieee od 245 emaptes 1e say PM SY SSNS ieee ter te tecaa teagan state E 246 Images generated during an experiment are auto acai ord naice 2 Database E E E E E N 251 tred databases 12 1 Directories for Data Storage cccceseceesseseeseseeeeseeeeeeseseeeesaeeeeees 252 analytical data can be 1 amp 2 Open DatapaSEnrarnesraroisann aaa A 253 added 129 NeW Database noaea a andes 254 Contents OLYMPUS 12 4 The Database Features cccccceccsccsseeesecseeseeeseeeseeeeeeseeeseeseeesans 255 124 1 General Remarks crair a Er EE 255 12 4 2 The Database WINKOW ccccccsccssccssceeeeseeeseeesseeseeeseeeneeeneesaes 256 1
97. 4R cell M For some microscopes cell4R cell4M can directly read out the magni fication 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 magnification of an image acquired in another application program Resolution The Resolution field displays image size in pixels and information depth bits pixel For example an entry of 768 x 576 x 16 would mean that the image width is 768 pixels the image height is 576 pixels and the image can have up to 2 different color values per channel Width Height The Width and Height fields display absolute dimensions of cell4R cell4M images These values are determined using the current image calibration For other non cell4R cell4M images these fields will simply display image width and height in pixels Comment The Comment field is for any comments you wish to make on an image 7 10 2 The Dimensions and Markers Tabs 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 Dimensions field Here the extension of a multi dimensional data set in the dimension Time Z and Color are listed number of points in time number of layers and number of color channels Color Channels The table lists the channels by name as
98. 6 6670 31 E 3 45875 37290 6264 31 ina 4 45897 36640 6626 31 4 Execute the command Focus List Correction in the context menu A message like the following will appear Olympus OY100 Y The maximum 2 shift is dz 52 5 pm Position 4 Do you want to continue 5 Click Yes and the Z coordinates of all positions in the list will be adjusted by the amount given in the message 4 5 3 Calibrating the Motorized Stage O In case the Stage dialog box does not show the calibration commands click on the button with the arrowhead pointing downwards in the bottom right corner Loading an existing positions list to use it anew is only practical if it is known where the origin of the coordinate system was set at the time the list was generated If for exam ple the same slides are used routinely it is commended to set the origin always at the same spot on the slide Set Origin Upon clicking this button the current stage and Z device position will become the origin of the coordinate system with the X Y Z values 0 0 0 The red status box Not calibrated turns green and states Calibrated cell s cell software Manual Chapter 4 Image Acquisition and Hardware Control 33 Positions Position Connected o e Set Origin Auto Calibrate Mot calibrated Set Limits Auto Calibrate Upon clicking this button the stage will always move to a certain corner position and set this as origin of the coordinate system The red s
99. 8 6 2 No Neighbor The No Neighbor algorithm is the faster algorithm as compared to Nearest Neighbors As a trade off in order to achieve this speed it is less accurate It works by deblurring one image slice at a time The principle An unsharpened image of the image that is being processed source image is cre ated and then subtracted from the source image The unsharpened image is taken as an approxi mation of the out of focus information Thus the unsharp image contents are being removed while the sharp contents remain Available All cell4R cell M image types can be deblurred single images time series Z stacks and 3 D time lapse series all either monochromatic or multi colored 170 Chapter 8 Image Processing OLYMPUS 8 6 3 Nearest Neighbors The principle The two neighboring layers one above and one below of the Z stack layer that is being processed are temporarily unsharpened and then subtracted from the layer that is being processed Similar to the No Neighbor approach the unsharp images are approximations of the out of focus information However in the Nearest Neighbors algorithm the supposed out of focus information in fact stems from out of focus layers and is thus considered as a superior input for the calculation Available Z stacks and 3 D time lapse series both either monochromatic or multi colored 8 6 4 Deblurring Images Requirements The images need to be calibrated in the X Y and Z dimensions see Chapt
100. A E a AEE E 169 6 6 2 NO NGIQNDOM oar ainan end aaeceiesatiGentetneeeardloiaveens 169 8 6 5 Nearest NGIGNDOIS ruis arainn E a awe 170 6 6 4 DEDIUMING IMAGCS wins totic ncieweit le dee eee 170 809 INVERSE TINGE eaaa EEROR aTa 172 8 0 6 3D Blind De convol tl O Me a a as 173 Chapter 9 9 Measurements sssssnsnunnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnmnnnn nnna 175 The software containsa 9 1 Measurements TOOlDar ccccccceeccecceccecceeeceeceeeeeneeeeeeueeaeeeeeeneenees 176 host of measuring tools 92 Drawing Tools for Length and Area Measurements c0sce0ee 177 in a special measurement g 9 4 E A ANE A ENE AN E O E A N E ATE AEE N de oeatece 177 SPON IEN ODD TOUGH COMING aia e colar Gore N 177 9 29 Lengi asna neteriias R 178 924A 6 6 riesenia EAE EEEE 180 920 A eaaa a a a oe BN alt al a a as 181 So Magie WV ANC oteeli EE EEEE e EEEa EEE EATE NEESS 186 93T Mage Wand ODON Sesso eee Racclelane 187 9 4 T ES EAE T TEA E EE PEERI PEE SILENE EE ERTE PA E CIAN TA E SUE Be 188 OAT Move Orgi serrie E 188 9 4 2 Create Measurement Sheet cccccccecceseeceeceeceeeeeeeeeeeeeeeevensees 189 9 4 3 Deleting Measurement Results ccccececeeeeeeeeeeeeeeeeeeeeeeneeeeaees 190 DAs MAMAS LINK senpia E RONCEA ANEA EEEO RETEK 191 OAS SHOW AICS SC a wend nese eaiwendencaeieniweneneen 191 9 4 6 Select MEASUrEMENTHS sasinan a 191 GAT Delne SIAUSTICS eenen E a A E A EEE EE 193 9 4 8 The Preferences gt Measure Tab
101. Add to Module Module Info and Close Module In addition you can enter the names of directories containing modules to be included in the two lists in the Define search path for modules dialog box Module Manager Loaded BMDLib A F ExcelDDE CleanReport Black Dicom efi i blia Module D cell ASM oduleU nmisingsUnmmixing ssu File Active Dialog box description This dialog box is non modal meaning that it can be kept open while you use other commands cell cell software Manual Chapter 14 Imaging C Loaded All loaded modules are listed in the Loaded list All functions of these modules are cur rently available to you To load modules from the Other list use the Load button To load executa ble SXU module files use the Open button or use the Open Module command O Most modules can also be loaded and closed in the Add In Manager Some modules will be automatically loaded for example when you load a configuration use user defined measurement or statistical parameters or activate an image import filter These modules will be shown in the Loaded list too Other All modules fundamentally available to you for use are listed in the Other list these mod ules are however closed The functions of these modules are not available to you currently To close modules click on the Close button or use the Close Module command The following modules are displayed in the lists e the active modul
102. Analysis gt Convert to Sheet to convert the coordinates of the displayed graph into a new datasheet 11 3 4 8 cell R cell4M and Excel ExcelDDE x Be i ExcelIDDE toolbar Use the Start Excel button left to open the spreadsheet program Excel if it is installed on your Imaging computer Use the New Workbook button center to open a new workbook in Excel This command can only be performed if Excel has already been started Otherwise an error message will be displayed Use the Transfer Data button center to transfer the data from the data sheet to the Excel work sheet 249 250 Chapter 11 Graph Display and Graph Analysis OLYMPUS cell amp cell Software Manual 12 Database Chapter 12 Database 251 The database is an integral part of the cell R cell M Imaging Software It allows an organized stor age of all acquired images created graphs and documents like sheets diagrams and texts The design of the database facilitates an easy and fast access to even a huge data stock The database is fully network compatible and can be used simultaneously by different users 12 1 Directories for Data Storage ccccccccccssseceeeeeeeeseeeeeesseseeeeseeeeessees 252 12 2 QOPEM DataDaS rreo e a a E E S ERARE 253 12 3 NeW Database rarna in E EE OETA E AEAEE NERE A Eh 254 12 4 The Database Features sacii enaa aaraa aei 255 124 1 Genesia Remarks nssr e ye is ivecourstch aiees events te 255 12 4 2 The Databa
103. BOOL bEnable Switch uMsg case WM INITDIALOG dlgCenter hDlg CheckDlgButton hDlg IDD CHECKBOX bCheck SetDlgItemText hDlg IDD EDIT szText EnableDlgItem hDlg IDD BEEP bCheck break case WM COMMAND Switch LOWORD wParam case IDD BEEP MessageBeep MB ICONQUESTION break case IDD CHECKBOX bEnable IsDlgButtonChecked hDlg IDD CHECKBOX EnableDlgItem hDlg IDD BEEP bEnable break case IDOK bCheck IsDlgButtonChecked hDlg IDD CHECKBOX cetDlgItemText hDlag IDD EDIT szText Sizeork szT ext case IDCANCEL EndDialog hDlg LOWORD wParam break default return FALSE switch wParam break default return FALSE switch uMsg return TRUE O If you have set the language to German before initiating the Module New com mand all comments of the source file of the new C module will be in German Lan guage is selected in Preferences in the Module tab in the Special menu 308 Chapter 14 Imaging C OLYMPUS OK Click on the OK button in the New Module dialog box to open a text document containing the source code of the text How to assign a new MAPI function to the T button You have generated a new Imaging C module called New using the New Module command You ve selected the Auto configuration check box in the dialog box This means that there is a new button bar containing the T button the SampleFunction can be initiated vi
104. Box Use this dialog box to have other modules from other directories shown in the module manager Define search path for modules Lookin same OE Folder name AMEA MEERE E Selected directories D cell A Moduleesample 0 cell ASM oduleerhtra Dycal RAS Module What s it for This is where you set up a list of directories containing the C modules cell R cell4M is to search for The directory itself and all sub directories will be searched All modules will be shown in the Other list in the module manager That s where you can load activate edit and or compile modules Look in Select the directory you want included in the Selected directories list from the Look in list and field Folder name The Folder name field shows the directory meaning the complete path of the direc tory you have selected in the Look in field You can keep the directory or enter another directory The complete path of this directory can be added to the Selected directories list Selected directories The Selected directories list contains the paths of all directories whose modules are shown in the module manager Add Click on the Add button to add the path in the Folder name field to the Selected directories list Delete Click on the Delete button to remove the selected directory from the Selected directories list 320 Chapter 14 Imaging C OLYMPUS 14 11 Browser Use this command in the Special C Module menu t
105. Chapter 4 Image Acquisition and Hardware Control The exposure time also deter mines the opening and closing of the shutter of the Illumination System MT20 MT10 Acquisition time This read only information gives the time necessary for the system to acquire the image according to the selected parameters and transfer the data to the cell4R cell4M Imaging PC Name Specify the name for the acquired image The image will be stored under this name in the database If this image becomes one of the color bands of a multi color image upon usage of a color frame see next Chapter 5 3 2 2 this will be the name of the band The default name is the name of the image type Get Current Camera Settings Click this button to read in the exposure time the binning factor and the ROI settings as currently set in the Camera Control dialog box see Chapter 4 2 Camera Control This is only necessary when modifying an Experiment Plan If an Image Acquisition icon is newly added to the Experiment Plan the current settings are used by default Properties Frame By default the current settings in the Camera Control dialog box See Chapter 4 2 Camera Con trol are read in at the time of placing the icon See the same chapter for a description of these parameters Binning The factor can be changed by selection from the pull down list Frame A sub frame that is a Region of Interest ROI to be read out from the camera can be defined by setting the X a
106. Configuration tings for example to minimize vibrations This can be done by using the sliders or typing the new values into the corresponding boxes The Velocity and Acceleration settings do not affect the joystick and are relevant only for experiments carried out with the Experiment Manager Calibration The calibration of the origin of the stage coordinate system and limits can also be done in the cell4R cell4M software and is described in Chapter 4 5 3 Calibrating the Motorized Stage in detail Position loop Z device It is possible that a cell4R cell M imaging station contains more than one motorized Z device for example an objective PIFOC or a nosepiece PIFOC Select the device from the shortlist that shall be used for the change in Z position when using the Stage loop com mand in the Experiment Manager 15 7 The UCB Control Box Light Panel The light panel on the UCB control box indicates the functioning of the various components It can provide valuable information in case of difficulties For a detailed description of the single LED s please see the Olympus user s manual Green signifies OK blinking signifies a problem A LED that is not switched on signifies the ab sence or incorrectness of the component The red ERR error light indicates that a component has been incorrectly plugged in An orange RMT remote light signifies control by the PC If it blinks the PC connection cannot be established
107. Consider a triple color image a 2x2 Viewport arrangement could be useful The image can be displayed in each Viewport via drag amp drop selection from the Image Manager list Then the All Color Channels view can be selected for one Viewport while each of the individual channels can be selected in one of the three others O If more than one Viewport is activated via lt Shift gt click navigation through time and Z as explained in the next chapters will be carried out for all Viewports simultane ously 6 3 4 Time Lapse Sequences Hl 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 Selection of a time se quence in the Image Manager enables the Navigate Time button in the Navigation toolbar K KW 8 b p First Previous Next and Last With the Previous and Next buttons in the Navigation toolbar you can navigate frame by frame backward or forward 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 directly go to a specific frame by typing the respective number into the Go to field and pressing the Return key Alternatively you can click with the mouse into the Viewport to update the image displayed ce
108. DPG cccccceseeeeeeeseeeeeeseeeeeeeeeeeaeeeseaaes 333 15 2 1 General Configuration cisini eaaa ta dair ia 333 15 2 2 Z Drive COnfiQuration ccccccsssscccsesceceeeseeeseeseeeseueeeeseeeeeeneeees 335 15 2 3 Configuration of the Objectives ccccceeeceeeeeeeeeeeeeeeeseaeeeeenees 336 15 2 4 Configuration of the Fluorescence Filter Turret c00008 337 15 2 5 Configuration of the Transmission Contrast Inserts 0 338 15 2 6 Configuration of the Filters of a Filter Wheel cccsseeeeeeeee 339 15 3 Definition of Image Types cccccccseccececeeeeeeeeseeeeeeeesaeeeseeesaeeeses 340 15 4 Configuration of Additional Shutters 0 0 0 0 ccceccsseeeeeseeeeeeeeeeeeeeees 342 15 5 Configuration of the PIFOC eeeccceseceeseeeeeseeeeeeseeeesssaseeensaes 343 15 6 Configuration of the Motorized Stage ccccccccseseeeeseseeeeeeeeeenaees 344 15 7 The UCB Control Box Light Panel cceccceesseseeeeeeeeeeeseeeeenees 345 15 8 Parfocality Correction Of Objectives ccccccsseeeseeeeseeeeeseeeeeeeees 346 15 9 Configuration of the Dual View Micro IMaget cccseeeeeeees 347 15 9 1 Configuring the Emission Filters ccccsssceeeseeeeeeseeeeeeseeeeeenees 347 15 9 2 Configuring the Image TYPeS cccccsesceessseeeeeeseeeeseseeseeeeeeesaees 349 329 330 Chapter 15 cell M cell4R Configuration OLYMPUS 15 1 The Illumination S
109. Edit FalS COlO Picco wsicss nde ni cedesec a oe ese oe 87 image NAVIGATION ustcsencentscacececwcoagh cteeacedn a Ea N 93 Generalaren a eee es a en eee 93 Multi Color IM Q S cccccsssccecseseeecseseeecseseeeeseuseeeseneeeseusessseaeees 93 Displaying Different Color Bands in the Tile View Mode 94 Time Lapse SEQUENCES cccsccccsseeeceeecceeeeceeeecaueecsueeesaueesseeeesees 94 ZODIAC KS sdaieiepise ic E tines Rican dicen ood ied ali pled cod alee ed raya 96 Multi dimensional SCQUENCES cccseeeccseeeceeeeeceeeecseeesseeeesaees 96 Parallel Navigation in Multiple VieWPOrts cccccceeceseeeeeeseeenees 97 Projections and Extended Focal IM Qing ccscceseeeeeeeeeeeeeeeeees 97 Projections Along the Z and Time AX S cceeeeeeeeeeeeeeeeeeeeaeeenes 97 EFI Extended Focal Imaging cccccccseccseecseeesensensersenseceeeses 98 Fluorescence and Transmission Image Overlay cccssscessseeeeees 99 Intensity Modulated Display ccccccseeceeeeeeeneeeeeeeeeeeeeeseeeseeenees 100 Image Data Handling sssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn n 101 Calibrate Mage Sannaa a aT 102 Why Calibrate Images sssi inia iia 102 Calibrating the Camera Channnel ccccssscccssseeeesseseeeseeseeseaeeees 102 XY Call atlOM oeenn oeno e EER Ee REER 104 ZC ANNA OM i sca a ee 106 Scie BI ge eee ENO ene tr CoN Ee et EMER Beene CEE ee en Ee een ects Sas 107 Gene
110. GW DOM seira n r ds tereses 76 602 mage DISHA V reinen aE E a Ea 78 G25 Genea iesr e a N 78 622 AGUSPDISHIAY irea a E a a EEEE 78 O 2 3 AUTO AGIUSE DISDIGY anasa slates el ae lee ete eee ois 82 6 24 NVAMG BANCO gng eepe a T 82 6 2 9 BIAK Bal AICS renen e a a a a aE 83 020 GV gt 0 eiea a a eR ee Mem Ty are ee 83 6 2 7 Fluorescence Coloreccanne see a a 84 6 2 8 Edit Fluorescence Color cccccccccsseeceeeeceeeeecaeeeceeeeeeeeeeseeeesaaees 84 629 IPalSC2 OlOnsc ccedccice e E E ee EEE 85 6 2 10 Edit FalSe Collor cccccccccsscccsseeeceseeceseeecaseeceuceecaseeceueessaeesseeeess 87 6 9 Image NAVIGATION sivicssceccntciedssiciadsanscdudsnnesiedsannbsdndssnedaddcinesdusacinnsuenedss 93 Gol GOMGi al sacs cacece esoteric cette a ete tdartas eects 93 632 MultizColor lIMmaGQeS esia a A 93 6 3 3 Displaying Different Color Bands in the Tile View Mode 94 6 3 4 Time Lapse Sequences ccccceececeeeceeeeceeeeeceeeeceaeeecauseeseeessases 94 G29 Z ola S aiae te al 96 6 3 6 Multi dimensional SEQUENCES ccccccecseeeeceeeeceeeeceeeeeseeeessaees 96 6 3 7 Parallel Navigation in Multiple Viewportts cccccsecseeeseeeneeeneees 97 6 4 Projections and Extended Focal IMaQING cceceeeeeeeeeeeeeeeeeeeees 97 6 4 1 Projections Along the Z and Time AX S cceceeeeeeeeeeeeeeeeeeaeeeees 97 15 76 Chapter 6 Image Display and Navigation OLYMPUS 6 4 2 EFI Extend
111. Graph Define Statistics Statistics Define Context Menu 11 3 4 1 Edit Column Header Use Sheet context menu gt Edit Column Header to change the title neader of the highlighted sheet column The header is limited to 31 characters Edit Column Header Column Header 11 3 4 2 Sort Ascending Use Sheet context menu gt Sort Ascending to rearrange the data in the highlighted column s in an ascending order 11 3 4 3 Sort Descending Use Sheet context menu gt Sort Descending to rearrange the data in the highlighted column s in a descending order 11 3 4 4 Create Copy Use Sheet context menu gt Create Copy to create a copy of the currently displayed sheet 11 3 4 5 Autofilter Use Sheet context menu gt Autofilter to activate the autofilter function If activated the Autofilter command in the menu shows a check mark and the column headers in the sheet feature pull down menus You can select some predefined functions from a pull down menu to filter the data within the respective column e g select Top 10 to display the 10 sheet cells with the highest value 248 Chapter 11 Graph Display and Graph Analysis OLYMPUS 2 Sheet9 Graph GB 2 Kinetics of Ratio Image Fura 2 Sele Time Teles Roa efi ROS 5 A Ratio Ratio 19 003 Empty 71823 696 06 20 003 Mot empty ITF 698 59 71 003 defined 71693 699 25 79 003 214m3 717 16 B98 70 2 Sigma 25 005 i at Sigma fio p95 04 24 003 hot Sigm
112. Green and Blue scrollbars Set Thresholds Manual ote C og Preview wis ole Diagram Smooth J ajal iel Fill down When you click on the Fill down button in the Edit LUT dialog box all values of the first selected row will be transferred to the other selected rows Using this command you can enter new values into one row and then have them copied to successive rows Sheet Click on the Sheet button to generate a separate sheet document using the dialog box s current LUT The columns of the sheet are Index Red Green and Blue Such a sheet can for ex ample be exported in the Excel format via File gt Save As 89 90 Chapter 6 Image Display and Navigation OLYMPUS 6 2 10 2 The Polygon tab Diagram Within the diagram lines or polygons of the respective colors represent the three primary colors of the LUT The horizontal axis represents gray values from 0 255 The vertical axis represents LUT entries i e color intensities from 0 255 Edit LUT Sheet Polygon Formula Linear Color Folygon points Bed Count a E Green Monochrome OB ate Mouse Functions A LUT polygon is defined by its points which are represented by small white squares joined by lines There will always be at least two points at the left or right edge of the dia gram 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
113. Imaging C 327 Debugging must be terminated before you can close cell R cell4M If you cannot close the Imaging C program proper then click on the Stop Execution button in the Debug button bar You cannot terminate debugging by closing the source text s document window nor by turning off the Debug button bar 14 17 Edit Breakpoints Use this command to edit the currently set breakpoints You may also simply press lt Ctrl b gt Defining a breakpoint Use the Toggle Breakpoints command to set a breakpoint What will happen at a breakpoint When you execute an Imaging C program or module and come to an expression where a breakpoint has been defined execution will be interrupted The Debug button bar will be displayed and the mouse cursor will be transformed into the shape of a hand The value of a variable can be tracked or execution can be continued in single steps Breakpoint list The Breakpoint list displays all breakpoints defined using the Toggle Breakpoint command Breakpoints can belong to varying source texts Once you close a source text its break points will remain undeleted only if the text has a source file Edit Breakpoints Breakpoint list E D cellASModuleSexampleSexample stm 1 The list shows the following on each breakpoint e breakpoint status e path of the corresponding source file if there is one otherwise the name of the corresponding text window e line number of the breakpoint w
114. Imaging C OLYMPUS on the file in the file list If the file has a file name extension of SFT H or DLG it is considered a source file and will thus be appended to the Header files list If the file has another file name ex tension it will be treated as a function source file and thus appended to the Source files list If the list position of the appended file is to be changed you can adjust it using the Up and Down but tons Remove To remove a file from the module select that file and click on the Remove button In this case Remove simply refers to the file being removed from the list The file has not been deleted from the disk Edit To edit a module file select that file from either the Header files field or from the Source files field and then press the Edit button Or you can simply double click on the file in the list 14 7 Build Module Use this command to compile the active module Available This command is only available when e aC module is active and if e there is a MKU make file for this module You may also press the lt F7 gt key Before you use this command activate the module in question in the module manager Then you can check and see whether the module has a MKU make file using the Open button in the Open Module dialog box What will happen All files of the active module will be compiled After the build process has been completed without errors an executable file will be stored on the hard disk with t
115. Insert Image dialog box by an exclamation mark on its left side 264 Chapter 12 Database OLYMPUS Define Fields Field properties Field list Mame Expenment Comment i Experiment Hame Sa Experiment Comment i Binning Input required E Color Channels lt 4 Comment Datatype Text Picklist E Exposure Time Graph Comment Image Dimension E Markers E Regions OF Interest ROIs E Sample Preparation Time Frames f u Size E Y 5ize Default G z Layers G Value of last input 2 Step width OL En Organizational field Mark this check box to identify a database field as an organizational field The content of an organizational field identifies all of the data files and documents that belong to this organizational element Picklist Edit Click here to open the Edit Picklist window Here you can define a list of possible entries for the user defined database field When inserting images select the desired entry from this list Default Value of last input Select this to indicate that the field entry entered with the last inserted record is to be used This option is the default setting Choose the second option of this group if you do not want to define a default value for the active database field The database field will then be empty each time you insert data and has to be filled out again cell s cell software Manual Chapter 13 The Special Menu and the Window Menu 13 The Special
116. It s a good idea to clear this check box to ensure that you do not unintentionally over write an existing configuration file Create backup when saving text files Select the Create backup when saving text files check box to have a backup saved automatically when you save text files This second file the backup contains the version of the text file as was last saved not the current text file This backup will be created when you overwrite the previous version of the text file The first time you save a text no backup will be created What you ll have is two files containing two different versions of a text document the current ver sion will be saved in the file format selected in the Files of type list of the File gt Save As dialog box e g report txt The previous version will be saved in the so called bak file format e g report bak Loading backups bak files can be loaded in cell4R cell4M To do this select the All entry from the Files of type list of the File Open dialog box Now the bak files will be shown as well They will not be shown via the Text Formats entry however Click on the Open button in this dialog box The Load File dialog box will be opened Click on Yes to open the bak file in a separate text document When do I need a backup Primarily backups make sense for macros You ll still have the previous version of the macro if it turns out that the new version is causing you proble
117. Manual Chapter 14 Imaging C 321 letters substitute a question mark Upper and lower case are not relevant when searching for a symbol Range The Range list contains the following search ranges for symbols Callable in Interpreter Mode Exported by Tool Exported by loaded modules Exported by Active Module Defined in Active Module Defined in Interpreter Mode If you have selected the Internal Function type of symbol the list will not appear The Exported by Tool range for example searches for predefined symbols Result The Result field indicates the number of symbols found according to the settings of the Query group This list contains the names of the symbol s Example The Internal Function type and the symbol will result in all internal menu functions Description The Description group displays more detailed information on the symbols selected in the Result list Edit The Edit button is only available if you ve selected a symbol from the Result list this symbol must have a source file Click on this button to load the source file of the selected symbol in cell4R cellAM This file will be opened in a separate text document window The cursor will appear right at the spot in the text where the symbol is defined and the name of the symbol will be selected Copy amp Paste The Copy amp Paste button is only available if a symbol has been selected in the Result list C
118. O Images acquired via the Experiment Manager will be stored automatically in a data base 3 4 Loading Images To load images that are not stored in a database and to display them in the Viewport use the com mand File gt Open short cut lt Ctrl o gt or click on the Open button in the toolbar As usually you have to navigate to the storage folder of the file and select it Open the experiment folder in the database to load an image or an image sequence from it see previous chapter 3 3 In the Structure Strip on the left hand side of the database window you will find the Image Icon and in the Gallery Field the Image Thumbnail Drag either the icon or the thumbnail into the Viewport or the Image Manager to load the image set 3 5 Conducting Experiments with the Experiment Manager oR Experiment Manager In general most imaging applications in life science are rather complex and go beyond taking sim ple snapshots for example multi color imaging time lapse imaging ion imaging with ratiometric fluorescence dyes multi dimensional imaging etc The cell4R cell4M Imaging Software includes the Experiment Manager an easy to use and intuitive tool to plan configure and execute even cell cell software Manual Chapter 3 Brief Introduction and First Steps 15 the most complex experiments without any programming knowledge The Experiment Manager is explained in every detail in chapter 5 In the following only a brief intro
119. OLYMPUS Se Olympus cell Family User Manual NY P h Y 4 S pa A S Software Life Science iC OLYMPUS cell amp cell Imaging Software for Life Science Microscopy Software Manual for Cell and cell Imaging Stations OLYMPUS Imaging Excellence We at Olympus Soft Imaging Solutions GmbH have tried to make the information in this manual as accurate and reliable as possible Nevertheless Olympus Soft Imaging Solutions 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 Olympus Soft Imaging Solutions 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 Olympus Soft Imaging Solutions GmbH be liable for any indirect special incidental or consequential damages arising out of purchase or use of this manual or the information con tained therein No part of this document may be reproduced or transmitted in any form or by any means elec tronic or mechanical for any purpose without the prior permission of Olympus Soft Imaging Solu tions GmbH 2003 2008 by Olympus Soft Imaging Solutions GmbH All rights reserved OLYMPUS SOFT IMAGING SOLUTIONS GMBH Robert Koch Strasse 9 D 82152 Planegg
120. Please check the status on the light panel of the UCB in case of software problems for an appro priate diagnosis 1 Exit the software and shut off the UCB controller 2 Switch on the UCB controller box ONLY 3 Check the status of the light panel Any problems indicated by blinking lights are of physical nature on the microscope itself Please refer to the respective Olympus manuals IX2 UCB2 or BX UCB to troubleshoot such microscope problems 4 Ifthe light panel displays no error then start up the software 345 346 Chapter 15 cell M cell R Configuration OLYMPUS 5 Click on the Log On button in the IX BX button bar and login 6 Check the microscope configuration and compare the entries with the components pre sent on the microscope correct if necessary 7 Check the definition of the key configuration adjust where necessary 8 Now you may continue working with the microscope via software control 15 8 Parfocality Correction of Objectives Parfocality Correction z i To calibrate the Focus difference between your LD microscope objectives 1 Select any objective 2 Use the Live mode and focus your sample 3 Press the Read button 4 Repeat the abowe steps For all remaining objectives Objective Correction Position oo PLAPO G UPLAPO 20x 0 0 0 0 The absolute position of the focal plane will always slightly differ from one objective to the next In case of microscopes with m
121. SLISTS XML in the cell4R cell4M folder To move the stage to one of the positions in the list select the position via mouse click then click Go to or simply double click the position 29 30 Chapter 4 Image Acquisition and Hardware Control OLYMPUS List Positions Positions a 596520 57020 5707 59470 50820 5695 566870 55920 5655 x 4 5 2 Correcting a Positions List It is possible to automatically correct all positions of an existing positions list This may be conven ient if a focus drift is observed if the microscope was shaken causing the sample to move or if a sample was removed and is put back onto the stage Corrections can be carried out in the XY plane and in Z Lateral List Correction If there were a lateral shift of the sample on the stage you can correct the list by doing the follow ing 1 Go to any list position and correct the position of the stage 2 Right click the position in the list to open the context menu and select Lateral List Cor rection 3 A message like the one below will appear 4 Click Yes and the X and Y coordinates of all positions in the list will be adjusted by the amounts given in the message cell amp cell Software Manual Chapter 4 Image Acquisition and Hardware Control 31 Copy ut Delete Set Current Position As Reference Olympus O 100 2 The list will be shifted by d 59 d 62 ym Do you want to continue Lateral List
122. Sa aca at 201 1042 Drawno ROS eaa a 201 10 4 3 ROI Measurements 2 D ccccseeccseseeceseeeceeeeseeeeeseuseeseeeessaees 204 10 5 Background S btracton resies ae a a aa 205 TOS fase 9 lt 1 72 o A Pe Tease re nt See or 205 10 5 2 Subtracting the Image Background ccccccseeeeeeeeeeeeneeeeeenees 205 10 6 Intensity Kinetics in Time and Z cccccceccseceseeeeeeeeeeeeeeeeeeseeseeenees 207 10 7 DeltaF F AF F Analy SiS wvec2sccciccd cidecdtcecedsciivecdidersdctiiecdiedineekt 208 TOT AGING Well a E a a E Wace ued 208 10 7 2 Generating a AF F SCQUENCE cccsceccceeeeeceeeceeseesaeeeeseeessaees 208 10 8 IRATIORANGIYSIS ainra a sncesiedveQeacivacaaciie 210 10 81 GMC i arssete echoes Seether ea a Gayla a 210 195 196 Chapter 10 Intensity Analyses OLYMPUS 10 8 2 Generating a Ratio SEQUENCE cccceecceceeeeeeeeeeeeenaeeeesnaeeeeeaaes 211 10 9 Spectral UNMIXING cccccecccsesececeeseeeeeeseeeseaeeeeeeaseeessaseeessaeeeensaes 212 109 1 PROPIA OM iee EE ER E 212 10 9 2 10e PODIO pesoen ORA 213 We TMe SOUNO aen a eeacacensenceseetanatomececuessseee 214 10 9 4 How Does it Work ccccccccecseseeeeeseeeeeseeeeessaeeeeesaseessaneeessaseessnaes 215 10 9 5 Spectral Unmixing with cell R cel M sssssssssssssesesnessnnresnnrnsnn 216 10 9 6 CalDratiOr werner renrn re mente tr re errr aa a rte errr enor 217 UO WIIG eae ine aE aE E EEE EEEE ENEE 218 10 9 8 Unmixing of Color Camera
123. T TROSC Lieto Gad ed see Secreted aie cee ica md ale ce ek ned ac pede all 2 8 Oe MOA ii asia artes sited tect el tien tinct a Ga wadst 278 gS 4 R WY ope ene aa ears ate eee art er e ere eae eee ee 279 LS PrelerenCES ainn a a a a a 280 13 5 1 The Preferences gt Image Tab s sssssssssssssssenrsrrrenrresrrnsnresrnesrnrennne 280 13 5 2 The Preferences gt View Tab cccccscccsscccseeceeeeseeeeeeeeseeeeneeees 282 13 5 3 The Preferences gt File Tab cccccccscccssecceseceeeeceeeeeeeeseeeeneeees 284 13 5 4 The Preferences gt Measure Tab cccscccseeceeeeseeeeeseeseeeeeeees 289 13 5 5 The Preferences Module Tab ccccssccseeecececeeeeeeeesaeeeneeees 291 13 5 6 The Preferences gt Graph Tab c ccscccsececeseeceseeeeeseeseeessaees 293 13 5 7 The Preferences gt Database Tab ccccscccsseeseeeeeseeseeeeeeeees 294 TOO NVIINOOW esseen EEA sata E A N 295 19 0 Mi iMZ amp Al orraa a aa 295 als is 6 Fag Close Allie ea eran e eet tee ne ne 295 13 0 3 DOCUMeNL Manager snake titan a eee eee cae 295 1310 4 WIGW DOR Manage iraa RRE AARE 297 1 0 5 Image VIAN AG SM opon r AT A NEA 297 Chapter 14 1360 Staus Bar ea a 298 A complete integrated Or SGCOMMMANG WINONA O 298 development environ ment for macros based 14 Imaging C PPT errr etree eee eee 301 on the programming TAT General arare aaO Nara 302 language Imaging C AA 2 NEW Mod l mana a a r a a a 303 cell amp c
124. The Image gt Extract command enables you to extract a subset of data out of a multi dimensional data set The ROIs tab It is possible to crop a single image or an image set in the XY plane The area to be cropped is defined via an ROI see Chapter 10 4 Regions of Interest ROIs If the image contains already ROls select one from the list Otherwise define a ROI with the ROI drawing tools via the Define button it has the same function as the ROIs button of the Complex Analysis toolbar If an ellipti cal or free hand ROI is used a rectangular area that circumscribes the ROI will be used for the cropping cell cell software Manual Chapter 7 Image Data Handling 117 Extract Ed Extract Dimensions ROIs Dimensions ROIs Color Channels Selected ROIs Full frame Z Layers Eels de tee Tle Time Frames From i Wela Eeh E Border ox Lert te To enlarge the cropping area by a number of pixels in the X and Y directions type a value into the Border pixel box Execute the cropping by clicking OK The Dimensions tab Here you have to define the Color Channels and range From to of Z Layers and time points Time Frames to be extracted By setting a step size higher than 1 you can opt to extract only every 2 3 and so forth layer or frame Execute the cropping by clicking OK 7 8 Combine 7 8 1 General cell4R cell4M allows to fuse individual data sets within the color time an
125. 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 the central pixel cannot influence the results What will happen The Sobel filter employs a non linear method for edge enhancement This filter is comprised of a set of differentiation filters Gray value modulations that are either horizontal or vertical will be especially enhanced The Sobel filter generally yields the magnitude and direction of the most significant intensity gradient Matrix a b Two matrices are applied independently and then their geometric mean is taken Comparison The results are comparable to those of the Laplace filters Laplace Il leads to a clearer accentuation of edges but increases the noise 8 2 12 Roberts Application Rapid edge detection What will happen The Roberts filter employs a non linear method for edge enhancement and registers transitions from light to dark pixels This filter is comprised of two basic differentiation matrices Of importance is the difference in brightness in the diagonal of the four upper left values Large differences result in high values areas with small gradients will be suppressed Due to the low number of computations this filter functions well as a rapid edge filter Matrix Comparison The results a
126. The unique all in one Illumination System MT10 or MT20 for fast wavelength switch and at tenuation to meet the experimental requirements for fast real time acquisition by highly sensitive digital cameras e cell4R The Real Time Controller a hyper precision control board to synchronize the all the hardware devices and modules This additional independent plug in CPU board assures highest accuracy in experiment timing temporal resolution 1 ms precision lt 0 01 ms In practice this ensures that illumination of the specimen can be strongly limited to the acquisition of the image As a consequence bleaching and photo damage of the specimen can be minimized e cell4M The System Coordinator a control board to synchronize the all the hardware devices and modules temporal resolution 1 ms It carries out all the tasks that cell4R s Real Time Con troller does but lacks its timing precision and ability to run all tasks in parallel e The sophisticated cell4R cell4M imaging software is a powerful all embracing platform that features an intuitive and user friendly graphical drag and drop interface the Experiment Man ager for setting up and executing even the most complex experiments in a convenient and con cise way A structured database for multi dimensional data handling xyz time color is also included as well as tools for image processing image analyses and more complex analysis like rationing AF F FRET and spectral unmixing The in
127. This is helpful when you no longer need the current configuration or when you want to create further configurations based on the standard configuration Saving current configuration If you have made alterations to a particular configuration which have not yet been saved before the standard configuration is loaded you ll be given the opportu nity to save your altered current configuration 13 4 2 Load Use this command to load a previously saved configuration Available If you ve already created and saved your own configuration you can activate it using the Load command making it your current configuration If you load a configuration that was created with an older cell4R cell M version the Update Con figuration scy dialog box will open Cancel If you exit this dialog box via Cancel the older configuration will not be loaded Use standard configuration If the configuration you wish to load contains essential user defined functions you would be well advised to load that configuration using the Use standard configuration cell s cell software Manual Chapter 13 The Special Menu and the Window Menu and include functions defined in selected configuration option In most cases this option will get you the desired result Update Configuration temp scy Tou are tring to load a configuration which was previously defined in an older version of this e program Choose how to update the Cancel
128. a 715 10 696 63 Qe 0NA TAG AG CUE ae string Top 10 User defined Use this command to define the lower and upper end of an interval for the rows to be displayed If Exclude rows is checked the rows within the interval will be excluded User defined ROI 2 Display rows with values in interval Lower end Upper end T P C cog Be E 1005 21 A Hel Exclude rows String Use this command to define a string to display only those rows that contain this string If Exclude rows is checked the rows containing the string will be excluded For example if 40 is set as a String for the sheet above only entries higher than 4000 will be listed Search String ROI 2 Display only rows containing E Exclude rows Use 7 to represent any single character Use to represent any senes of characters 11 3 4 6 Create Graph The command Sheet context menu gt Create Graph is active only if a column of the sheet is acti vated via click on the header Use this command to make a new graph from the active column From the dialog box you can edit the Title and select the X and Y axis for the newly created graph cell a cell software Manual Chapter 11 Graph Display and Graph Analysis Create Graph from Sheet Title gt Kinetics of Ratio Image Fura 2 are i F Urut Start value 19 Increment 1 Y aniz Title Unit 11 3 4 7 Convert to Sheet Use Graph
129. a homogeneous object with high noise What will happen The algorithm examines all pixels within an area of a set size and calculates the average value of all those pixels which are within a certain intensity range l enra 20 relative to the central pixel All pixels with gray values outside this intensity range very likely belong to another set of object information and are therefore ignored If the number of pixels within the intensity range is less than or equal to half the square root of the number of all neighbors the central pixel will be replaced by the mean value of the four directly adjacent pixels Comparison Unlike the Median filter the Sigma filter does not remove isolated bad pixels with intensities that differ strongly from the surrounding pixels 145 146 Chapter 8 Image Processing OLYMPUS Define Sigma Filter Define Sigma Filter oe EE ance C Ereview Size Enter the radius of the area to be considered You can select radii from 1 to 11 at intervals of 0 5 The preset value is 3 Sigma Enter the value that defines the intensity range to be considered 8 2 20 DCE Differential Contrast Enhancement This filter is based on the definitions set in the Processing gt Define Filter DCE dialog box Application selective enhancement of weak differences in contrast What will happen The DCE filter renders image structures visible that are barely distinguishable from one another in the original image
130. a left sided blue node to a right sided one If a command symbol is activated connections cannot be drawn the mouse tip does not turn into a cross To deactivate a symbol click on an empty area of the editor sur face In a valid Experiment Plan all commands need to be connected 5 3 2 The Command Symbols and Their Properties Pages 5 3 2 1 Image acquisition 5j Image Acquisition P command icon color and subtitle depend on the Image Type Fite the exposure time is displayed in the little box on the right 44 Chapter 5 Experiment Manager Use for the acquisition of an image OLYMPUS Execution of this command in an experiment will cause the following actions 1 The illumination of the sample is switched on by opening the shutter 2 Immediately afterwards image acquisition starts upon a trigger pulse to the camera 3 Immediately after image acquisition is terminated the illumination of the sample is switched off by closing the shutter 4 The acquired image data are transferred to the PC RAM and eventually to the database on the hard disk Properties Image Properties Image Frame Display Image Type Dapi Light Intensity 100 Objective PLAPO 60x Contrast Insert Do not change Exposure Time 40 Acquisition time 121 ms Get Current Camera Settings Name Dapi EJ Properties Image Frame Display Binning Doo v Frame x 0 w iaz
131. a sample is newly placed onto the microscope and is found to be totally out of focus Autofocus fine In this mode the range set in the Autofocus options see previous chapter is divided by three and the step size is adjusted by default This mode can be used if the sample is already relatively close to the specimen In this case the settings of the Autofocus normal mode would probably first move the specimen way out of focus and it would cause an unnecessary loss of time to bring it back into focus again With the Autofocus fine mode the user does not have to reduce manually the range in the Autofocus options Autofocus very fine In this mode the range set in the Autofocus options see previous chapter is divided by five and the step size is adjusted by default Similar arguments as for the Autofocus fine mode apply The Autofocus fine mode can be useful for example if a slight loss of focus is caused due to a movement of the specimen with the microscope stage cell a cell software Manual Chapter 5 Experiment Manager 5 The Experiment Manager is a universal and easy to use tool for planning preparing and executing Experiment Manager experiments with the Imaging Station cell R cell M This chapter explains the general concept of setting up experiments lines out how the different system modules are integrated into experiments describes different types of experiments and guides through the system preparation and the execu
132. a this button Within the source text of the module you have defined a new function called An_other 1 To compile the new C module use the Build Module lt F7 gt command 2 Select the Edit Button Bars command in the Special menu 3 Select the module name in the Button Bars list Then click on the Edit button example sfm Seles 21 02 2006 define IDM_SAMPLE IDM_USER 1 o BEEEREEHEHEHEHEEHEREHEEREHEEHEHREHEHEEREREREHREEHREHEEHHEEREE COMMENTS The Add In manager browses for this function to display a long and a short description of this module BHREEREHHEEXHEREERHHHRHHEHEHHHRHHREXEREERHEHEHREHEHEEHHRERERHHREXEHES void ModuleComment f eu Version st1 0 100 2 n Copyright st 21 02 2006ra Description Mt Imaging C Module exanple Ls Y BER EXEEXEHHEHHHHEREHRERHHHREHEKREHEHEHHEHHHRHHEHEREHEXRERHHHRERHHEHHEHHREHE Created Changed Comment Sample MAFI function HEHEHE HEEHREREEHHEEREHEEEHHEHEERE HEBER EEREE HERE HEERERERERHRRERERES export UINT SampleFunction t HAPI IDM_SAMPLE Info in status bar Sample amp function gt dlgQutput Hello orld return IDOE 4 Select the New C module from the Command groups list The Commands list will now contain the SampleFunction entry as well as the name of the new function called An_other 5 Select the T button in the New Button field 6 Select An_other in the Commands fields cell s cell software Manu
133. ace If the ZDC detects any drift of the said interface position during an experiment the motorized Z drive will compensate it Properties Properties Range 200 00 H Sample offset 20 00 Linn Tol Objective UPLAPO 60x w Range Set here the scan range the ZDC may use when searching for the interface The larger it is the longer will the detection take Make sure however that the range is larger than any expected Z drift Sample offset This is the absolute difference between the interface and the focus position of the sample It is set automatically by the software To determine the offset focus the specimen and then click the Determine Offset button in the properties window The system will start a ZDC scan and set the value accordingly Objective Select the objective to be used by the ZDC from the shortlist 5 3 2 12 Additional atomic commands the Microscope Commands toolbar Microscope Sr LL Microscope Commands 58 Chapter 5 Experiment Manager OLYMPUS a n command icons Command icon Sa Filterwheel oo tr 120 al a Bott part l Side aah bats bb al DSU in DSU out ey ll pe Camera Ocular a me Tran close Tran open gt ie gt PLAFO 60 tq ty cet loos pA Parameter to set relative movement left icon or movement to absolute position move PIFOC move stage relative movement or movement to absolute position
134. active C module will be searched Lastly the prede fined symbols in the Exported by Tool range are searched Upper Lower case Wild cards When making a search request upper or lower case is of no rele vance Any parts of a symbol s name you don t know can be substituted with an asterisk letters you don t know can be substituted with a question mark 14 13 Goto Definition Use this command to view the definition of a symbol Available This command is available when a text document is open and active You may also use the lt F12 gt key What s it for This command can give you rapid access to the definition of a symbol when you re programming in Imaging C To do this position the cursor within the symbol s name in the text document and then press lt F12 gt The source file will be opened in a separate text document win dow The cursor will appear at the spot within the text where the symbol is defined and the sym bol name will be selected Nothing will happen if cell4R cell4M does not find the source file Just as when using the Find Symbol command all you have to do is enter the letters the symbol name begins with say if you don t know the precise name of the symbol or if the name of the symbol is extremely long Continue pressing lt F12 gt until the definition of the symbol you re looking for is shown Where are symbol names searched for Firstly symbols are searched for among the symbols def
135. age 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 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 considered 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 The Manual tab 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 cell cell software Manual Chapter 8 Image Processing Set Thresholds Manual Auto Settings Preview g None Color igh E Current All a x 4 Include Pixel Backgroun File C Transparent Auto Diagram 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 O and 100 this can be expressed in the following way 0 lt G
136. ages containing the two emission channels generated by the Dual View Micro lmager 4 Select the correct Illumination Excitation Filter and Filter Cube 5 Select Image Splitter as Observation Emission Filter and confirm the settings with 6 OK OBS System Configuration Illumination System Image Type Illumination Shutter Filter Cube Observation Fluorescence General Excitation Filter l Emission Filter l Color Excitation Filters Dapi 403 DAFI MT Shutter DapiFitcTxRed empty OoOo l A Full Control Fitc 492 FITC MT Shutter DapiFitcTxRed empty D T Errors JL L a oe oS a Microscope 572 TxRed MT Shutter DapiFitcTxRed w empty 2 T General 5 ADe CFP Split 430 CFR MT Shutter crpe l crre iw H i l Objectives A D _ ieee a Piter kubes YFP Split 500 FP MT Shutter CRPIYFP CFRIVFP JF Tl Contrast Inserts Filters Eime Toes M 2T Shutter E Image Splitter i Tl Pifoc i be i Stage a Al Ti i OptiGrid 350 Chapter 15 cell M cell R Configuration OLYMPUS cell amp cell Software Manual 16 Software Chapter 16 Installing the cell M cell R Software 351 Installing the cell M cell R The cell R cell4M Imaging Station will be delivered with the software installed However in ca
137. aging C programs are concerned cell4R cellAM will switch over to the debug mode when it is executing a function and it encounters a breakpoint With regard to macros cell R cell4M will also switch over to the debug mode if you execute the macro using the Single Step command instead of the Run Macro command You ll know the debug mode has begun when the mouse cursor transforms into the shape of a hand What s it for When you execute the function or the macro the expression will be selected in the line containing the breakpoint Now to have a look at the value of the expression or variable use the Quick Watch command or simply press lt Shift F9 gt You can then set another breakpoint and continue execution of the program by pressing lt F10 gt To have a look at the value of that expres sion simply press again lt Shift F9 gt The above is how you can work through the source text of Imaging C programs and macros and be able to follow what values an expression occupies while the program is being executed It s how to find programming errors The specified expression and its current value If the expression selected consists of a variable the current value of this variable will be shown If a value is undefined the following will appear This will apply e g to the value of a non initialized pointer variable QuickWatch The specited expression and its current value i digQutput Hello World Token Sy
138. aia 311 14 5 Save Module Configuration cccccccccssscecssssceeseeseeeeeseeeesseseeeseees 311 146 Edit Mod le siana a 313 WAP Bald Mogule s eon se cesoseccutcostngcscexeceseansesdcs esnccsernee icecesecdcasaetedce 314 14 8 Glose Modules sues a nea 2s 315 149 ADOT WIOGUIG opii cian cusencsnidic Nan EE E 315 1410 Module Manage sinnini tata eiaeie tie tleacshe ee amet eal aa 316 14 10 1 The Define Search Path for Modules Dialog BoX cccccceee 319 WA Vile SOW Side wees sass te cceinte ruc tinde E aans noe tack ene race ETa 320 Wael 2 AIMCO VIMO beno a a adbadacanibnci ERER 321 14 13 Goto DETNIMON es aes ces ices e deine ee eee 322 TAs A QUICK WY AUC iat ict Sxcenete det cenedetientticedecsaededeataecetccanededeedeedetecteedeteatest 323 T4 tS WV AUCH WV ANADICS eia a hac siees a Ea aa 324 14 16 Toggle BreakpOlNt nosiann aa 325 VAT Edit BIGAKDOINIG sirean sssr in iE aE E a EELER 327 302 Chapter 14 Imaging C OLYMPUS 14 1 General The Special C Module menu contains commands for the administration of Imaging C modules A C module provides one or more C module functions C modules are written using the macro lan guage Imaging C translated so that the computer is able to understand it and stored as files ex ecutable by cell4R cell4M extension SXU C module functions enhance the functionality of cell4R cell M and are seamlessly integrated into the user interface It is not necessary to learn Imaging C if you want
139. aining the Image Acquisition icons within a Multi Color Frame 2 Second an agonist will be injected automatically with a micro injector device that is able to receive trigger pulses from the cell R Real Time Controller cell M System Coordina tor via the Digital I O interface at the front of the cell R cell4M imaging computer To set this up in the Experiment Plan a TTL Out ON icon and a TTL Out OFF icon will fol low the Time Loop Frame The switching of a trigger port is very fast if the two icons immediately follow each other in the Experiment Plan the entire pulse lasts about 50 us in case of cell4R only the cell4M System Coordinator causes a jitter of 10 20 ms In case such a pulse is too short for the connected device to read it a Wait command that lasts say 20 ms can be inserted between the two trigger commands 3 Third after the injection the image acquisition shall continue but with an increased speed In the Experiment Plan a Time Loop Frame similar to the first one will follow the trigger commands however with a shorter Cycle Time The entire Experiment Plan would look like this F Fura340 Fura3s0 Fura340 Fura3a0 m m L Lal m Ll z L m m m 10 x 500 00 ms 50 x 500 00 ms cell s cell software Manual Chapter 5 Experiment Manager It is not necessary to draw the second time lapse acquisition part of the Experiment Plan anew Pressing the lt shift gt key and simultaneously c
140. al Chapter 14 Imaging C 309 7 Click on the Modify button 8 Once you click on OK the T button will be connected to the name of the new function 9 Use the Save Module Configuration command to save this altered module configura tion Using this command will result in the previous module configuration being overwritten 10 Use the Build Module lt F7 gt command to now compile the C module again When you do this any alterations made in the C module configuration file SCX will be included in the actual SXU of the C module O If you change the function description of a MAPI function the configuration file does not have to be adapted accordingly All you need to do is have the C module re compiled using the Build Module lt F7 gt command 14 3 Open Module Open Module Look in ExcelDE e go A My Recent Documents Desktop My Documents PE File name My Computer Files of type Module Formats Add module to module search path _ hy Network 310 Chapter 14 Imaging C OLYMPUS Use Special C Module gt Open Module to load and activate a C module Application The standard Windows dialog box for opening and saving files will be opened Select the path of the module desired in this dialog box You can use either the MKU make file or the SXU executable file to load and activate the module You may also use the Module Manager command to load and or activate a module D
141. al Unmixing How ever while in the latter dual excitation images are taken exclusively here a dual emission image of the donor and a dual excitation image of the acceptor are taken 10 12 2 1 First correction factor donor emission This calibration determines the distribution of the donor emission between the two channels that is the fraction of the donor fluorescence transmits through the acceptor emission filter A dual emission image of the donor only reference sample has to be acquired using the FRET equipment either the observation filter wheel or the Dual View Micro lmager as described above The following filters have to be used donor exciter dual band dichroic mirror donor emitter Fdon channel acceptor emitter Ffret channel i 1 Activate the donor sample image and click the FRET Correction button 2 Make sure that the Ffret and Fdon channels are selected correctly on the Donor Sam ple tab of the FRET Correction window 3 Two ROls have to be defined The first one has to be within an area where the fluoro chrome is visible while the second ROI is used for background correction To do so press Define ROls to open the respective dialog box The ROls are set in the same way as in the Image gt Define ROIs menu Chapter 10 4 Regions of Interest ROIs Afterwards close the Define ROIs window 4 The ratio of the average intensities within the first ROI after background subtraction se
142. ally Coloring This specifies the coloring of the online image display during image acquisition within the previously defined intensity range e Gray Scale The image intensities are displayed in a linear gray scale e Image type color The image intensities are displayed using the Fluorescence color defined in the Image type dialog box of the cell4R cell4M Configuration Software See Chapter 15 cel AR cell M Configuration For example it may be set so that GFP images will be displayed in green scale 5 3 2 2 The Multi Color Frame B Multi Color Frame Use for the acquisition of multi color images of multi labeled specimen or samples with dual exci tation or dual emission dyes e g Fura 2 Indo This command causes the storage of the acquired images of different Image Type coded by dif ferent Image Acquisition icons encircled by the multi color frame within one file in the database e Any number of single images within the Multi Color Frame is combined into one multi color image e Each image within the multi color frame will make up entirely and exclusively one color channel of the resulting multi color image O The multi color frame can only be used if all Image Acquisition commands inside the frame have the same binning factor and ROI settings Frame properties Otherwise an error message will be generated upon execution of the Check command Properties Multicolor The only parameter to be set here is the Name to
143. alter average 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 W 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 Example The application of a filter for a numerical example is calculated as follows Gray values of the original image Gray values of Matrix the filtered image 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 realization Derivative filters Negative weight factors are admitted in the filter matrix of derivative filters and the sum of the weight factors equals zero The result for homogeneous gray value areas is 0 a black area On the other hand side gray value edges i e the edges of objects within an image and intensity gradients become accentuated These filters can aid you as far as edge extraction is concerned The occurrence of differential terms will however increase image noise Rank Filters The Rank Filters comprise a filter class of their own An environment of the central pixel is defined here
144. an be preprocessed using a mean filter before separation In the Sigma field you enter the half width values 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 O means the mean filter is a binominal filter Smooth Use the 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 Sigma gt 0 the mean filter is a sigma filter for Sigma values greater than 0 Smooth 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 O means that no averaging takes place at all The Sigma field will not be available Fine Coarse Adjust the scroll bar such that 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 Result The result provided by the separator is an image containing the outlines of the objects to be sepa rated The width of the separation lines is
145. and Z will always have the same size Interpolation Resizing images will result in a reduction in image quality This is because the color of each original pixel must be redistributed across a different number of pixels and the result will be less precise than the original There will be an inevitable loss of sharpness regardless of whether the image is enlarged or made smaller Nearest Neighbor This method doubles or removes pixels The appearance of the new image may be somewhat coarse Trilinear This method interpolates the color of the original pixels to come to the new pixels Bicubic XY Linear Z This method interpolates the color of the original pixels to come to the new pixels using a different algorithm in the X and Y dimensions as compared to the trilinear method The appearance of the resized image will be slightly less blurry 153 154 Chapter 8 Image Processing OLYMPUS 8 4 2 Rotate The function Process gt Image Geometry gt Rotate allows rotating single images and entire data sets in the XY plane By doing so it creates a new data set so that the original images remain un touched Rotate Properties Angle 315 lal Degrees 1 359 Set Angle Direction Options Interpolation Keep XV ratio Angle Set the rotation angle in this box Set Angle This command allows setting the rotation angle interactively using two lines initializing from the same point 1 After clicking the button pos
146. and intruders IFyou re not sure how to set these properties use the Network Setup Wizard instead What else should know about Windows Firewall 362 Chapter 16 Installing the cell M cell R Software OLYMPUS cell amp cell Software Manual Index Acquisition 12 20 43 3 D Time lapse 65 FRET 224 Kinetics 52 65 Multi Color 46 Not synchronous 59 Ratio image 51 65 Single image 60 61 Time lapse 49 63 Z Stack 47 62 Add Ins 273 Arithmetic Operations 151 Autofocus 34 55 68 AVI Recorder 21 Background subtraction 205 Binarize 167 Camera Binning 13 22 Calibration 102 Control 12 21 Exposure Time 22 Gain 24 Offset 24 Selection 355 Subframe 23 White Balance 24 Colocalization 222 Command window 298 Configuration 329 Dual View Micro lmager 347 GUI 277 Illumination system 330 Image types 340 Chapter 16 Installing the cell M cell R Software 363 Microscope 333 Motorized stage 344 PIFOC 343 Shutters 342 Database 13 69 74 252 Preferences 294 Query 262 Deblurring Inverse Filter 172 Nearest Neighbor 170 No Neighbor 169 Deconvolution 169 173 Display 78 Adjust 78 Auto adjust 82 Black balance 83 Brightness 23 False color 85 87 Fluorescence color 84 Full screen 77 Gray scale 83 Grid 109 Histogram 23 Images 16 Intensity 16 78 Intensity modulated 100 Markers Time and Z information 109 Overlay 111 Properties 10 Sequences 17 Tile view 76 94 White balance 82
147. and the NxN filter certain parameters have to be set in the respective Define dialog boxes other wise the settings of the last usage will be taken by default Sharpen I Define Filter User Filter Sharpen II Filter r MoM Differentiate Pseudo Arithmetic Operations Arithmetic Operations Image Geometry Differentiate Y Reimer RuSB Studio Laplace I Image Geometry t Edge Enhance Laplace IT RGB Studio Rank Mean Sigma Median 3D Images T Pseudo Sobel Roberts Reimer User Filter MaM Lowpass Edge Enhance Rank Sigma 3D Imaqges F 8 2 2 Sharpen Application Detail emphasis The Sharpen filter belongs to the class of derivative filters and amplifies the central pixel relative to the X and Y axes Image details become emphasized by an enhanced contrast Thus the image the will seem to have increased focus The Sharpen filter enhances however noise as well Matrix Comparison The overall effect is opposite to that of a smoothing filter cell a cell software Manual Chapter 8 Image Processing 135 8 2 3 Sharpen II Application Detail emphasis Matrix Comparison This filter is similar to the Sharpen filter but has a more pronounced effect 8 2 4 Differentiate X Application Extraction of gray value edges in the Y dimension Use this derivative filter to detect gray value edges parallel to the Y axis gray value mod
148. ands in one Experiment Plan However same as for the offline analysis only one graph with several ROI curves of one image set can be displayed at a time The graph to be displayed can be chosen and changed in the Graph gallery of the Image Manager box at the left side of the user interface during the experiment The calculation of a kinetics analysis is a time consuming command for the PC proces sor The software estimates the additional CPU usage and might change the minimal cycle in dependence of online analyses However in rather fast and complex experi ments it still may happen that the image acquisition is faster than the generation of online analysis results by the PC In such a case the image acquisition timing has the highest priority and the analysis will remain incomplete That means while certain data points will be missing in the graph all images will be acquired as designed in the Ex periment Plan and stored in the database The analysis has then to be repeated offline 54 Chapter 5 Experiment Manager OLYMPUS 5 3 2 8 The digital port switch commands TTL Out ON and TTL Out OFF I Digital Port Tye ra ar ee T L ray command icons with and without eoa Port 1 onsi ports t23 right conditional option activated Use for respectively switching on trigger pulses to external devices that are connected to the 3 plugs on the computer front panel or switching them off The pulses mentioned are call
149. annel OK Confirm the settings and return to the Deblurring dialog window 3D Deconvolution Filter Selection Select either No Neighbor or Nearest Neighbor from the Filter Selection shortlist O In case of single images and time series only No Neighbor is available 3D Deconvolution Filters Projection Filter Selection Nearest Neighbor Mo Neighbor Nearest Heighbor Inverse Filter Haze Removal Factor 4 Sub Y olume Overlap Microscope C Transmitted Brightfield Channel Settings Haze Removal Factor This parameter is expressed as a percentage value and determines the power of the deblurring The higher the number the more blurring is removed from the images The effect will hardly be visible with values below 50 while the default of 85 gives moderate results Very high values like 98 or more result in rather grainy images with very pronounced contrasts and can introduce artifacts Be sure to carefully check the results Transmitted Brightfield Mark the check box if you use a brightfield setup The check box Phase Object becomes available Phase Object Mark the check box if the specimen is a phase object A phase object is any specimen with little light absorption e g living Mitochondria chromosomes bacteria or cyto plasm 171 172 Chapter 8 Image Processing OLYMPUS Execute This button in the Deblurring dialog window starts the calculation A window opens that shows the progress of the
150. aph Database Report Save image files Sheet format Tagged Image Format tif s Standard Oo C Replace blanks Decimal symbol PCD Format List separator TES x 512 w Use Windows regional settings General Save configuration on exit without confirmation Create backup when saving textiles Keep file type in file inpuboutout dialogs Humber of entries in recent file list AE JPEG JFIF jpg JPEG200 jp2 LEAD Compression cmp In order to achieve a stronger data reduction you may choose to export images using these formats However they go along with loss of quantitative information in dependence of the compression quality JPEG does result in information lost i e an image you had saved using the JPEG method is no longer 100 identical to the original image A compressed image is no longer of any use for pre cise quantitative analysis You may have trouble making out the difference with the naked eye depending on the degree of image compression Some image information loss may be acceptable for applications where the most important thing is the visual appearance of the image JPEG compression provides fine results for gray value and true color images of a photographic quality For applications where color or gray value distribution is important we recommend to avoid JPEG compression JPEG compression is not suitable for synthetic images or for images containing fine lines or inscriptions which
151. ar chy display This button is only available if you ve selected a menu or submenu entry When for example the main menu is being shown you can select an entry and then click on the Show Sub Menu button to have a look at all commands belonging to the entry selected Then once all com mands of a specific menu are being shown you can select a submenu and then click on this button to have a look at all the commands contained by that submenu An easy alternative is simply to double click on the entry in the Menu list Show Main Menu Click on the Show Main Menu button to move up a level in the menu hierarchy in the Menu list This button will not be active when the main menu is being shown in the list you cannot go any higher This button will become available when you click on the Show Sub Menu button moving you down a level in the menu hierarchy Command groups Specific groups of commands can be selected from the Command groups list If you select e g All Groups all available commands will be displayed Commands The Commands list shows available commands depending on which group of com mands was selected in the Command groups list Description The Description field provides a brief description of the menu or command selected Up or Down After you have selected an entry from the Menu list you can adjust the position of the element by clicking on either the Up or Down buttons A main menu will be moved either to the left or right
152. are divided by those of the 380 nm im age pixel by pixel The result is a new image the ratio image Any change of intensity in a time sequence of ratio images is directly correlated to a change in calcium concentration cell s cell software Manual Chapter 10 Intensity Analyses 211 10 8 2 Generating a Ratio Sequence a Ratio Open the Ratio dialog window with the Ratio button or via Measure gt Ratio Settings Dimensions Calibration Output Image C calibrated Background Subtraction None Select ROIs from Image 2 Fura 3 ROIs wt Constant Furaz40 ___ _ roal ROLL ROIL ka Furas o ROLI ROT 1 Ww Kinetic Select ROIs From Image 2 Fura 3 ROIs Available ROIs ROL1 ROI1 ROI 2 ROIZ ROLLS ROIS Sheet Highlight selected ROIs Thresholds Fura34o Furaseo so Output Scaling Scale Factor 1000 A Ratio Settings tab Output field The selection of the options Image Kinetics and Calibrated determine if a ratio im age sequence a graph or a calcium concentration image sequence are generated Any assortment of selections is possible Background Subtraction field The option None is of limited value because the unavoidable background intensity leads to skewed results when dividing pixel values If Constant is selected a fixed background value is subtracted from all pixel intensities in all images of the two channels The constant for the two chan
153. are in the image itself and not in an overlay This is because JPEG compressed images do not reproduce abrupt gray value transitions satisfactorily In fact false color images cannot be directly JPEG compressed This is why cell4R cell M will transform false color images into 3x8 bit true color RGB images before saving along with JPEG compression If there is an overlay it will be burned automatically before conversion Warning Try to avoid compression in general Compression always results in image artifacts an exception being the png format Images should only be compressed after the analysis is completed Options This opens the context sensitive Save Image Options dialog box 285 286 Chapter 13 The Special Menu and the Window Menu OLYMPUS Save Image Options TIFF Save Image Options Preprocessing C Convert 16 bit images to 8 bit JPEG C Burn overlay into image Compression O Lossless Compression a E E Progressive pasos EE Output Format Standard we Color space Convert into packed RGB aaa i TIFF Preprocessing Before saving the image you can adjust whether the image should be Con verted form 16 bit to 8 bit and whether the Burn overlay into image should be done by clicking the respective check boxes TIFF Compression The TIFF format also supports a series of compression methods Packed Bits JPEG JPEG 2000 for reducing the file size of an image you are saving The methods can b
154. arger than 0 Often it is around 128 counts The Offset option allows to automatically subtract the value set in the Offset box from each pixel intensity Set the value by using the slider or type in a number Color control These functions are available for color cameras only R G and B gain values These functions are available during Live View image acquisition only and allow setting the gain value for each color individually to adjust the color balance Set the values by using the R G and B sliders or type in the numbers White Balance This function is available for color cameras only It is primarily useful in brightfield imaging to provide accurate color rendition regardless of illumination variations Upon clicking the button a snapshot is taken automatically the color balance is analyzed and the appropriate color adjustments are being conducted the R G and B gain values are changed Finally another snap shot is taken using the new settings WBalance ROI For many specimens better white balance adjustment may be achieved by manu ally selecting a white or neutral gray area for reference within the specimen image area Clicking on the button allows setting a region of interest first Once this is done the color adjustments are Carried out as described above Shutter If this option is activated the shutter opens and closes in synchronization with the camera exposure If it is deactivated the shutter has to be opened by clicking t
155. art of the system 15 2 3 Configuration of the Objectives Nosepiece Check here if a Motorized nosepiece is available to be able to use it via the cellAR cell4M software Objectives slider You can use the slider to move any of the mounted objectives into position with out having to start the cell4R cell4M software Magnification Set the objective magnification by selecting from the shortlist Name Select the objective name from the shortlist or type in a name N A Type in the numerical aperture of the objective If a standard objective is used the N A will be set automatically once Magnification and Name have been set Correction If a camera mount with optics that change the magnification is used type in the correc tion factor here By default the value is 1 0 Magnification changer The IX71 and IX81 microscopes have a manual slider in the frame that allows increasing the objective magnification by a factor of 1 6 Check the 1x 1 6x option if this feature is available cell s cell software Manual Chapter 15 cell4M cell4R Configuration 337 E ops System Configuration Illumination System Nosepiece General Motorized Nosepiece Available Excitation Filters Burner Objectives Full Control Magnification Mame A Refraction Correction Errors 60 i A 4 ty ar a Microscope V ns General uao UpLAPO Contrast In
156. ary format all bits of n that are not identical to those of the constant will be ignored e n 64 4 sawtooth signal the constant here 64 will be subtracted from n multiple times as long as no negative value results e n 32 32 step function unless the divisor is converted into a floating point number by typing a decimal point see above e n lt 128 0 n 128 2 different formulas section wise read if n lt 128 then set to O other wise Limit 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 marked the values over 255 will all be set to 0 Values below 0 will be set to 255 File Click on the File button to open the standard dialog box for the opening and loading of files Files will be saved using the LUF format Linear When you click on the Linear button all formulas will be reset to n This corresponds to the linear standard LUT coordinates of 0 0 and 255 255 O The LUF format cannot be used by Imaging C functions to alter the lookup table of an image Therefore it is necessary to save LUF type palettes as LUT files as well see above Again only LUT files but not the LUFs will be listed in the False Color dialog box see above cell s cell software Manual Chapter 6 Image Display and Navigation 93 6 3 Image Navigation 6 3 1 General In most cases the acquired images are multi dimen
157. ase has been defined currently the Binarize Color Image command will automatically use the three pairs of color thresholds of the three channels RGB or HIS 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 see above cell s cell software Manual Chapter 8 Image Processing 169 8 6 Deblurring and Deconvolution 8 6 1 General Deblurring and deconvolution both are software tools to remove out of focus light from images to sharpen the contours of the imaged structures They should however not be confused with each other i 3D Deconvolution 3 D deconvolution is an image restoration method which moves out of focus light from each slice of a Z stack back into the slice where it originates It thus conserves all light information of the Z stack sharpens the resolution improves the signal to background ratio and allows and improves quantitative intensity analyses Deblurring however is not a restoration method but works subtractive and doesn t allow any meaningful quantitative analysis afterwards but should be regarded as a purely cosmetic method Side effects are increased background intensity increased noise decreased signal to background ratio and possible sharpening of out of focus structures
158. ated Dimensions tab See the description in Chapter 10 5 Background Subtraction 208 Chapter 10 Intensity Analyses OLYMPUS 10 7 DeltaF F AF F Analysis 10 7 1 General While few fluorochromes allow the determination of absolute ion concentrations via ratiometric methods the majority of ion sensitive dyes only allow the qualitative observation of changes in concentration A convenient way to monitor such changes is to relate each image of a time series to the state at the beginning of the experiment and to analyze dynamic processes with a AF F plot pronounced Delta F over F The actual algorithm is rather AF F const1 const2 Here F refers to the reference fluorescence intensity usually the first image or the average of the first couple of images AF is the difference between the current intensity and the reference F urew F The ratio is usually normalized const1 const2 100 or 1000 so that changes in intensity are given in percent or tenth of percent respectively with a value of 100 or 1000 10 at the start Why AF F The advantage of this plot is that it often more clearly displays the change of signal intensities regardless of the absolute value of the fluorescence Hence it makes it easier to com pare the variations in weak and bright parts of the sample A graphical analysis kinetics of the raw data will often clearly show changes in the intensive parts while shifts in dimmer structures al though rel
159. atively of the same magnitude might be overlooked A side effect of this type of plot however is often the loss of structural details 10 7 2 Generating a AF F sequence a Delta F F Open the Delta F dialog window with the Delta F button or via Measure gt DeltaF F Delta Settings tab Output field The selection of the options Image and Kinetics determine if a AF F image sequence and or a graph is generated Background Subtraction field The option None is of limited value because the unavoidable background intensity leads to skewed results when dividing pixel values If Constant is selected a cell a cell software Manual Chapter 10 Intensity Analyses fixed background value is subtracted from all pixel intensities in all images The constant is set in the box that is only visible if this option is chosen ROI The typical approach to background sub traction is to mark a ROI in an image area that contains only background intensity and use the av erage intensity in this ROI as background value This value is determined individually in each image of each channel Image The last option is to subtract a predetermined background image from each of the images of the data set Delta F Delta Settings Dimensions Gurpue Images Background Subtraction Mone O Constant 2 Fura 3 ROIs wy ROI Select ROI Image ROLL ROT 1 w kinetic Select ROIs from Image Select ROTs from Image 2 Fura 3 ROIs
160. ayed in individual Viewport O The Images window is limited to 4x4 Viewports by default This setting can be in creased to 5x5 as maximum see Chapter 3 1 3 The Viewport If the Experiment Plan calls for more Viewports for example if the positions list contains ten positions and the Stage frame three Image Acquisition commands an error message that states Could not allocate requested number of displays will be generated upon experiment evalua tion see Chapter 5 4 2 Executing an Experiment 5 3 2 6 Online ratio image sie bo STA e Ratio _ 2 _ command icon with store option activated Use for the calculation of the ratio image of two images acquired during one cycle of the experi ment This function is mostly used for calcium ratio imaging with the calcium sensitive dye FURA 2 and images acquired with 340 nm and 380 nm excitation wavelength For more details see Chapter 10 8 Ratio Analysis Properties Ratio Background Subtraction Three different modes are available a constant a background image or the average intensity of a ROI can be subtracted See Chapter 10 5 2 Subtracting the Image Back ground for more details Thresholds and Output Scaling See Chapter 10 8 2 Generating a Ratio Sequence for details Properties Calibration See Chapter 10 8 2 Generating a Ratio Sequence for details Properties Store If the option Store is marked this is indicated by the storage symbol in the top right corner
161. ayers This command deletes the entire overlay The command Image gt Delete Overlay has the same function 113 114 Chapter 7 Image Data Handling OLYMPUS S Load Objects This command enables you to load a previously stored overlay file ovl from a storage medium Other object types that can be loaded are bitmaps bmp icons ico and enhanced meta files emf hii Save Objects This command stores all objects selected into an overlay file ovl The button remains disabled if no object is active dt fs Cut Objects Copy Objects Paste Objects These commands work only on the overlay objects but otherwise have the same functions as in any common software The typical key combinations lt Ctrl x gt lt Ctrl c gt lt Ctrl p gt do NOT work here but start the Cut Copy and Paste commands respectively for texts and single images If no object is selected the entire overlay will be cut or copied De Lh Oh Bring to Front Send to Back Bring Forward Send Backward If several layers of overlays exist positions can be changed with these commands in case of over laps This only applies if several objects have been loaded Objects created with the overlay draw ing tools are always located in the same layer abl Text This command allows writing a text into the overlay Initially a rectangle is displayed on the image to indicate the size and position of the text This box has to be positioned by mouse With the press
162. 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 Intensity 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 The RGB tab sets thresholds based on the RGB model Set Color Thresholds E rem one _ Current me Os Background Include pixel New 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 O Be sure to give each phase a specific name If you do not and continue using the standard phase names then when you delete a phase the phase name and the actual phase itself will no longer correspond to each other If you use specific phase names name and phase correspondence will remain unchanged Color ranges are defined separately for each color channel The Red Green and Blue fields indi cate where the lower and upper limits of each channel are Each color channel can be located 163 164 Chapter 8 Image Processing OLYMPUS anywhere between the values 0 and 255 The easiest way to defi
163. but they can be pasted into the current line for editing and execution This can be useful e g if you ve received an error message and you would like to make a correction Simply position the cursor at the relevant preceding command line This line will be then selected in its entirety Then press Enter to have this line pasted into the current command line After editing the incorrect entry you can re execute the function cell cell software Manual Chapter 13 The Special Menu and the Window Menu Saving Command Lines In contrast to other document windows the contents of the Command Window cannot be saved using the Save As command However you can circumvent this prob lem therefore open a new standard text document and simply paste the desired command lines via the clipboard into this text document The text document can now be saved using the Save As command O You can determine whether the Command Window is to be automatically activated by an Imaging C output Select Special gt Preferences or press lt F8 gt In the displayed dialog box select the Module tab and mark Activate on output in the Command window field This ensures that no output goes unnoticed e g because another document is covering it up On the other hand some Macros or C Modules use the ac tive sheet document for their calculations If the Command Window is activated by an output this can interfere with the running of your program because instead
164. calculation OK Exit the Deblurring dialog after the calculation is finished 8 6 5 Inverse Filter The Inverse Filter is a one step non iterative deconvolution method based on inverse filtering the ory It utilizes optimal linear filtering and is a simple deconvolution method It is very useful for ob taining quick results but is not as accurate as Blind Deconvolution as offered for example in the AutoDeblur software package by AutoQuant Inverse Filtering is typically more robust than the Nearest Neighbor or No Neighbors deconvolution methods The execution speed of the Inverse Filter is between that of Nearest Neighbors and Blind Deconvolution Available Z stacks and 3 D time lapse series both either monochromatic or multi colored Requirements The images need to be calibrated in the X Y and Z dimensions see Chapter 7 1 Calibrate Image otherwise an error message will be generated 3D Deconvolution Filters Projection Filter Selection Inverse Filter w Filter Parameters Sub Y olume Overlap E Noise Level The picklist offers the Auto Low and Medium options Select the option according to the level of noise in your image stack In most cases the Auto parameter produces very good results Should however the convolution only be faintly visible results with the Low or Medium options can be better Tiling The algorithm produces huge temporary data the size of which will easily exceed the com puter RAM even
165. cale Bar Properties dialog box The scale is part of the overlay plane See Chapter 7 5 1 Overlays General and does not interfere with the image data thus it can be activated and deacti vated any time 7 2 2 Setting the Scale Bar Properties Open the Properties dialog for the Scale Bar via Image gt Scale Bar gt Properties Display Scale bar Selection field Here you select if a Horizontal and or a Vertical Scale Bar are to be 108 Chapter 7 Image Data Handling OLYMPUS shown A Palette bar depicts the color gray scale coding of the light intensities and is especially useful for images displayed with a pseudo color palette Scale Bar Properties Display Format Size Scale bar selection Horizontal scale bar Vertical scale bar Palette bar C false color palettes only Show scale bar for View port Clipboard Show scale bar for field In case images are to be printed or exported to the clipboard with the scale the respective boxes have to be checked Format and Size Here the style and size of the bar and the dimension units can be set 7 2 3 Show in Viewport Activate and deactivate the display of the scale bar by clicking on Image gt Scale Bar gt Show in Viewport or using the short cut lt Shift F4 gt The same can be done in the Show scale bar for Viewport box on Image gt Scale Bar gt Properties Display 7 2 4 Draw into Overlay As opposed to the Show in Viewport option with the Draw
166. calibration automatically Nevertheless here it is explained how to adjust the calibration The software offers the possibility to show a scale bar and a color palette bar in an image overlay Objects and texts can be added manually to an image overlay It is possible to extract sub sequences out of multi dimensional image Image Data Handling sets or to combine images 7 1 7 1 1 7 1 2 7 1 3 7 1 4 7 2 7 2 1 7 2 2 7 2 3 7 2 4 7 3 7 4 1 9 Calibrate IMAJ eSa n a r E 102 Why Calibrate Images cccccecssseceessseeeeeeeeeeeseeeeessageeeeeaeeeesnees 102 Calibrating the Camera Channnel cccccccecssseeeseeeeeseeeeeeseeeeeens 102 AY Ge 9 2 18 0 0 sarea a a 104 ZAM AVIOMN erescoceset oie cated acest E tt ata as 106 SC al Bal oera Na 107 CCl all easeesecrascst cece tet cent EEE EE 107 Setting the Scale Bar PropertieS ccccccecsesseeeseeseesseeeeeesaeeeeens 107 Show in VieWport 2565506 cecct os fecuetinroesececcesedior nnen eninai iniia n nienia a nukean 108 Draw into Overlay xcn22ciicccdicigecsiieiecs edi eset canned eects ee 108 Show Markers Time and Z Information ccceececeeceeeeeeeeeeeeeeees 109 CN AG 5 tan cbc a a tua ip eased eens 109 OU GIA ere a e a a e a 111 General roroa an N as 111 Activating the Overlay TOOIDar cccccccccseseeeeseeeeeeseeeeesseeeesaees 111 Creating and Editing Overlays ccccsssceeeeeeeeeeeeeeeseaeeeesnaeeeens 112 DO DANAUS enna a ieee eee acest 1
167. can have objects located in selected color ranges displayed in any false color 222 Chapter 10 Intensity Analyses OLYMPUS 10 10 2 Phase Analysis This command evaluates area 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 Allow operations on false color images check box within the Special Preferences gt Image tab is selected Phase Analysis is the quantitative analysis of the area s of various gray value ranges called phases Chapter 8 5 Thresholds and Binarization explains in detail how to set thresholds to define phases The Phase Analysis command generates a measurement sheet that contains the absolute areas of the gray value phases as well as the area of each phase relative distributed by percentage to ei ther the total image area or the area within the active frame Sheet column headers contain phase name and lower and upper thresholds The column header s color corresponds to its respective phase Phase analysis of various gray value ranges will enable you to e g determine the surface area distributed by percentage of a particular material on a background Surface area can be calcu lated in true color images using selected color ranges Phase Analysis can be applied to other images and or other areas within the same image usin
168. ceceececeeeeceeeeeceeeeeeeeeeseeesseeessees 20 AVITICCOIG GF ceonrona rE E E 21 Camera Control nannnannnnnnnnnnnnnnonnennnnnnnrnnnnnnnrnnnnrnnnrrnnrnnnrennrenneenne 21 IMAG CONTO Sesa a RNAS 25 Micr sc pe Conmrol nssr anaa a A 26 Motorized Stage Control esiis e ations 29 DGTMING as OSIIONS LIST ices ces eateries 29 Correcting a Positions List x sissacesdsnsacascesse lace seenseccesseceecsnsarcccnandecnas 30 Calibrating the Motorized Stage ccccssscecseseeeeseseeeeeeseeeeaeeeeeenees 32 PRI OT OCS sas silane essere tiered anata a a a oats 34 AUITOTOCUSODUOMNS ai 5 2555 stag eras ienrecccsiteas a E niece E E AE 35 Executing an Autofocus SCAN ccccseeceeeseeeeeeeeeeeeeeeeeeenaeeeenaeeeeens 36 EXPeniment Vial aC Sl iisissatescvnae vaccaticwes davneeuauat ianeadasiapsnentasetvacsine 37 AC Graphical TOO assis net eel tattoo ats tacit Sosa o ly Sone ON 38 CONGEDE Ol USAC men aaa E 39 Experiment Manager Components sscccseeeeeeeeeeeeeeseeeeeeeeeens 39 Arrangement and CuStomiZatioOn ccccccccccsseeeeeseeeeeeseeeeesneeeeeens 40 Setting Up Experiment Plans ccccccsececeeeeeceeeeeeseeseeeesseeeesaaees 42 Types of Graphical Icons and the General Principles of Usage 42 The Command Symbols and Their Properties Pages 00 43 Contents Chapter 6 Touching up the image display by brightness contrast and color ad justment and navigation through multi dimensional image sets Cha
169. cell software Manual Chapter 5 Experiment Manager Fitc ail Image Acquisition deactivated left and active right Icons can simply be moved by drag amp drop Frames can be enlarged or diminished by dragging the black knots at the corners and sides that appear upon activation Both can be deleted by activation via mouse click and Edit Cut or by pressing the Del key They can be copied and pasted via Edit Copy and Edit gt Paste or lt Ctrl c gt and lt Ctrl v gt In order to generate an unequivocal Experiment Plan the icons and frames have to be intercon nected to give the order of execution of the commands To specify the sequence connect the blue connecting node arrow head on the right side of a command symbol with the blue node on the left side of the successive one When pointing with the mouse tip over a right sided connection node the mouse tip turns from an arrow or the symbol you selected in the MSWindows settings into a cross Press the left mouse button drag the mouse tip to the blue arrowhead on the left side of the successive command icon and release the left mouse button The two commands are now con nected with an arrow indicating the succession of the commands see the image below Arrows can be deleted after activation as above b FF s B PL Dapi Port 1 100 ms Port 1 O The time axis within Experiment Plans runs always from left to right i e commands cannot be connected from right to left that is from
170. check box a sub directory of the current direc tory will be created using the module s name You will see the current directory name displayed in the Current directory field Directory The Directory button is for opening a standard Windows dialog box where you can select an other current directory In addition you can create a new directory or delete one you no longer need Main Function If you have selected the Main Function check box the new C module will contain an initialization function of this same name The Main function of a C module is used when loading the C module If this function returns a value of 1 the loading procedure will be interrupted and the C module closed In general however Main is used for the correct initialization of a C module If an initializa tion of the C module is not necessary you can skip this function i e the function does not even have to be present Example Imaging C Code int Main return TRUE Exit Function If you have selected the Exit Function check box the new C module will contain a de initialization function of this same name The Exit function of a C module is used when a C module is exited If a de initialization of the C module is not necessary you can skip this function i e the function does not even have to be present Example Imaging C Code int Exit return TRUE MAPI Sample Function If you have selected th
171. ck on the Directories button to include more modules from other directories in the Other list The Define search path for modules dialog box will be opened Open Click on the Open button to load a C module and activate it The Open Module dialog box will be opened Using the Open button however the previously active C module will be only deactivated and not closed 317 318 Chapter 14 Imaging C OLYMPUS Build Click on the Build button to compile the module selected The button s function corre sponds to the Build Module command This button is only available if you have selected a module in either the Loaded or the Other lists Furthermore there has to be a MKU make file for this module To interrupt compiling simply use the lt Ctrl c gt keys Files Click on the Files button to edit the module you ve selected The Edit Module dialog box will be opened This button is only available if you have selected a module in either the Loaded or Other lists Fur thermore there has to be a MKU make file for this module Add File Click on the Add File button to add the active text document to the module selected The File field indicates which text document is active The text can comprise for example another source text and contain definitions and or a description of module functions This button is only available if you have selected a module in either the Loaded or Other lists Fur thermore there has to
172. ck the Set button to set the X position interactively A dashed line connects the text frame which is initially placed in the center and the X position of the cursor Left click to con firm the setting and return to the Set Labels window The new X value has been transferred into the Position field To fix the label at the new position click the Align button To move the text frame in the vertical direction click the Set button next to the Label text field not the Set button of the Data position group Draw line Select the Draw line check box to connect the text frame and the X position on the X axis by a line Draw arrow Select the Draw arrow check box to connect the text frame and the X position on the X axis by an arrow 244 Chapter 11 Graph Display and Graph Analysis OLYMPUS 11 3 1 3 Modify Use the Graph gt Markers and labels gt Modify command to change the text and or the position of the previously set annotations in the active graph Alternatively right click within a graph docu ment to open the context menu and then click Modify Labels Labels Select the label to be modified from the pick list in the Labels field Delete Use this command to delete the label currently listed in the Labels field Delete all Click this command to delete all labels in the active graph Alternatively select Data Analysis gt Markers and Labels gt Delete All from the file menu or use the respective command in the context men
173. command is based on the definitions set in the Processing gt Define Filter gt Pseudo dialog box Application Relief like appearance with shadows Filtered images will appear topographical as if a relief like surface is illuminated by an oblique light source Dark objects will appear as depressions and light objects as elevations This pseudo three dimensional display of image structure gives the filter its name Matrix 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 normal ized such that the zero level is assigned to the value 128 This means that gray values between 127 and 128 are available Comparison The pseudo filter is a derivative filters but not be compared with the other derivative filters Sharpen Differentiate and Laplace filters Define Pseudo Contrast Set a contrast factor 0 01 100 this will be multiplied by the weight factors of the filter matrix The preset value is 2 The larger the factor the larger is the 3 D effect Define Pseudo fx Contrast E a Preview Preview If this option is selected the image will show sub frame window with a preview of the filter result The sub frame can be set with the Window function cell a cell software Manual Chapter 8 Image Processing 139 8 2 11 Sobel Application Edge detection
174. connection remains un changed Otherwise the system may become disabled 1 To control the settings click on the Properties button in the CTR Status window to open the CTR Properties window 2 Select the Internet Protocol TCP IP connection and click on the Properties button to open the Internet Protocol TCP IP Properties window 3 The Use the following IP address has to be activated 4 The IP Address must be 42 42 42 17 5 The Subnet Mask must be 255 255 255 0 360 Chapter 16 Installing the cell M cell R Software 4 CTR_R Status General Support Connection Status Connected 00 56 49 10 0 Mbps Duration Speed Activity Received Packets 6 355 Properties Internet Protocol TCP IP Properties General You can get IF settings assigned automatically if your network supports this capability Othenwse you need to ask your network administrator for the appropriate IP settings O Obtain an IP address automatically IP address Subnet mask 299 255 2559 D Obtain ONS server address automatically Default gateway Use the following DNS server addresses Cid Preferred DNS server Alternate DNS server OLYMPUS CTR_R Properties General Authentication Advanced Connect using Eg Realtek RTL8139 810x Family Fast This connection uses the Following tems El Client for Microsoft Networks Internet Frotocol TCF F Install
175. consequently increases the readout speed Therefore binning is recommended if weak signals have to be detected at high acquisition rates or if spatial resolution is of minor im portance Exposure Time The exposure time determines the period of time during which the CCD chip is sensitive to incoming light in other words during which photons are collected and converted into charges to be read out afterwards The exposure time can be changed by 1 ms increments using cell amp cell Software Manual Chapter 4 Image Acquisition and Hardware Control either the slider or the mouse scroll wheel or by directly typing the value into the Exposure time box The slider limit can be selected via its context menu right click Exposure time w Adjust with binrin L Show saturation 2 wills 10s Standard Extended 305 Brightness adjustment Adjust with binning This feature automatically adapts the exposure time if the binning is changed See also http www olympus europa com medical 39_MicroGlossary cfm Show saturation This applies a Cold Warm Hot lookup table to the live image see Chapter 6 2 9 False Color It is only really useful if the Automatic brightness adjustment option see below is NOT activated In this case saturated pixels will be displayed red close to saturated pixels green and very dim pixels blue All others remain in gray scale Standard tab Brightness adjustment After a snapshot acquisition or during live
176. crosoft Excel Select the Microsoft Excel option to save sheets in the XLS file format These sheets can be loaded into and further processed in the MS Excel sheet calculation program cell4R cell M is not able to read this file format Lotus123 Select the Lotus123 option to save sheets in the WKS file format These sheets can be loaded into and further processed in the Lotus123 sheet calculation program cell4R cell4M is not able to read this file format Text Select the Text option to save sheets in the TXT format Sheets will be saved as text Semico lons will separate data within individual columns from one another Each text line represents a sheet row This text file can be read by any text editor or word processing program The advantage here is that sheet data can then be read into many other application programs This file will be opened as a text document in cell4R cell4M not as a sheet document Replace blanks Select the Replace blanks check box to automatically have all spaces replaced with an underscore symbol _ when saving sheet data in the TXT format This check box is for making the reading of sheet data from text files in other application programs much easier When reading in strings it s generally easier to deal with underscores than with spaces Use Windows regional settings If this box is checked the Decimal symbol and the List separa tor will be used as corresponds to the regional settings of the operating s
177. ct All w Select None A mouse click toggles between the two buttons used to select or deselect all objects z Object Properties Object Properties Object Properties Text Font Colors and Lines l Object Properties Test Font Colors and Lines Tp Agency FE Bold Italic Bald Italic FP Allegra BT Sample lt New text gt Colors and Lines Fill diy Arial Baltic Color F Arial Black Fp Arial CE Ine Text alignment Fp Arial CYR AaBbCel Color C Autosize bound Fr Aral Greek Word wrap Effects Dashed weight LE Qutine C Shadow Strikeout E Smooth Color C Underline Use as default C Center vertically Zoom with image Arnos Begin style End style This button opens the corresponding dialog which is different for Text Lines and Highlight fields The tools are self explaining and similar to those in other common software programs and need not be explained here cell cell software Manual Chapter 7 Image Data Handling a a a c Image nd Overlay Image Only Overlay Only e All Layers Annotation Layer Data Layer w Lock Data Layer Layer This button opens a pull down menu with the following commands Image And Overlay Use this command to display the image and the overlay simultaneously Warning This is a global setting i e this flag determines th
178. d The saturation and the intensity can be set between 0 and 1000 on an arbitrary scale A Brightness of O results in the color black while a Saturation of 0O causes the color to be in a shade of gray It is advisable to select the switch button color according to the filter properties i e according to the effective sample illumination color Edit Fluorescence Color Hue 196 Saturation 1000 Brightness 1000 437 nm Example The selected GFP filter has a center transmission wavelength of 492 nm and thus trans mits excitation light of turquoise color Set the hue to a value of 185 The corresponding wave length will be 492 nm and the switch button will be cyan Before the filter wheel can be rotated via software to another position to exchange insert addi tional filters the filter flap needs to be closed After closing the filter flap the filter wheel is initialized this takes approx 5 sec After this is done the green STATUS diode on the MT20 MT10 front is turned on Then the filter wheel can be rotated into the next position for insertion exchange of another filter as described above 15 1 2 Burner Configuration The Illumination System gt Burner dialog is used for configuration and optical alignment of the burner after installation and or exchange This is described in detail in chapters 7 3 1 and 7 3 2 of the Hardware Manual Part B Display message You can activate a display warning message which reminds y
179. d below Satu ration can be seen as the difference between a set color and white Similarly Intensity is the dif ference to black Selected areas may differ considerably in multi color fluorescence images as compared to the RGB selection e Hue Hue Hue is the color tone in a scale of pure rainbow colors ranging from red over yellow green cyan blue to magenta and back to red Onscreen these colors are composed of binary mixtures with different proportions of the monitor colors red green and blue These conditions are often visualized by a colored ring with red at 12 o clock 0 green at 8 o clock 120 and blue at 4 188 Chapter 9 Measurements OLYMPUS o clock 240 With hue as similarity criterion artifacts may result in fluorescence images because the pixel intensity is not considered at all e Intensity Intensity Here the similarity criterion is the overall pixel intensity added up from all color channels In other words the image is analyzed as if it was a monochromatic black amp white image 9 4 Results 9 4 1 Move Origin ig Move Origin Use this command to change the origin of the coordinate system By default the origin of the co ordinate system can be found in the upper left corner of the image Click the Move Origin button to move the coordinates origin to a desired position on the image All values already measured are adapted to the new origin 1 As soon as the Move Origin button is clicked the mou
180. d on the editor surface and certain parameters have to be set in context sensitive Properties pages either by typing in values or by reading in current settings from the cell4R cell4M software 5 3 1 Types of Graphical Icons and the General Principles of Usage se a pai cal Hed di amp Standard icons to drop Il E AA Ea frames to drag There are two types of buttons in the Standard Commands and Additional Commands toolbars to be used when setting up an Experiment Plan one sort serves to place command icons into the editing surface and the other enables dragging command frames around groups of icons To place a drop icon activate the respective button via mouse click and place it on the editor sur face by clicking at its designated position Frames are used to encircle one or more icons similar to a bracket in a mathematical function To draw a frame activate the button and click in the des ignated top left corner and then drag the frame towards the designated bottom left corner When a new icon is placed or a frame drawn the corresponding Properties Pages opens immedi ately This will be explained in detail in the following chapters The blue squares on the left and right side of inactivated icons and frames are connection nodes their use is explained below Frames and icons can be activated via mouse click Activation is indicated by black dots on the icon frame as shown on the right side of the following image cell s
181. d z dimensions to gener ate 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 118 Chapter 7 Image Data Handling OLYMPUS contain the same number of color channels On the other hand let s assume the two series are monochrome a combined time series of 50 dual color images could likewise be generated 7 8 2 Combining Data Sets The Combine window is opened with the Combine button or via Image gt Combine 24 Combine Combine Mode field Determine here in which dimension the combination is to be performed For example to create a multi color image out of several snapshots select Color 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 field 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 with the active data set have to be selected by mouse click The active data set will become the first part of the new set with the other sources being merged according to their data slot number Combine Combine Mode Feasible image objects Sor
182. ded automatically by the opening and succeeded by the closing of the transmission shutter In other words it is not required to consider the shutter movements when setting up an Experiment Plan Such a system allows combining transmission and fluorescence imaging in one experiment 5 3 3 3 Z Stack acquisition monochromatic or in multiple colors To setup a Z stack acquisition a Z Stack Frame has to be drawn around one or several Image Acquisition icons see the examples below The result will be data files one or several respec tively that contain all the images taken at the different focal planes Monochromatic Z stack The Image Manager indicates a Z stack acquired with a single excitation wavelength with a black amp white stack icon E SE m m C on a a bapi Fo papi Fo Fie ol tTRed Aa 21 x 0 50 um 2i x 0 50 um The order of image acquisitions in the triple color experiment plan is the following First a DAPI image is taken at the lowest stack position then a FITC image at the same position then a Texas cell s cell software Manual Chapter 5 Experiment Manager Red image Afterwards while the TxRed image data are still readout from the camera the PIFOC or motorized Z drive moves to the second lowest position Once the system is ready again a DAPI image is taken first then a FITC and finally a TxRed image This sequence is repeated for each layer After the last image of the last layer the
183. duction will be given To open the Experiment Manager click the Experiment Manager button in the acquisition toolbar Experiment Plans are set up by assembling and connecting diverse command icons and frames The most frequent ones can be selected via buttons in the Standard Command toolbar and placed into the editing area by drag amp drop Standard Commands x Bows Image Acquisition Multi Color Z Stack and Time Loop A Properties page to specify certain settings accompanies each command icon that is placed into the editing area In case of Image Acquisition for example parameters such as exposure time and excitation filter have to be set The scheme below is just one example of an experiment plan It shows the command icons that define a multi color time lapse experiment There are three Image Acquisition icons each with a different excitation filter surrounded by a multi color frame for combination of the three Image Types monochrome images to one multi color image The outer frame represents a Time Loop to repeat all commands contained a certain number of times 30 x 1 505 The Experiment Manager is not only a tool to design the experiments but also the command center to acquire the images according to the Experiment Plan The necessary icons are grouped in the Control Center toolbar Control Center EJ Ys gt 1 E Check System Ready Start Pause and Stop The execution of an Experiment Plan involves t
184. e Execute SQL Recreate Thumbnail Image Change Data base Password and Settings in the Database gt Administration menu become enabled To close an open database select Database gt Close Database or press the standard MS Window Close button 254 Chapter 12 Database OLYMPUS O The Exclusive option is only possible if the database is currently not opened by an other user On the other hand if the database is opened in the Exclusive mode no other user has access to it 12 3 New Database A database assistant makes it easy to create a new database New Database New Database Databaze name select Finish to let the database assistant create a database with the Name example Database files apl Location of TT files D VArchive exarnple Browse Location of images and documents D Archiveserample Organized by E spermen Organizational ID Experiment name 1 Select New Database in the Database gt Administration menu to open the New Da tabase dialog box 2 Write the name of the new database into the Database name field The database will be created by default in the directory specified in the preferences see Chapter 12 1 Direc tories for Data Storage To select a different directory press the Browse button 3 Click Next gt and then Finish if you accept the settings displayed in the dialog cell s cell software Manual Chapter 12 Database 255 12 4 The Database Feature
185. e chapter 10 5 is computed automatically and its value displayed in the dialog box cell a cell software Manual Chapter 10 Intensity Analyses FRET Correction Donor Sample Acceptor Sample Ffret Channel 3 CFP only FFRET yfp em wt Don exc Acc amiss Fdon Channel i3 CFP only FOon cfp em w Don exc Don amiss Please define 2 ROIs ROI 1 should be an area where the Fluoro chrome is visible ROT 2 should be a dark background area Define ROIS Computed Ratios 0 697060 Select or type label 10 12 2 2 Second correction factor acceptor excitation This calibration determines how strongly the donor excitation light excites the acceptor as com pared to the acceptor excitation light A dual excitation image of the acceptor only reference sample has to be acquired using the FRET equipment either the observation filter wheel or the Dual View Micro Ilmager as de scribed above The following filters have to be used donor exciter Ffret channel acceptor exciter Facc channel dual band dichroic mirror acceptor emitter Continue from step 4 Go to the Acceptor Sample tab activate the acceptor sample im age and make sure that the Ffret and Facc channels are selected correctly O If a Dual View Micro Imager is used only the long wavelength channels of the two resulting split images are needed for the calculation This means the Ffret and Facc channels have to be select
186. e e all loaded modules e all modules whose directories you ve selected using the Directories button in the Define search path for modules dialog box e all modules contained by the current configuration e all modules registered in the Add In Manager e all user defined modules containing measurement and statistic parameters registered in the Install Modules with Statistics Parameters or Install Modules with Measurement Parameters dia log boxes Remove Click on the Remove button to close the module selected After clicking on this button the functions of this module will no longer be available to you This module will then be removed from the Loaded list and placed in the Other list if the module is included in a search path or comes under one of the above list of criteria This button is only available if you ve selected a module from the Loaded list Remove All Click on the Remove All button to close all modules Module functions will not yet be available to you All modules will then be removed from the Loaded list and placed in the Other list if the module is included in a search path or comes under one of the above list of criteria Load Click on the Load button to load the module you ve selected The module s functions will be currently available The module will be moved from the Other list into the Loaded list This button is only available if you have selected a module in the Other list Directories Cli
187. e selected from the list in the Compression field This allows you to save more images than you could without compression an important point as far as image archiving is concerned Compres sion can be relevant as far as long distance image data transmission is concerned transmission time is reduced due to smaller file size thus reducing costs To return to the default settings no Preprocessing and no Compression press the Default button TIFF Output Format Select Standard to have all additional information possible in the TIF format included in the file Select Copy of Image Display to convert the image as shown in the image document to a standard 24 bit TIF image All additional information is lost in the process Select Cell4R 1 1 to allow third party applications to import 16 bit TIFF images JPEG Compression Here you can define the options for saving the image as JPEG format The file size of the image file generated is inversely proportional to image quality If you re dealing with realistic images and you enter a quality value of 85 you won t be able to distinguish the com pressed image from the original onscreen At 75 there is minimal deviation at less than 50 you ll be able to see the loss in quality You ll notice this loss in quality at higher values when work ing with synthetic images The image quality set should in the main depend on the properties of the available output device Say you print out a gra
188. e MAPI Sample Function check box the new C module will contain a MAPI function of the same name i e Sample function cell a cell software Manual Chapter 14 Imaging C Various functions can be defined within a C module It is only the MAPI functions however that can be used aS menu commands or inserted into a button bar You can assign the description of the MAPI function which will appear in the status bar when the function is selected to the respective function description string The first thing you enter into a MAPI function s Function description is the MAPI constant This function description is located between the head and foot of a function It is enclosed by the following symbols lt and gt Example Imaging C Code export UINT SampleFunction lt MAPI IDM SAMPLE Info in status bar Sample amp function gt dlgOutput Hello World return IDOK Available The check boxes beneath the MAPI Sample Function check box are only available when the MAPI Sample Function check box has been selected OninitMenu function If you have selected the OnInitMenu function check box the new C module will contain a func tion of this same name The OnlnitMenu function of a C module is used when the program needs to know whether a certain MAPI function is valid or not This is important for MAPI functions that have been integrated into a menu If OnInitMenu gives you MENU_DISABLE concern
189. e View image display thus a final snapshot is taken which remains in the image memory Snapshots are displayed with the false color defined for the currently active Image Type see Chapter 4 4 Microscope Control The Live View image is displayed in gray scale unless the option Image gt Image Dis play gt Fluorescence Color is activated In that case the false color defined for the currently active Image Type is used cell4R cell4 M uses the current camera control and display settings for image acquisition It re members the settings from your last session after program shutdown and restart Please be aware that in case the properties of your object dye intensity background etc have changed signifi cantly it might be necessary to adjust the camera control and display settings accordingly before you can see a reasonable image This is explained in the following chapters The following settings should be checked or corrected before the image acquisition is started e Exposure time Camera Control e Binning factor Camera Control e Brightness adjustment Camera Control e Image size i e full frame or area of interest partial frame Camera Control e Excitation filter Illumination Control e Light intensity Illumination Control O By default snapshots are not being stored and also a new snapshot overwrites the older one These settings can be changed however see Chapter 13 5 1 The Prefer ences Image Tab cel
190. e buttons located in the Include pixel group to define color ranges within the image interac tively 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 cell a cell software Manual Chapter 8 Image Processing Use the options located in the Preview group to see whether all the image structures you re inter ested 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 1 Left click to have a circle appear around the mouse cursor 2 Keeping the left mouse button pressed you can alter the radius of this circle 3 Confirm the size and position of this circle by right clicking 4 You can select several image areas in this way To return to the dialog box simply dou ble click using the right mouse button Click the Include button to add color values to the current color values comprising the active phase This will enlarge the color range the phase covers Click the Exclude button to
191. e display of the image overlay for all images loaded It is recommended to remove this flag only at specific times and for particular purposes Image Only Use this command to show the image without the overlay When this command has been selected only the current image will be displayed on the screen without any overlay This is a global setting i e this flag determines the display of the image overlay for all images loaded Effect on other commands The overlay display mode has an effect on the following functions If the Only Image display mode is active all images will be printed without overlays The image only will be copied into the clipboard i e if you paste the contents of the clipboard into another appli cation the image will be inserted without overlay Use the Copy command in the Edit menu or the lt Crtl c gt keys to copy the image into the clipboard Overlay Only Use this command to show only the overlay without the image When this command is selected the defined overlay will be displayed without any background image You will see the overlay superimposed on a black screen Warning This is a global setting i e this flag determines the display of the image overlay for all images loaded The overlay display mode has an effect on the following functions If the display mode Overlay Only is active cell4R cell4M will only print image overlays OY Burn Overlay This function is currently disabled ab Delete L
192. e example example mku i K i Application analyS1S Last change 21 02 2006 15 33 Hel TOOL version 460 3 05 10 2005 8 44 Imaging C Module example File 0 cell ASM odule example example su Version 1 0 100 Copyright 21 02 2006 Description On close remove module functions from configuration Available The command is only available when a C module is active 316 Chapter 14 Imaging C OLYMPUS Before you use this command activate the module in the module manager On close remove module functions from configuration This check box is only available if the C module contains a configuration file SCX and there is a MKU make file Select this check box to have C module functions automatically removed from the Graphical User Interface GUI configuration when you close the C module Any alterations you made will only become effective once you ve compiled the module 14 10 Module Manager Use this command to manage C modules You may also use the lt Ctrl Shift F5 gt keys When do I use the Add In Manager The Module Manager command is for developers of Imaging C modules If you wish to load finished C modules then use the Add In Manager command in the Special menu What s it for Both dialog box lists show all C modules All these modules can be either loaded or closed Modules can be activated or deactivated The following functions are available Open Module Build Module Edit Module
193. e property page from the Experiment Manager s win dow and position them anywhere on the screen or to re arrange them according to your personal preferences within the Experiment Manager s main window To detach a toolbar point with the mouse tip on the double line at its left side and drag amp drop it to the new position To detach the properties page click on its title bar and than drag amp drop it to a new position Afterwards the size of the new independent toolbar window or properties window has to be adjusted via mouse drag cell s cell software Manual Chapter 5 Experiment Manager To reposition a detached component to the original location double click on the title bar or drag amp drop the window back into position 5 2 2 3 Minimizing the Experiment Manager The Experiment Manager window can be minimized with the standard MSWindows Minimize but ton or by clicking on the Experiment Manager button in the cell4R cell M toolbar or in the MSWindows task bar This is often desirable during image acquisition or experiment execution when the monitor space is required for the display of images in the Viewport The Experiment Man ager window can be reopened by pressing the Experiment Manager button in the cell4R cell4M toolbar or the MSWindows task bar again O Closing the Experiment Manager instead of minimizing it will not cause the loss of the plan it will reappear upon opening the Experiment Manager again 5 2 2 4 Minim
194. e range will not be assigned a phase it will be considered part of the background Select the High option if image structures are dark and the background light 162 Chapter 8 Image Processing OLYMPUS 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 indi vidually The values in Underflow and Overflow are for you to define what percentage of an im age s pixels at the upper and lower end of the image s histogram 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 value 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
195. e text document line Debug x THP a ote 4 eN Debug toolbar Text document and selection If cell4R cell M is not in the debug mode and the active text document does contain a selection the interpreter will not be reset Only commands that have been selected will be executed The Debug button bar is displayed and the mouse cursor will be transformed into the shape of a hand The first executable step will be indicated by the footprints symbol at the beginning of the text document line Debug mode If cell4R cell4M is in the debug mode the program will continue with the next command independently from the active document s type The line containing the command will be indicated by the footprints symbol Syntax error If a syntax error is found in the macro text the first incorrect line will be selected and a warning indicating the type of error will be displayed 13 1 7 Reset Interpreter Use this command to re initialize the macro interpreter Definition Each time a command is executed it is compiled in the interpreter mode cell R cellAM makes use of this mode when a command is executed via the command window when the text of a window is interpreted by the Run Macro command or when a macro is defined using the Define Macro command cell amp cell Software Manual Chapter 13 The Special Menu and the Window Menu 271 What will happen All variables and functions defined in the interpreter mode will be deleted
196. e text frame in the graph document Note that the text cell a cell software Manual Chapter 11 Graph Display and Graph Analysis 243 frame can only be positioned in the visible part of the graph You cannot scroll or zoom the graph while you set the labels Anchor text Check this box to position the text frame at a fixed position of the graph document The position of the text frame in the graph area thus remains fixed upon changing the zoom factor or scaling Set Labels Label text Anchor text Centered text Data position IE Clear the Anchor text check box to position the text frame at a certain XY position of the graph When you shift the graph or change the zoom factor the position of the text frame inside the graph area will change accordingly Centered text 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 rectangle Clear the Centered text check box to left align the text in the text frame Select the check box to center the text Data position Use the features in this group to set a label to a defined X position of the graph The options are disabled if the Anchor text position is deselected Position Set the X position where the label shall be placed at in this field The minimal and maxi mal X values that can be entered are the left and right margins of the current graph display Set and Align Cli
197. easurements OLYMPUS 9 2 5 8 Fitted Polygon 2 Fitted Polygon This command is identical to the Interpolated Polygon command with the difference that the segmented polygon will circumscribe the interpolated line instead of vice versa A right click auto matically draws a line to close the structure 9 2 5 9 Free Hand Polygon Z Free Hand Polygon This command is identical to the Interpolated Polygon command with the difference that the out line of the area has to be drawn freehand with the left mouse button pressed A right click auto matically draws a line to close the structure 9 3 Magic Wand A Magic Wand Use this command to measure objects defined based on similarities in intensities or colors of neighboring pixels Default measurements are area and perimeter Several measurement series can be conducted successively 1 Click on an image point The software determines its intensity and color and compares it with the neighboring pixels Pixels with values that match the selection criterion See next chapter will be included into the selected area 2 Right click to confirm the selected area 3 Select another area or terminate the measurement via right click or lt Esc gt The result will be displayed in the overlay and listed in the measurement display cell s cell software Manual Chapter 9 Measurements 187 9 3 1 Magic Wand Options Measurement Properties in the Measurement View The Magic Wand Options a
198. eatures after the movement and the resulting numbers may become meaningless The Process gt Image Geometry gt Auto Align Z function performs an object detection and detects movements of the objects in X and Y from one image to the next It then corrects the movement by shifting the images so that the objects are positioned in the same XY position again The resulting images are smaller than the original ones because the areas vacated by the shift are cut off cell cell software Manual Chapter 8 Image Processing 8 4 6 Shift Correction Use this function to move individual color bands interactively in multi color image sets in X and Y relative to the other bands A new image is generated the original data remain unchanged The Shift Correction is useful when multi color images are acquired with several single band filter sets instead of multi band filter sets It may be that the dichroic mirrors in different cubes do not have exactly the same angle relative to the optical path As a consequence the resulting images may be slightly shifted relative to each other The function is also especially useful for dual emission images acquired with the beam splitting device DualView Microlmager Shift Correction oO oe Shift Horizontal 2 kl vertical le ial C save values for other uses The use of the Display Channel Adjust Channel Load LUT The Adjust Display tab buttons is described in Chapter 6 2 2 1 The Adjust Disp
199. ecommend you to use the TIF format as default for the reasons already described in Chapter 13 5 3 The File Tab In the Location field you can determine the pathname for the Database files and the Temporary storage directory as well as the Backup volume capacity 13 6 Window 13 6 1 Minimize All Use this command to minimize all open documents and arrange the document icons What will happen Document icons will be aligned along the lower edge of the documents area Using this command can help you keep track of all the documents you re working with This com mand is for text sheet diagram and database documents as well as for the Image Document 13 6 2 Close All Use this command or the short cut lt Ctrl d gt to close all open documents This command is for text and sheet documents It does not affect Image or Graph Documents or any images within the image buffer box Database documents will not be affected either 13 6 3 Document Manager Use this command to open the Document Manager dialog window This is helpful in case your workspace contains many open and or minimized windows As an alternative you can open this dialog window using the short cut lt Alt 3 gt 295 296 Chapter 13 The Special Menu and the Window Menu OLYMPUS E Document Manager A 1018x aaam g Document Name EA Sheet Sheet Graph GBT Kinetics of Graph Kinetics of Fura dey Database LifeS cenceD emos TAR apl dey Database tr
200. ecorder Click on the second button in the Recorder toolbar or select Special Recorder gt Stop Macro Recorder after you have executed all commands or image analysis steps to be included in the macro 13 1 5 Run Macro Click on the right button in the Recorder toolbar or select Special Recorder gt Stop Macro Re corder or press the lt F5 gt to execute the commands in the active text document or the selection in the active text document as a macro What will happen The exact function of the Run Macro command will depend on the current context Text document The active document is a text document containing a macro text cell4R cell4M is not in the debug mode and the active text document does not contain a selection in this case the entire text document will be interpreted as an Imaging C program and executed in the inter preter mode Before doing so the interpreter will be reset This means that all variables and func tions defined in the interpreter mode are internally reset They remain however in the text Text document and selection The active document is a text document containing a macro text cell4R cell4M is not in the debug mode and the active text document does contain a selection In this case the interpreter will not be reset Only the commands selected will be executed Debug mode If cell4R cell4M is in the debug mode Special C Module gt Quick Watch see below the program will continue with t
201. ect a name from the shortlist 340 Chapter 15 cellM cell4R Configuration OLYMPUS OBS System Configuration Illumination System Filterwheel General Excitation Filters Burner Full Control Errors Microscope General 2 Drive U IFSS0 Objectives Filter Cubes Skip Contrast Inserts 3 gr Filters Skip Image Types Shutter Skip Image Splitter Pifoc Skip Stage Observation UFFWO Filters Skip cancel 15 3 Definition of Image Types The definition of image types corresponding to certain excitation wavelengths is important for the set up of experiments with the Experiment Manager and for the display of images onscreen Image Type The Experiment Manager will use the names given in the Image Type fields of the dialog box Also the resulting color bands of multi color images will have the image type name Illumination Excitation Filter An image type has always to be connected with one of the excita tion filters defined in the Illumination System gt Excitation Filters page see Chapter 15 2 1 Con figuring the Excitation Filters f to follow the example in the screenshot a TxRed image icon is set in an experiment plan the filter wheel automatically moves to position 3 which holds the corre sponding filter named 572 TxRed before the image acquisition starts during the execution of the experiment An exception obviously is the image type Transmission which does not rely on the fluorescence il
202. ed Focal Imaging cccccseeeeeseeeeeeeeeeeeeeseeeesseeeenaes 98 6 5 Fluorescence and Transmission Image Overlay cccsssceeseeeeeees 99 6 6 Intensity Modulated Display ccccccseccseceseeeeeeeeeeseeeneeeneeeneeeeeees 100 6 1 The Viewport Image s acquired with the Experiment Manager or loaded from a database are displayed onscreen in Viewport s within the Image window This window features a number of buttons the functions of which will be explained below io iv J is Cancel Arrange Viewports You can set the number of Viewports to be displayed and their arrangement using the Arrange Viewports button in the toolbar of the Image window Just mark the columns and rows by moving the mouse cursor over the schematic grid with 16 Viewports that open The selected Viewport dis play will be shown in the field beneath the grid 2 x 2 Images in the example an Single View Tile View The favored images to be displayed can be selected via drag and drop from the Image Manager You can easily switch between the display of a single Viewport or the tiled display of all selected Viewports by pressing the Single View Tile View button a Zoom In f Zoom Out Clicking on the Zoom In button doubles the size of the displayed image while the Zoom Out but ton reduces the size by half cell cell software Manual Chapter 6 Image Display and Navigation 77 Zoom Factor From the shortlist you can
203. ed TTL Out pulses TTL stands for transistor to transistor logic Out refers to the pulse going from the controller to a peripheral device The command icon changes in dependence of the selected pulses The icons in the four examples above indicate the following Port 1 high Port 1 low Port 1 and Port 2 low Port 1 low Port 2 high Port 3 low conditional option selected Properties Properties x Properties Multi Digital Port Condition Multi Digital Port Condition Pork 1 Enable High level Once AL Every Nth Port 2 Port 3 Multi Digital Port Port 1 2 3 Select one or more of the three BNC ports at the front of the cell4R cell4M imaging computer They are labeled 1 2 and 3 High level This command TTL out ON sets a voltage of gt 2 4 V to the selected port Low level This command TTL out OFF sets the voltage of the port back to the default of O V cell s cell software Manual Chapter 5 Experiment Manager 55 Condition Enable If this option is selected this is indicated by the traffic light symbol in the icon one can set the cycle or cycles during which the trigger command shall be executed Once At The command is executed only in the cycle set in the Cycle field Every Nth Set the interval between triggers in the Cycle field if cycles with and without triggers shall alternate 5 3 2 9 The Wait command g g F Fa d Wait _ 110 ms_ com
204. ed because an active dll file will be detected Make a back up copy of the ObsConfig xml in the cell4M cell4R folder before start ing the software un installation Use it to replace the blank ObsConfig xml created when installing anew You thus avoid having to reconfigure the entire hardware 1 Run the Setup exe that is to be found on the cell4M cell R 3 0 DVD Use the existing cell4M cell4R folder on the hard disk usually on the large D partition as destination for the installation 2 Select Install software in the setup window 3 Accept the License Agreement in the next window by clicking Yes 4 Select cell4M cell4R in the Package Selection window and click Next gt 5 The setup routine will detect an existing cell4M cell4R program You can choose be tween Update and New Installation The Update installs new files and new versions of existing files but keeps unchanged files The New Installation overwrites all existing files cell cell software Manual Chapter 16 Installing the cell M cell R Software 353 S Olympus Cell Setup OLYMPUS Software Documentation Acrobat Reader Network software protection key Explore this CD Quit O If you select New Installation make a back up copy of the software configuration file ObsConfig xml that contains the information about excitation filters image types etc Thus you avoid having to go through parts of the configuration steps described in Chapter 15 2 The
205. ed data set will contain only the selected subset of the source data If the option is selected a copy of the original Source data will be created in which the selected subset range is processed background subtracted while the rest is identical to the source data cell a cell software Manual Chapter 10 Intensity Analyses 207 10 6 Intensity Kinetics in Time and Z ed Kinetics Open the Kinetics dialog window with the Kinetics button or via Measure gt Kinetics This tool is used to quantify the changes of fluorescence intensity within ROIs over time or within a Z stack and display the results graphically Kinetics Intensity Profiles Dimensions Dimension Parameters Select ROIs from Image i2 Fura 3 ROIs Available ROIs kOIl ROD ROIZ ROI ROIS ROIG Sheet Highlight selected ROIs Intensity Profiles tab In case of a multi dimensional data set you have to select in the Direction field if the intensity changes are to be calculated over Time Kinetics or as a Z Profile For simple time series or z stacks no choice is necessary The software recognizes the dimensions automatically In the Parameters field you have to select from which data set the ROls shall be taken and for which ones the analysis shall be performed If the option Highlight selected ROls is activated those ROIs are shown with bold lines If Sheet is activated in addition to the graph also the corre sponding spreadsheet is gener
206. ed from different source images 6 f 8 Define two ROIs as described above for the first correction factor The calculated ratios are displayed as Computed Ratios Enter a name in the Select or type label box and Save the ratios 227 228 Chapter 10 Intensity Analyses OLYMPUS FRET Correction eh Donor Sample Acceptor Sample Fret Channel iz yfp only cfp exc FFRET yfp ern Don exc Acc amiss Facc Channel il yfp only yvFp exc Face yfp em ACC EXC ACC amiss Please define 2 ROIs ROI 1 should be an area where the Fluoro chrome is visible ROI 2 should be a dark background area Define ROIs Computed Ratios 0 697060 0 031351 Select or type label Seve Gas 9 Exit with Close 10 12 3 FRET Analysis E FRET Analysis The cell4M cell4R FRET module offers four different methods for the calculation of FRET images The simplest is the Ratio Method that does not correct for the channel bleed through and relies exclusively on images acquired with the donor excitation filter It is the only single wavelength exci tation method only images taken with the donor exciter are needed The three other methods re quire images taken with two excitation filters and differ in the way of correction and normalization They require single labeled donor and acceptor samples for calibration A click on the FRET Analysis button opens the dialog box 10 12 3 1 Ratio Method
207. ed left button the size can be adjusted by dragging A right mouse click opens the Object Properties dialogs for Text where the text can be typed in and the fonts colors etc chosen oO Rectangle Enables rectangular boxes to be drawn Position the top left corner of the rectangle as desired then depress the left mouse button to create a rectangle of the desired size Right click to super impose the box cell cell software Manual Chapter 7 Image Data Handling E Ellipse Enables ellipses to be drawn Position the top left corner of the rectangle that describes the ellipse as desired then depress the left mouse button to create an ellipse of the desired size Right click to superimpose the box E Line Enables straight lines to be drawn Position the mouse cursor at one end of the desired line left or right click and move the cursor to the opposite end then click again Arrow Enables variable arrows to be drawn Position the mouse cursor at the desired start position of the arrow Pressing the left or right mouse button and moving the mouse the arrow length and its di rection can be varied Click the mouse button again to superimpose the arrow Polyline Click on this button to draw a polyline into the overlay Keep the left mouse button pressed and move the mouse to draw freehand Define the polygon point by point by clicking the left mouse button on each point Straight lines then connect points automatically Righ
208. ed microscope IX81 or BX61 select the serial port of the imag ing PC that is connected with the universal microscope control box UCB from the shortlist 334 Chapter 15 cell M cell R Configuration OLYMPUS I OBS System Configuration Illumination System TYPE General Excitation Filters Burner Full Contral Errors Microscope y General Shutter 2 Drive Objectives Transmitted Reflected F Filter Cubes Contrast Inserts Lightpath Ix 81 Connectivity com Filters Image Types Bottom Port Zt Frism BI Shutter Image Splitter Pifoc Stage Transmission Lamp Lamp Shutter If the transmission light path of an IX51 71 81 microscope is equipped with a shutter se lect Transmitted This is not available for the upright microscopes BX51 61 cell4R and cell4M do not feature a reflected light shutter at the microscope frame The corresponding shutter is integral part of the illumination system MT20 MT10 Consequently the option Reflected should remain deselected The selected shutters can be operated with the respective buttons Lightpath In the IX81 microscope it is possible to switch the image light path between a standard camera side port and a camera bottom port To activate this option select Bottom port here Like wise in the IX81 it is possible to switch electronically between the camera and the ocular To acti vate this option select Prism here The selected modules can be operated with the re
209. ediar Standard Deviation Skewness Sum Yaiance The total number of all values C Mean is known a priori N variance Description Color rows of result sheet E Line Chapter 9 Measurements Length 19 64 pm E Line Length 14 33 pm a h Line Length 18 19 pm El fo Line Length 28 93 pm Elf Line Length 33 65 pm a h Line Name Count Mean Minimum Maximum Standard Deviation Value 124 2404 um 10 47 pm Ay 2 um 12 23 um Available All available statistics parameters that are not yet selected are listed here Current All statistics parameters to be shown in the Statistics field of the Image Manager are listed here Add gt gt Select a parameter from the Available list and click this button to add the parameter to the Current list lt lt Remove Select a parameter from the Current list and click this button to remove the parame ter from the list Description This gives and explanation of the parameter currently selected 193 194 Chapter 9 Measurements OLYMPUS 9 4 8 The Preferences gt Measure Tab Measurement Properties Use this function to open the Preferences gt Measure Tab For a detailed description refer to Chapter 13 5 4 The Preferences Measure Tab The Magic Wand Options are described in Chapter 9 3 1 Magic Wand Options cell cell software Manual Chapter 10 Intensity Analyses 10 Intensity Analyses The
210. eeecue 139 POD GING asictaserincdaui ees a e chest eden E E E dent veadgaany 139 ROIM sca ctivbccuieteb teteet out a a 140 SS Ira Me ote te bata cete recente E csi tne tuleec dante ens 141 NXN Secsesstt ieee ae eee 142 OW DAG Sea cancennres scat taceeeecenccaen E 143 EOO6 EMMA CC ismin EAT ena NEN 143 PAE et alars lie eae ersten erste en acest cena ra E a ee nae akan 144 SIOM Aeae E anncdadiasadearnenadhie vedsdeasteraitessonsaeacees 145 DCE Differential Contrast Enhancement cccssecesseeeeeeees 146 SEPO o asics ahi r E e E E O ee 148 Arithmetic Operations ccccccseccsseeceseeeceeeecseeeeceesessueeesaeeesseeeess 151 IMAGE GSOMOUY wesseit ste esut snubs EE NE duns tenants seuuaules 152 Contents OLYMPUS 8 4 1 FROSIZ Ce weh cates cen i died add Oi ee eich td tatu ibe al iaaa 152 0AA in Cc gt Pe ae Oe OR oO Oe ee ee Oe ee ee eee 154 oe DG Pama UL zene a er E a rN en ae oer ee 155 GAA ANO teak cet ote atte etl ene Mt ev at nee oa ene ore cre ee oa ON 156 84o FUORAIIGIN Zoeren geer aE EE EEN EEE 156 GAO alii GCOMCCUIO I riar ene E e E E 157 8 5 Thresholds and Binarization cccccceceeeceeeeeeeeeecececeveueueusuaeneeeeanes 158 Dor SCE O OO A TD 158 8 5 2 Set Color ThresholdS asssnsnsnsenenennnnnnnnennnnnnnnnnnnnnnnrnrnrnrnrnrnnnnne 163 Goo BAO aa O E E enc chen endaeeneees 167 8 6 Deblurring and Deconvolution ss sssssssseeseesserrersennrerrernrnrrernennne n 169 8 60 Generala
211. eesseeeess 20 PAN WIRE C OFC h asiasi a eee eho 21 camera Control sirsie e EEA E LEE suecacentiode 21 H mnaton Controls tiene tenntent te Ghent teeter ten ite en tert erie 25 Microscope Control cccccsececseeeceeecseeeeceueeceueeecaueecsueessaueesseeesaes 26 Motorized Stage Control cccccsccccssseeeseeeeeseeseeeeeeseeeseaeeeessaneeeees 29 Defining a Positions LISt ce eccssececeseeeeseeeceseeeeaeeeseeeenaeeessaess 29 Correcting a Positions LiSt ccccccccecsseeeeeeeeeeeseeeeeeeeaseeesneeeeenaes 30 Calibrating the Motorized Stage ccccscccecseeeeeseeeeeeeeeeeeneeeeeenees 32 PUR OTO SUS wacciiuelacelsahnc ciel abn a a a N tied 34 AUTOTOCUS ODON Se saias 35 Executing an AUTOIOCUS SCAN sesicsasetnaceadssiietacadsnesetrctiveereiemesteetens 36 Chapter 4 Image Acquisition and Hardware Control 19 20 Chapter 4 Image Acquisition and Hardware Control OLYMPUS 4 1 Simple Image Acquisition 4 1 1 Snapshot and Live View i Snapshot TR Live View There are two ways to achieve simple image acquisition The first one is the acquisition of a single image with the Snapshot button in the Acquisition toolbar or the Camera Control or Acquisi tion gt Snapshot The second one is the live view started with the neighboring Live View button or Acquisition gt Live Here the displayed image is updated continuously and the previous image discarded Pressing the Snapshot button stops the Liv
212. eesseeess 191 Detinne SlAlISUCS t2022ccecocencicsansndenzeseccecanse dintesssceacnes cateecescencosg size 193 The Preferences Measure Tab ccccccccacececeececcaeacsceaeaeeceaeaes 194 175 176 Chapter 9 Measurements OLYMPUS 9 1 Measurements Toolbar aF Me Measurements List Read i Measurements Click on the Measurements button at the bottom of the Image manager to open the Measure ments View Select Measurement gt Measurements Bar or press lt Alt 4 gt to open the measurement button bar The measurement functions of the button bar and the different analyses in the menu are ex plained in the following chapters 3 Points Angle 4 Points Angle Rectangle Rotated Rectangle 3 Points Circle Circle with Center and Radius Ellipse Measurements X eR EZEHSADAAAA OO OOO RGRIN KS E 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 All the measurement functions can be found on the Measurements button bar There 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 a right click or the lt Esc gt key Several measurements can be conducted successively The results will
213. efinition The reason you load a module is so that its functions are available to you in cell4R cellAM Which modules do I have loaded The modules you ve already loaded are listed in the Loaded list in the Module Manager What will happen The module will be included in the Loaded list in the Module Manager All global and static variables of the C module will be initiated as well as the C module s main function If this function results in a value of 1 the loading of the C module will be stopped The C module will be closed Definition To be able to apply the following menu commands to the module activate the module the commands are Edit Module Save Module Configuration Add to Module Build Module Close Module and Module Info Which module is the active one Although several C modules can be loaded at the same time only one can be active To check which one is currently active have a look in the Active field of the module manager or use the Module Info command What will happen The Open Module command will deactivate the C module that had been active and will close it This means that the previously active module is no longer loaded The module will be withdrawn from the Loaded list in the Module Manager and placed in the Other list If you use the Open button of the Module Manager however the currently active module will not be closed it will simply be deactivated Activating a module without having to load A mod
214. eld Digital images displayed on computer screens are based on integer numbers representing intensities Division of two of such integers generates so called floating point num bers which cannot be displayed e g 438 316 316 0 3869759 These numbers have to be mul tiplied by a Scale Parameter with the positions after the decimal point being clipped With a factor of 1000 which is the default the above number would give 387 However in case of a decreasing signal a negative number which again cannot be displayed would result Thus an offset is added to each resulting value usually 1000 A pixel value of 1000 thus stands for 100 In the example 1387 results indicating a signal increase of 38 7 Dimensions tab See the description in Chapter 10 5 Background Subtraction 10 8 Ratio Analysis 10 8 1 General This analysis tool is used for ion imaging with dual excitation or dual emission fluorochromes the fluorescence intensity of which is dependent on the ion concentration Calcium imaging with the dual excitation dye Fura 2 as in the example in this chapter is probably the most common such technique The principle is that the sample is imaged simultaneously at two different illumination wavelengths typically at 340 nm and 380 nm Simultaneously means here that the two images of a pair are acquired immediately one after the other Afterwards a background correction is per formed and then the intensity values of the 340 nm image
215. ell Software Manual Contents TASS Open Modules isere Sse EAEEREN 309 144 AGG TO MOGUI Cie icvcec ote cced adedavehce ds ennaa 311 14 5 Save Module Configuration ccccccsececsseeeeeseeeeeeseeeeeeeaeeeeeneeeeeens 311 WAG ECMO GUIG a icc a a a aeeoteeatl aceaeenedneenae 313 VAST JB UNG Module oes EE ER 314 14 8 Close Module ssrin a a a LEi EI ESER 315 149 GADOUU IWMICOCIUIG nnani aea 315 14210 MOGUIC Manage i aioin Er sexo db acceestetaceastccmitacdaraecsies 316 14 10 1 The Define Search Path for Modules Dialog BoX cccceee 319 TAA BOWS ET arn e EEEE enti Ghie ea E EE A 320 1412 Find Sym DON siaa a AE S E a E EES Ea EENAA 321 14 13 Goto DETIAINION sswictivesncctatnsattiucsnteekutadesiennsteesintaces tun eeeetaiaub say lous 322 14TA QUICK VW ALC Mier e 323 TA VS Watten NV ANADISS aa sueds nansaiserscel poetic ees E 324 1416 TOOGIE Break Polner e T ane ian 325 14 1 SG PEAK OOINTS rse EE CE EEEE ELEDE 327 Chapter 15 15 cell M cell4R Configuration s sssssnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn 329 Telling the software 15 1 The Illumination System MT20 MT10 ccceeccsseeeeeeeteeeeeeeeaees 330 which hardware modules 15 1 1 Configuring the Excitation Filters ssssssssseenssssesnnnnnrenessrnnnnnees 330 are available and what tost 2 Burner ConnguratioN esed oen nnn E N 332 the current installation is 45 4 3 Using MT20 MT10 without the Imaging Software c0s00 333 for example what filters
216. en saving an image cell4R cell4M will automatically suggest the image name for use as the name of the file Warning Image name and file name are not the same within cell4R cell4M For example say you name an image yeast 02 25 2003 image 23 its image name you can save this same image un der a file name such as 02250323 tif Then when you later re load this image the original image name will appear within the image buffer Image buffer The Image buffer field displays the number of the image buffer currently containing the image This number will of course change when you for example put the image into another image buffer cell s cell software Manual Chapter 7 Image Data Handling 123 Memory size displays the size of the computer memory used by the image File size gives the information about the file size if the image is stored as a file Frame The Frame field displays the number of the current image and the total number of images of a data set Created The Created field displays the time and date at which cell4R cell M images were cre ated the date and time an image file was last altered will be displayed for other non cell4R cell4M images Channel The Channel field displays the camera used to acquire a cell4R cell4M image This field will be blank for images not acquired via cell4R cell4M Magnification The Magnification field displays the magnification used when the image was ac quired via cell
217. ent fluorochromes from the respective pick lists Before performing the linear unmixing it is additionally necessary to define a ROI for background correction To do so press the ROI Background button to open the Define ROI dialog Draw a ROI as usual to determine the background intensity Afterwards close the Define ROIs window Figure 5 Images of a GFP YFP double labeled sample GFP fused with H2B histone protein and YFP with tubulin taken with GFP exciter YFP exciter YFP dichroic and emitter Left original right after spectral unmixing 219 220 Chapter 10 Intensity Analyses OLYMPUS 7 Press the OK button to start the unmixing algorithms The processed image is automati cally loaded into the current destination image buffer Acknowledgement The images were acquired and unmixed with a cell4R Imaging Station and kindly provided by Dr Paulo Magalhaes and Prof Dr Tullio Pozzan University of Padua Italy 10 9 8 Unmixing of Color Camera Images It is possible to unmix color images taken with a color camera in a very similar way System re quirements are dual or triple band filter sets including dual or triple band excitation filters Calibration RGB images of the mono labeled reference specimen have to be acquired always using the same filter set The calibration is identical to the procedure described in steps 2 5 in Chapter 10 9 6 Calibration O The unmixing of a color image which is a three channel image
218. er 15 cell M cell R Configuration OLYMPUS O By carefully setting the stage limits one can avoid driving the objective accidentally into the specimen In case of an inverted microscope for example the topmost position of the Z drive should not lift an oil immersion objective with short working distance more than a fraction of a millimeter above the level of the stage Otherwise damage to the objective and the specimen could result If the upper limit is too low however focus cannot be reached In case of the IX81 the nosepiece is the moving part in case of the BX61 it is the stage For both the lower limit is uncritical while the upper limit has to be set carefully For the BX61WI with fixed stage the moving part is the nosepiece and the situation is vice versa the lower limit is the critical parameter Lower Limit For both the inverted microscope IX81 and the upright BX61 the lowest possible position 1 can be set without causing any problems see the note above In case of the fixed stage upright microscope BX61 the value has to be set so that focusing is possible but the objective cannot be driven into the specimen Upper Limit For both the IX81 and BX61 this value has to be set carefully see above so that fo cusing is possible but possible damage to the objective is avoided For the BX61WI the highest possible position can be set without causing any problems ZDC Available Click here f a Z Drive Compensation ZDC device is p
219. er 7 1 Calibrate Image otherwise an error message will be generated Channel Settings Click the Deblurring button or select Process gt 3D Images gt Deblurring If deblurring was not applied before on the active image the Channel Settings dialog will open Channel Settings for Deconvolution Calibration Scale 4 Color Channel Scale r Scale Z A bea A r A r Microscope Parameters Emissive Wave Length A nm Mumercal Aperture hoo E Refractive Index ir 1 0 Ww Color Channel Select the channels one after the other in order to set their Microscope Parame ters Calibration This field lists the scaling in the three spatial dimensions as set in Chapter 7 1 Cali brate Image If necessary the settings can be changed in the Scale X X Z boxes cell cell software Manual Chapter 8 Image Processing Emission Wavelength This is the maximum intensity wavelength of the light emitted from the specimen but also dependent on the transmission properties of the emission filter in the fluores cence cube of the microscope If running a brightfield setup enter 540 nm Numerical Aperture This parameter is printed on the objective It determines the light gathering capacity and resolution of the objective The theoretical upper limit of is the Refractive Index Refractive Index This is an optical characteristic of the immersion medium and can be selected from the pick list for each Color Ch
220. er into the currently opened database folder Select the images to be added from the Insert Images window that opens If the option Always is selected in the Prompt for data input field the insert dialog will appear every time before an image is inserted Insert Images OF 2 Dap Fite TsAed unmimed 3 Tv3 Cancel Select All Unselect All Help Prompt for data input Always Once Database gt Insert gt Image File This command imports stored image files for example from the hard disk into the database Database gt Insert gt Data This command opens a dialog box that enables the user to add a comment to the database folder This comment is not an independent document but is an entry in the Field table Database gt Insert Documents This command inserts Graphs Sheets or Text documents Macros into a database folder Select the documents to be added from the Insert Document window Insert Documents Sheet Sheet3 Fura f OK Sheet Sheet2 Graph GB 2 Kinetics of Fura Fura Sheet Sheet Graph GBT Kinetics of Fura Fura Cancel Graphi Kinetics of Fura Graph 2 Kinetics of Fura Select All Unselect All Help Prompt for file names 262 Chapter 12 Database OLYMPUS Database gt Insert Document File The command allows to import stored document files e g from MSWord MSExcel etc into a database folder 12 5 3 Query al Query by Example Database
221. er is a term for a folder in the tree structure of the cell R cell M database This folder is no file folder on the level of the system software and thus is only visible in the data base window but not in a file manager program like MS Windows Explorer Database folders are created automatically when an Experiment Plan is executed and carry the name of the experiment They can also be created via Database gt Insert gt Experiment Database fields The database fields are entries in the table of parameters and information listed for the active document in the Form view of the database window see next chapter 256 Chapter 12 Database OLYMPUS Organizational fields An organizational field is a database field associated to a database folder and thus to all data re cords within that database folder as well The organizational field has the same value for all records created in a respective database folder The cell4R cell4M template database has the predefined organizational fields Experiment Name Experiment Comment Date and Time They are automatically stored with each data record Organizational ID This is the name of the database folder i e the Experiment Name The other Organizational fields cannot be chosen as Organizational ID 12 4 2 The Database Window After opening or creation of a new database the database window will be displayed The title bar shows the name of the database apl Important database commands a
222. erences b File Tab ccccccececseceeeeeeeeeeeeeeeeeueeeeeeueenenens 284 13 5 4 The Preferences gt Measure Tab cccccececeecececeeeeeeceueeseeeneeneeens 289 13 5 5 The Preferences Module Tab c cece cceccccecececcececeueaesceneaeeees 291 265 266 Chapter 13 The Special Menu and the Window Menu OLYMPUS 13 5 6 The Preferences gt Graph Tab ccccsscccsseeecseeceeseecaeeeeseeeesaees 293 13 5 7 The Preferences gt Database Tab ccccccesecseeeceeeeeeeeeeeeeseees 294 jE Fe Mame 6 0 Gemeente eee reer ee lee neers Ree Rete ea eter mee Seren nae on ee te EEES 295 TGT MEA Seen eee nee le ee lee nee S 295 T62 GOSS Al Pannen Seen eet eee eine ere re ee ee eee 295 13 6 3 Document Manager ccccsecsseecesececseeceseteseeeeseaeeeseneeeeaeeseaens 295 13 6 4 Viewport Manager ccccceceeeeeeeeeeeeeeeseeeeesaeeeseeeeesaeeeseneessaneenaees 297 13 6 5 ANIM BUNA Ol ces etic tsetse ace eee eect weeaneeadseccanesececeacecase 297 13 6 6 Status Bar cccccccccccseececseeeccenceceeeecceneeceseeeceneecoasesceseeseasescess 298 13 6 7 Command WINCOW ccccssccceeeeceeeeeceeeeceueeecaueecseeesaneeseeeesaaees 298 13 1 Macros 13 1 1 General Macros and C modules commands under Special Recorder and Special C Module respec tively are similar In general macros and C modules fulfill the same tasks C modules are often complex and make use of all elements of a
223. es and entire data sets at four predefined axes of choice the central Vertical the central Horizontal or any of the two Diagonal axes Check the radio button of the desired axes and then click OK 8 4 4 Align The Process gt Image Geometry gt Align function performs a rotational alignment of the Source 1 image relative to the Source 2 image Make sure that the Source images are set correctly in the Operand box of the Imager Manager After starting the function set the first reference point by mouse click in the active image Source 1 Upon doing so source 2 becomes active automatically Set the corresponding reference point there Source 1 becomes active again for you to set the second reference point Again Source 2 becomes active again for you to set the corresponding second reference point You may continue setting reference points or finish the process by a right click Upon finishing a rotated copy of Source 1 will be generated with its reference points lined up as good as possible with those of Source 2 8 4 5 Auto Align Z Especially in long term time lapse experiments it may happen that the sample is moved in the XY plane This may be caused by an accidental push of the microscope or the table or upon intention ally manipulating the sample may be after injection of an agonist Such a movement makes certain analyses difficult for example intensity kinetics An ROI defined in the first image will no longer contain the same structural f
224. ese two view options In the Structure Strip mode only the Tree view and the Preview are displayed Select Gallery Strip to reduce the database window to the Gallery view in form of a single column of thumbnails ca Update Views Select the command Database gt View gt Update Views use the key lt F5 gt or press the Update Views button in the button bar of the database window to refresh the display of the database win dow info 9 Pin Info Window Select the command Database gt View gt Info Window or click on the Info symbol on the bottom right corner of a thumbnail in the Gallery to open the Info Window Record Hame Dap Fite TA ed Sample Preparation Document Creation _ 31 05 2005 Document Creation _ 14 57 Image Type Multi Dimension Binning 1 File Size Baes4 i 4 The information displayed can be defined in the dialog Arrange fields gt Info Window Usually the window closes if a different icon or document is activated To prevent this press the Pin button in the Info Window the button turns into a counter sunk pin Now the window remains open showing 260 Chapter 12 Database OLYMPUS the information about the active item The Info Window can be closed as usual with the Close but ton 12 5 Working with the Database 12 5 1 Loading Documents Any document contained in a database can simply be loaded by a double click on the thumbnail in the Gallery However this does not work for the ic
225. esponding labeling of measurement objects or series of measurements of objects being measured Corresponding sheet entries Background This sets the color of choice of the background underneath the labels of the overlay objects Without such background they would easily be overlooked in non gray scale images depending on the color O This field is inactive when the option Show labels None is selected Magic Wand options See Chapter 9 3 1 Magic Wand Options for details cell s cell software Manual Chapter 13 The Special Menu and the Window Menu 291 13 5 5 The Preferences gt Module Tab The settings in this tab apply to users who wish to create their own macros or Imaging C modules for use in cell4R cell4M Preferences Image View File Measure Module Graph Database Report Modules Masinum number of modules 2 E Reload on startup Language version Debug mode Command window Macro recorder Activate on output Named arguments C Display string pointer values C Image buffer arguments C Issue eror message for invalid pointers Modules Auto save on build When selected the text document is automatically saved using the Run macro command when you start up your macro When compiling a C module using the Build Module command or using the identical function Build in the Module Manager dialog box all files used in the C module will automatically be saved For this to hap
226. esulting image according to the above mentioned formula Positive whole numbers greater than zero are permis sible The sum of all weight factors is used for image normalization when user defined filters are in use SS 299 Square Hexagonal lattice lattice ia Image row 2n E image row 2n 1 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 will be replaced by a hexagonal matrix The diagram above shows determination of neighboring pixels using either a square or hex agonal filter matrix 8 2 15 NxN This filter is based on the definitions set in the Processing gt Define Filter gt NxN dialog box Application Reduction of statistical noise What will happen 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 average When determining filter size N you should keep generally the following in mind the smaller the matrix the finer the details you can edit These details can include artifacts 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 is this smoothing effect will be You may end up blurring edges Comparis
227. esults folder If a new query is carried out the contents will be actualized Database gt Query by Free Filter This command allows to perform queries with several criteria combined with AND or OR links Database gt Query by SQL This command enables the usage of the SQL database language to define search criteria 12 5 4 Administration Defining Organizational and Database Fields Create a new database or open a database in the Exclusive mode and select the command Data base gt Administration gt Define fields to open the Define fields window It contains only a few active control elements when you have set up a new database and you use this command for the first time Field list All predefined database fields in a new database appear in the Field list You cannot edit rename or delete predefined database fields or the entries in them The one exception is the data base field Record Name an entry in this field can be changed when you insert an image or when you edit records New button Click here to define your own database fields The Add New Field window will open where you have to set the Field properties Field properties Edit Click here to edit the field properties The Edit Field Properties window will open it has the same features as the Add New Field window Input required Mark this check box to make an entry mandatory when you insert data in the data base This database field will be identified in the
228. etic 100 x 1 00 5 The image taken with the donor exciter and the acceptor emitter is the FRET channel However its signal is contaminated by two factors One is the donor emission that passes the acceptor emission filter This contribution is calculated by multiplying the image taken with the donor exciter and donor emitter with the first correction factor The other is the acceptor fluorescence caused by direct excitation with the donor excita tion light This contribution is calculated by multiplying the image taken with the acceptor exciter and acceptor emitter with the second correction factor 232 Chapter 10 Intensity Analyses OLYMPUS The algorithm performs the following calculation MicroFRET Ffret a Fdon b Facc Create a purified FRET image as follows E 1 Click on the FRET Analysis button to open the dialog window 2 Select the Method NFRET 3 Make sure that the Fdon Facc and Ffret channels are selected correctly 4 The Background Subtraction Thresholds and Output Scaling functions are described in Chapter 10 8 2 Generating a Ratio Sequence 5 Confirmation with OK creates a corrected FRET image 10 12 3 4 MicroFRET Experiments with a Dual View Micro Imager In case a Dual View Micro Imager is used to monitor the two emission wavelengths a dual ac quisition Experiment Plan with two split images one with each excitation filter has to be setup For this it is necessary that tw
229. etneeaes 163 8 5 9 BINAE s a A Po nC Oe a en a a 167 8 6 Deblurring and Deconvolution ss sssssssssssssnesrrrrnnnrrrrrnnnrerrerneernne 169 86 Generais a aE EE EDI A 169 O02 WO NGSIONDO Ana EA 169 8 6 3 Nearest NeiQhbors cccccsccsscessesscesecsecseeeecsesseeessesseeessntenees 170 6 64 DEDIUGNG IMIAOSS rras sexes seteontecatecacnecaneastacecess A 170 8 6 5 Inverse Filter andes seca asa eh ciecn te ese ehevanetee ene eeanen cama nesncaaaes 172 8 6 6 3D Blind DECONVOIUTION cccceccecececececeeeeeeceeeeeceeeveveueusueeeeeeeenes 173 8 1 Shading Correction 8 1 1 General Gradual noise and sensitivity variations across the CCD chip and the corresponding inherent inten sity gradients in the images are termed shading The cause is a gradual mismatch of photodiodes and on chip microlenses Shading correction is a type of background correction in an image meant to compensate for irregular illumination effects 8 1 2 Define Shading Correction The Define Shading Correction dialog box is opened via Process gt Define Shading Correction and used to set the parameters for a shading correction cell cell software Manual Chapter 8 Image Processing 127 Define Shading Correction Assumed detenoration Preparation of shading image Additive G MaN average filter O Interactive zero level Source for shading image Polynomial fit Source 1 Save shading image Source 2 Neh average filter
230. ext chapter O Select an empty buffer in the Image Manager if you want to acquire a new image oth erwise the current image will be overwritten and lost snapshots are only stored tempo rarily and need to be saved 3 3 Saving Images The Database To save a snapshot in the most basic way select File Save from the menu bar or use the short cut lt Ctrl s gt The snapshot will be stored in a 16 bit tiff format by default Other data types can be chosen as usual in the Save Image As window As with other files you have to give a name and select the destination path of the storage cell4R cell M features a database module for the storage of images and entire experiments in cluding Experiment Plans data sets analyses and so on The module is explained in detail in Chapter 12 Database 13 14 Chapter 3 Brief Introduction and First Steps OLYMPUS The command Open Database to load an existing one can be found redundantly in the menus File Acquisition and Database Database files carry the extension apl New databases can be created with the command New Database to be found in the same menus In order to be able to store your images in the database first you have to create an experiment folder via Database gt Insert gt Experiment or use an existing one To store an image in the database just drag it from the Image Buffer Box into the experiment folder You will be asked in a dialog box to give it a name
231. f 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 dia gram plots the number of pixels versus color saturation or intensity The saturation curve the dis tribution of color saturation in the image The whole image area is used to calculate these curves no matter whether a global frame has been set or not 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 represent each of the color channels Color bars display phases that have already been defined Each bar displays its corresponding color range in the cell a cell software Manual Chapter 8 Image Processing same color as in the Preview Activate any of these color channels for a particular phase by simply left clicking 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 within the histogram The blue line represents the lower threshold the red line the upper threshold Threshold
232. ference images of the reference samples under the same imaging conditions as used later for the experiments with your multi labeled samples It is possible to use multi labeled specimen for reference purposes in case mono labeled structures are clearly resolved in the images For example the following filters have to be used to unmix CFP and YFP CFP exciter for one color channel YFP exciter for the other color channel dual band dichroic mirror dual band emitter 2 Load the acquired reference images into the image buffer and click on the Calibration button This opens the Measure Ratio Unmixing dialog window Measure Ratio Unmixing Please define two ROIs ROT 1 should be an area where the fluorochrome is visible ROI 2 should be a dark background area Computed ratiofs 0 52 Select or type label 3 Press Define ROIs to open the respective dialog box in order to define two ROls The first one has to be within an area where the fluorochrome is visible while the second ROI is used for background correction The ROls are set in the same way as in the Im age gt Define ROIs menu Chapter 10 4 Regions of Interest ROIs 218 Chapter 10 Intensity Analyses OLYMPUS 4 Afterwards close the Define ROIs window The ratio of the average intensities within in the first ROI after background subtraction see chapter 10 5 is computed automatically and its value displayed in the dialog box 5 Label
233. from the pixel intensities to generate a new image where the intensities result exclusively from energy transfer processes Images or image channels have to be taken with three different filter combinations all using the dual band dichroic mirror Donor exciter amp donor emitter Fdon channel Donor exciter amp acceptor emitter Ffret channel Acceptor exciter amp acceptor emitter Facc channel 10 12 3 3 MicroFRET Experiments with an Observation Filter Wheel In case an observation filter wheel is used to monitor the two emission wavelengths a standard triple color image acquisition Experiment Plan has to be setup see Chapter 5 3 3 4 Time lapse experiments monochromatic or in multiple colors For this it is necessary that three Image Types with the three different filter combinations be defined in the ObsConfig software An example is shown below see also Chapter 15 3 Definition of Image Types and Chapter 15 2 6 Configuration of the Filters of a Filter Wheel Image Type Illumination Shutter Filter Cube Observation Fluorescence Excitation Filter Emission Filter Color 430 CFP CRPIYFR CFP 470 an vie lt CFP YFP 430 CFF y CFP YFR se YFP 535 a eel 500 EP CFP YFP w YFP 535 vif E As usually it is possible to calculate online kinetics of one or several channels see Chapter 5 3 3 6 Experiments with online analyses An Experiment Plan would look like this for example Fdon ee CFP gt YF 40 Kin
234. g the same thresholds Measurements taken will be appended to the measurement sheet This inser tion 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 10 11 Colocalization Colocalization xX me we Colocalization toolbar Colocalization refers to two or more different fluorescent labels being in close proximity in a multi labeled specimen often due to binding to the same sub cellular structures resulting in image areas pixels that display intensities above background in two or more color channels This should not be confused with artifacts due to channel bleed through as explained in Chapter 10 9 Spectral Unmixing cell cell software Manual Chapter 10 Intensity Analyses Colocalization is of great interest in certain applications because it may for example indicate that the different molecules bind to the same target Thus qualitative and quantitative analysis is of importance The analysis is available for dual color images as well as for multi color images if only the two channels of interest are being displayed see Chapter 6 3 2 Multi Color Images The procedure consists of four steps to be executed with the four buttons of the toolbar from left to right 1 Thresholding of the first color channel 2 Thresholding of the second color channel 3 Gene
235. g a C module When you close a C module all functions of the C module contained by the current configuration will be removed This will happen if you have not cleared the On close remove module functions from configuration check box in the Module Info dialog box cell s cell software Manual Chapter 14 Imaging C 313 14 6 Edit Module Use Special C Module gt Edit Module to define the make file of the active C module Available This command is only available when e aC module is active and if e there is a MKU make file for this module Edit Module example mku E example sfm Files of type Macro sim ka Header files Ladd Remove Source files O cellA4Moduleexample example stm D AcellAModuleLexample example scx Before you use this command activate the module in question in the module manager Then you can check and see whether the module has a MKU make file using the Open button in the Open Module dialog box What s it for This dialog box is for adding or subtracting files to the list of C module files You can also load files from the module list into the text editor Files of type Add Header files Source files Up and Down To add a file to the C module first select the file type located in the Files of Type list to appear in the Look in field Select the file to be added to the module from the file list and then click on the Add button or simply double click 314 Chapter 14
236. g box it may have to be moved per mouse to be able to view results of setting changes in the image docu ment Interactive button Click on the Interactive button to be able to determine grid frame size and position interactively within the image document A red rectangle will appear in the overlay display cell cell software Manual Chapter 7 Image Data Handling ing 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 pressed move the mouse to enlarge or diminish the rectangle to set the size of the grid frames If the Use starting point check box has been se lected 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 but rather where the red rectangle has been positioned Right click 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 Grid size group 7 5 Overlays 7 5 1 General The Overlay is used to add information in form of text markers or other graphical elements to an image Although image and overlay form a unit the data are stored independently on the com puter s hard disk Imagine the overlay as a transparent foil covering the image Drawing and chang ing the overlay does not affect the image data Moreover you can fade out or delete the overlay at a
237. ge Control The cell4R cell4M Imaging Software features a module for the easy control and efficient usage of motorized microscopes stages by Marzhauser GmbH and Prior Scientific Instruments Ltd in imag ing experiments via the Experiment Manager see also Chapter 5 3 2 5 The Stage Frame and Chapter 5 3 3 8 Experiments with image acquisitions at different stage positions While simple movements of the stage are conveniently done via joystick automated experiments require posi tion lists This chapter describes how to define these and how to calibrate the stage O Proper operation of the stage via software is only possible if the stage is calibrated See Chapter 4 5 3 for details 4 5 1 Defining a Positions List EA 4 Motorized Stage A click on the Motorized Stage button opens the Stage dialog window The last Positions list used will be loaded automatically and can be modified at will To create a new one select lt New List gt from the short list To load an existing list select it from the shortlist To delete a list load it and then click Delete To add stage positions to the list move the stage to the desired position using the joystick focus and click Add Positions can be deleted copied and pasted with the usual MSWindows commands and moved within the list via lt Alt up down gt Once the list is complete type in a name in the List box and click Save All positions list are saved in one file called OBSPO
238. ging The Mean filter is a particular NxN filter based on a 3x3 neighboring pixel area Matrix 8 2 9 Median Application Removal of bad pixels The Median filter removes isolated bad pixels with intensities that differ strongly from the sur rounding 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 What will happen 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 ranked according to their magnitudes The mid dle value the 5th one out of nine 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 They thus disappear from the image This suppresses noise points and non extreme unevenness will be smoothed out Comparison 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 numerically more stable i e it reacts less sensitively to individual greatly deviating values 138 Chapter 8 Image Processing OLYMPUS 8 2 10 Pseudo Filter This
239. graphs In the following section you will means to modify and fur ther analyze these graphs and the underlying data spread sheets The Kinetics tool Chapter 10 6 Intensity Kinetics in Time and Z and the line profile tools Chapter 10 3 Line Profiles Intensity of cell R cell M generate analytical results of fluorescence intensity changes in form of graphs and spread sheets They can be either exported into other programs such as MS Excel or further analyzed within cell R cell4M Pied Graph DOCUMEN S asas iE EREE 236 IEZ TAG Graph Window eenaa es ee aod 237 11 2 1 The Cursor Changing the XY Scaling in the Diagram 237 11 2 2 The Cursor Measuring Individual Graph Points 0cccee 238 11 2 3 The Graphs Button Bar cccccccseeccseseeceeeeeseeeeeeeeeesaeeesseeessaees 238 11 3 The Graph Men nescicracesnnna diene ee 242 RSi Makers and Lapes minisiesnamar bieie ni tant anacostcdnediaandes 242 11 3 2 Protecting and Deleting a Graph sssssssssrsresrrrrsrrrennrrresrrenen 245 T33 Graph INPOPMANON ss siano e a Eaa EN EAER 245 AoA OM OCU aia i e A E E E eE 246 235 236 Chapter 11 Graph Display and Graph Analysis OLYMPUS i Several functions do not work on Kinetics graphs 11 1 Graph Documents For displaying and processing one dimensional data cell4R cell4M contains an additional docu ment format the graph document Similar to images newly generated Graph documents o
240. gt Query by Example The database architecture of cell4R cell4M allows to search the contents of a database for docu ments that match certain criteria Click the Query button or use Database gt Query by Example to open the search mask The fields in the dialog box are listed by default but can be changed as explained in the next Chapter 12 5 4 Administration Defining Organizational and Database Fields Query by Example Cancel Image Type DO E Y Resolution Data type Ca E speriment 2 linn Graph Document E Data Type in search parameters and click Search The results will be listed in the database in a new folder called Query results and indicated by the query icon Name fragments can be searched as common in other programs as well if the fragment entry is framed by asterisks e g lt name gt cell s cell software Manual Chapter 12 Database 263 If the active entry in the database is a folder the query will only scan the contents of this folder Activate the database icon in the Tree view to search the entire database A query can be further refined by marking data types in the Advanced gt gt view of the dialog box Documents that do not match the selected types will not be considered in the search To find the location of an entry in the database first mark the thumbnail then right click to open the context menu and select the command Goto Record A database can contain only one Query r
241. h line into two segments The angle is measured clockwise between the longer segment of the first and the longer segment of the second line Thus the order of drawing and the length of the lines are decisive The angle will be displayed in the overlay and listed in the measurement display 1 Position the mouse cursor at the origin of the first line and left click 2 Move the mouse cursor a straight line will appear in the overlay 3 Left click at the end of the first line 4 Draw the second arm in the same way starting at any given point 5 Right click to terminate the measurement O Note again that the angle is defined according to the length of the line segments and measured clockwise exclusively 9 2 5 Area 9 2 5 1 Rectangle mj Rectangle Use this command to measure a rectangular object Default measurements are area and perimeter Several measurement series can be conducted successively As soon as the button is clicked a red rectangle appears in the image overlay 1 Drag the mouse to move the rectangle 182 Chapter 9 Measurements OLYMPUS 2 Drag the mouse with the left button pressed to change the size and shape of the rectan gle 3 To fix the rectangle right click and a new rectangle will appear Proceed as before 4 To exit the measurement press lt ESC gt The result will be displayed in the overlay and listed in the measurement display 9 2 5 2 Rotated Rectangle Rotated Rectangle Use
242. hapter 10 Intensity Analyses OLYMPUS 10 4 3 ROI Measurements 2 D O If the ROIs are already drawn when any of the measurement commands is called you have to select the ROIs to be measured by mouse click If no ROIs drawn the start of any of the commands will automatically open the Define ROIs dialog described in the previous chapter 10 4 3 1 Area Measure gt ROI gt Area Use this command to measure the area of a Region of Interest The line that defines the border of a ROI is counted as part of it The results are listed in a sheet that is gen erated The area unit depends on the image calibration see Chapter 7 1 Image Calibration 10 4 3 2 Perimeter Measure gt ROI gt Perimeter The area unit depends on the image calibration see Chapter 7 1 Image Calibration 10 4 3 3 Average Intensity Measure gt ROI gt Average Intensity Use this command to determine the average intensity of all pixels within a Region of Interest including the pixels that define the border of it Pixels with O intensity are ignored because they result from thresholding background subtraction processes and would skew the result in a meaningless way This avoids having to be extremely careful when drawing a ROI close to the border of a cell for example in a ratio image The measurement results are listed in a sheet that is generated 10 4 3 4 Average Value Measure gt ROI gt Average Value This is the same function as Average Intensity w
243. he Open Database window select a cell4R cell4M database with the extension apl and click the Open button Recently opened databases can be selected from a list at the bottom of the Data base menu Open Database File name LifeScienceD emoS TAR apl Files of type Database ap vw To create a new cell4R cell4M database select New Database in the Database menu Type a name in the Database name field of the New Database window and click on the now enabled Next gt button In the following dialog box select Finish to create the new database with the high lighted parameters 70 Chapter 5 Experiment Manager Hew Database Database name Database files api D NAarchivestest 5 4 2 Executing an Experiment Control Center X Use these commands to run an experiment Control Center OLYMPUS Select Finish to let the database assistant create a database with the following settings Location of database files D WArchivestes Location of images and documents D 4rchivetes Organized by Experimer Organizational ID Experiment name Save settings as new template Execution of an Experiment Plan compiled with the Experiment Manager involves three steps 1 Check 2 System Ready 3 Start cell s cell software Manual Chapter 5 Experiment Manager 5 4 2 1 Check TA Check Before an Experiment Plan can be executed the Experiment Plan needs to be validated The s
244. he Shutter button in the Illumination control window before an image is taken and closed again in the same way after wards By default the image acquisition rate in the Live View mode is as fast as possible in dependence of exposure time binning image size and camera type The slider that is only active if the Shutter option is selected allows reducing the acquisition rate in the Live View mode This is a useful fea ture in case of light sensitive specimens because the overall photo exposure is reduced corre spondingly cell amp cell Software Manual Chapter 4 Image Acquisition and Hardware Control 25 4 3 illumination Control si a Illumination Settings To adjust the parameters of the illumination open the control window with the Illumination Set tings button or via Acquisition gt Illumination settings The Illumination system MT20 MT10 window can be moved freely across the screen to your convenience ia Illumination System MT E DR ge Main Switch Burner OM OFF Excitation 403 DAPI 500 YFF Intensity 5 A Burner type MT_ARCIXE 150W xenon Burner hours 63 Burner tJ Sample illumination J Main switch In this control window you find the Main switch Burner ON OFF button to ignite the arc burner or switch it off again At the front of the Illumination System MT20 MT10 housing the green LED labeled BURNER ON indicates that the burner is turned on 26 Chapter 4 Image Acquisit
245. he active C module This will result in commands and buttons of the active module being arranged in the correct order with regard to the commands and buttons of the other modules This is relevant for example if you have two modules sharing the same button bar Definition The term Configuration refers to the appearance and functions contained in the Graphi cal User Interface GUI e menus and menu commands e button bars and buttons contained therein e key functions of the keyboard e macros and e external programs On the other hand the configuration does not contain the settings of the Viewport Manager or the Image Manager Individual dialog box settings and which modules are currently loaded are not part of the configuration What s it for Use the command Save Module Configuration to assign a configuration to the cur rent module or to replace the module configuration with the current configuration if a configura tion has already been assigned Loading a C module When loading a module the MAPI module functions contained by the mod ule configuration will be added to the current configuration The C module will have to be recom piled in order for the alterations to the module configuration to be considered when you load the C module Just as with all other alterations made on C module files you will have to recompile the C module so that these alterations will be considered when you load the C module Closin
246. he current graph Window Mode The Window Mode button is currently without function cell a cell software Manual Chapter 11 Graph Display and Graph Analysis La Show Legend Click the Show Legend button to display legends of the individual curves in a graph containing the kinetics of more than one image ROI or more than one line profile 11 2 3 5 Overlay Functions O These functions do not work on Kinetics graphs The remaining elements of the button bar refer to overlay functions The display of more than one curve in a graph document is termed overlay Use the Graph Analysis gt Overlay Selection command to create such an overlay graph Ovi Overlay Mode If the Overlay Mode switch is set the scroll bars and all buttons of the button bar concerning the axis scale apply only to the selected curve These functions can now be used for alignment of the overlaid curve relative 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 blue 7 Line profile 1 of Overlay Use the Overlay list to select one of the overlaid curves The original graph cannot be activated because it always remains fixed with respect to the X and Y axes The active overlay is affected by the overlay operations explained below F Fi Click the Fit button to adjust the Y scale of the overlay to the scale of the actual graph This func tion is a quick and c
247. he next command independently from the active docu ment s type Syntax error If a syntax error is found in the macro text the first incorrect line will be selected and a warning indicating the type of error will be displayed 13 1 6 Single Step Use the Special Recorder gt Single Step command or the lt F10 gt key to execute a macro one step at a time 270 Chapter 13 The Special Menu and the Window Menu OLYMPUS The Single Step and the Run Macro command are similar Single Step however executes the macro one step at a time under user control Application This command is very useful for running a systematic error search in a new macro You can follow the flow of the program and have the content of variables and expressions dis played simultaneously What will happen The exact function of Single Step will depend on the current context Text document If cell4R cell4M is not in the debug mode and the active text document does not contain a selection the entire text document will be interpreted as an Imaging C module and exe cuted stepwise in the interpreter mode Before doing so the interpreter will be reset This means that all variables and functions defined in the interpreter mode will be internally reset but remain in the text The Debug toolbar is displayed and the mouse cursor will be transformed into the shape of a hand The first executable step will be indicated by the footprints symbol at the beginning of th
248. he same base name as the modules make file and with the extension SXU It will be stored in the same directory as the make file MKU of the module If no errors are reported the build has been successful All functions constants types and variables that are exported from the module can now be tested from the Command Window or can be included in a configuration e g in the button bar Canceling compiling To interrupt compilation use the lt Curl gt keys cell s cell software Manual Chapter 14 Imaging C 315 14 8 Close Module Use this command to close the active C module Available This command is only available if a C module is active Before you use this command activate the module in the module manager What will happen If the module contains an Exit function it will be executed Values of all vari ables will be reset MAPI functions will be removed from menus and button bars These functions can then no longer be executed via the command window Effect on other modules Data and functions of other modules remain unaffected by this com mand Reloading modules To reload the module use the Open Module command or the Module Manager 14 9 About Module Use this command to view information on the active C module Further you can set whether mod ule functions are to be removed from the Graphical User Interface GUI configuration or not when you close the module About Module File name O cell A Modul
249. he shorter will be the time required 56 Chapter 5 Experiment Manager OLYMPUS for the Autofocus scan to finish However the focus cannot be found if it is outside the range Thus certain cautiousness is necessary when setting the range Properties Autofocus Image Frame Display 2 Device Range Big Step Fine Step Uze entire range Ensure constant time Low Signal mode Big Step Set here the step width to be made between neighboring Z positions during the first of the two Autofocus scans Whenever the Range is newly set a default step size is being set in dependence of it Fine Step Set here the step width to be made between neighboring Z positions during the second of the two Autofocus scans Whenever the Range is newly set a default step size is being set in dependence of it Use entire range If this box is NOT checked the following will be done if the focality initially in creases during an Autofocus scan and then worsens again the process will be terminated and the found focality maximum will be set as reference position for the second finer scan If the box is checked however the process will not be terminated after a local focality maximum is determined but the entire range will be examined Obviously this causes the Autofocus scan to last longer but reduces the possibility that a wrong focal position is being detected Ensure constant time It is not always possible to foretell how many po
250. hen press Delete Macro description In the Macro description field you can insert a brief description of your macro Macro text Enter the program code of your macro into the Macro text field Normally you first record and test a macro in a text document Use the Copy and Paste commands in the File menu to copy the macro text into this field A macro text can be no longer than 1000 characters Definition of more comprehensive macros If you wish to define a longer macro you should remove the macro text for safe storage in a file via the include command see below or have the macro implemented via a C module To do this first save the program code of the macro e g in a file named C cellR macro MyMacro sfm Enter the following line into the Macro text field include lt C cellR macro MyMacro sfm gt When you use the include command within a macro function you ll have to reset the interpreter every time you make an alteration to the file linked to the macro in order to have the function re defined To do this use the Reset Interpreter command in the Special menu The interpreter will be automatically reset when you exit the Define Macros dialog box via OK Define as macro function If the Define as macro function check box has been selected a macro function will automatically be defined using precisely the same name with which it was en tered into the Macros field You may then use this new function within other macros
251. his configuration at any time Configurations can be designed for different tasks and or users and saved as individual settings File format A user configuration file is saved using the scy file name extension 280 Chapter 13 The Special Menu and the Window Menu OLYMPUS 13 5 Preferences Use this command for setting preferences within cell4R cell4M The Preferences dialog box can alternatively be opened with lt F8 gt 13 5 1 The Preferences gt Image Tab The settings on this tab apply the handling of images Preferences Image View File Measure Module Graph Database Report Imaging functions Confirm operations Available image butters Em Drag amp Drop Circular switch C Delete image 4llow operations on false color images C Copy image Overlay Image acquisition Delete overlay Sequence Burn overlay None Mi Tab Frefis for images a a ames C List Incremental number al O Gallery Both Overview e You can adjust the number of available image buffers in the image manager e You can activate the circular switch and set the cycle width e You can select whether you want to receive a query message when you delete or copy images from the image buffer via drag amp drop e You define whether most of the menu commands are also allowed for false color images cell s cell software Manual Chapter 13 The Special Menu and the Window Menu e You can predef
252. hree steps 1 Check The system verifies if the execution of the Experiment Plan is feasible or if there are invalid parameters or logical faults 2 System Ready The Experiment Plan is downloaded to the Real Time Controller and data storage space is allocated on the hard disk 16 Chapter 3 Brief Introduction and First Steps OLYMPUS 3 The experiment itself is started paused and stopped with Start Pause and Stop O Any image or image sequence acquired with the Experiment Manager is automatically stored on the hard disk in a cell R cell4M database 3 6 Displaying Multi Color Images F i E All Color Channels wv 1 Furas4o w 2 Furased Select Transmission Select Intensity Modulation Select Color Channel Multi color cell4R cell4M images consist of several color channels in principle the number is unlimited Each channel contains a monochrome image of predefined color resulting from one Im age Acquisition command in the Experiment Plan When loaded from an archive all color channels are displayed together in an overlay resulting in a color image The Select Color Channel button in the Navigation toolbar provides a tool to navigate easily through all color channels and to display selected ones Display Intensity Adjusting the single color intensities with the dialog box of the Display Intensity command can optimize the look of the multi color image For details see Chapter 6 2 2 Adjust Display
253. ht source is in the exchange position i It is not possible to run the cell4M cell R Configuration Software and the cell M cell4R Imaging Software at the same time Open the filter flap on the right side of the illumination system s housing The flap has a magnetic closure no tools are required for opening closing The filter slot 6 points to the flap opening in the example above The filter position number is labeled right above the filter opening In case an exci tation filter in this filter slot has to be exchanged this can be done easily without any tools the filter is Clamped with a metal clip onto the filter wheel Pull out the filter with your hand while taking care not to touch the filter s glass surface O Only optical filters with a diameter of 25 mm may be inserted into the filter wheel All fil ters purchased from Olympus Soft Imaging Solutions are suitable There are templates on the inside of the filter flap which can be used to check the appropriate size of an optical filter Insert the new excitation filter into the filter wheel simply by pushing it underneath the metal clips This can also be done by hand Again be careful not to touch the filter s glass surface The correct orientation of the filter with respect to the direction of the light path is in most cases marked with a little arrow on the frame of the filter The arrow has to point in direction of the light fiber Excitation filter In the configurat
254. humbnail creation size Small w Save images File type Compression None tif kai Locations Database fles a P A DMArchive s T mp5 torageLir ia Backup volume capacity sos ME Record name Synchronize with image name Auto Record Name Format Overview On this tab you can decide e the current size of the thumbnail display for all database documents and e the preferences for new databases These settings comprise actual thumbnail size the standard image format maximum size of image drive or directory compression method and the paths for system files and images To adjust the actual Thumbnail display size you can select between the predefined sizes Small Medium and Large from the pull down list Database defaults You can set the preferences for new databases Thumbnail creation size You can adjust the size of newly created thumbnails from the pull down list to Small Medium and Large In the Save images field you can preselect from the pull down list the File type for saving images acquired in the database Selecting Force TIF type for documents inserts images as documents with the TIF format The read only information Compression indicates if and which compression method you have selected The Options button has the same function as the one on the File tab for details see Chapter 13 5 3 1 Save image files cell cell software Manual Chapter 13 The Special Menu and the Window Menu O We r
255. icolor apl m Activate Activate This button activates the window selected in the Document Name list The same com mand can be executed with a double click on the window O Minimize Maximize Restore Close Minimize Maximize Restore Close These buttons represent standard Windows commands and can be executed on the presently active documents with the exception that the Image and Graph documents cannot be closed a Bm Cascade Tile Horizontal Tile Vertical Cascade Use this button to have all selected text sheet diagram and database documents ar ranged staggered one atop the other What will happen The order in which the documents are arranged will depend on which one was most recently active The currently active document will be the one displayed in front Title lines containing the names of all documents will remain visible so that you can easily activate any of them they will then appear in the foreground Tile Horizontal Use this button to have all open text sheet diagram and database documents arranged horizontally and above and below each other What will happen All open documents will be displayed at the same level and in the same size using up the whole documents area Document windows will be enlarged or reduced as needed If up to three documents are selected they will be displayed one above the other With four and more they ll be arranged in rows and columns so that windows w
256. id size check box to have height and width of grid frames automatically adjusted to fit the minimum number of grid frames Enter this value in the minimum of fields box This number is based on the width of the whole image The lowest value possible is 2 frames the highest 50 With the Automatic grid size the grid frames shape will cor respond 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 Grid size group Not all ad justments to the minimum number of fields result in a differing frame size because height and width are restricted to standard sizes such as 1 2 5 10 20 in the unit of image calibration and so on For example an image of 125 mm in width will have frames of 20 mm width if the minimum value entered is 3 6 For values between 7 12 the frames will be 10 mm wide Colors group Here you can determine the color of the grid and the overlay text Draw button Click on the Draw button to have the grid at its current settings drawn into the over lay In addition cell size or alternatively numeration of the grid frames will be written into the over lay 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 dialo
257. idiaries remain owners of the software and it s documentation With the purchase the User obtains ownership of the diskettes or other physical storage devices excluding the software and other data contained thereon the manuals and the software protection key 2 OLYMPUS SOFT IMAGING SOLUTIONS reserves the right to all publications duplication editing and marketing of the software and the software documentation Without prior written permission the User may not change translate de compile or de assemble the software copy any of the written or printed documentation of the software rent lease or license the software to a third party use the software protection different than described in this contract 3 The license property and user rights to the OLYMPUS SOFT IMAGING SOLUTIONS software disks and manuals may only be sold or transferred to a third party on a permanent basis if the third party agrees to abide by the conditions in this contract 4 OLYMPUS SOFT IMAGING SOLUTIONS is the legal owner of all copyrights and trademarks of the OSIS cellAM cell4R SOFTWARE PRODUCT and documentation Copyrights and trademarks are protected by national and international law OLYMPUS SOFT IMAGING SOLUTIONS reserves all rights which are not explicitly expressed in written form 5 Warranty 1 OLYMPUS SOFT IMAGING SOLUTIONS guarantees for the period of 12 months after the date of purchase that the software works
258. ility 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 10 9 2 The Problem 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 applications The considerable overlap of excitation and emission spectra of different fluorochromes is exemplified by the spectra of en hanced green fluorescence protein eGFP and enhanced yellow fluorescent protein eYFP two commonly used variants of the green fluorescent protein Fig 1 Even with the use of high quality optical filters it is not satisfactorily possible to separate the spec tral information See Figure 2 The consequence is the excitation and imaging of YFP labeled struc tures with a GFP filter set and vice versa eGFP eYFP 1 er absorption 450 500 550 nm Figure 1 Absorption and emission spectra of eGFP and eYFP The HeLa cells shown in Figure 2 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 cytosol However it is evident from the images in Figure 2 that there is a significant contribution of the YFP labeled structures to the GFP image Similarly you can 214 Chapter 10 I
259. ill tend to be rather squatty and broad Title lines containing the documents names will remain visible you can thus easily activate any of the open documents Tile Vertical Use this button to have all open text sheet diagram and database documents ar ranged vertically next to each other cell cell software Manual Chapter 13 The Special Menu and the Window Menu What will happen All open documents will be displayed at the same level and in the same size using up the whole documents area Document windows will be enlarged or reduced as needed If up to three documents are selected they will be displayed next to each other With four and more they will be arranged in columns and rows so that windows will tend to be rather long and narrow Title lines containing the documents names will remain visible you can thus easily activate any of the open documents El Close All Close All Use this button to close all Text and Sheet documents It does not affect Image and Graph documents or any images within the image buffer box 13 6 4 Viewport Manager Use this command to turn the Viewport Manager On or Off You may also use the short cut lt Alt 1 gt What is the Viewport Manager The Viewport Manager also displays the image of the active Viewport Additionally the Viewport Manager is surrounded by a red rectangle The size and posi tion of this rectangle can by changed using the left mouse button The area within
260. imal cycle time depends on the time requirements of all commands in the Time loop frame 5 3 2 5 The Stage Frame ime 4 Stage frame Use for experiments that are to be repeated at different stage positions Execution of this command will cause the following e The commands within the Stage Frame are executed at the first position of a specified posi tions list and then repeated at each other position e The image sets are stored in separate files for each position but within the same experiment folder The sets have the same name but carry the stage position number as prefix Properties Properties Stage Position List All positions in one display Each position in separate display Position List Select the positions list see Chapter 4 5 Motorized Stage Control from the shortlist The following options are only relevant if the online Display option is chosen for the Image Acqui sition commands within the stage loop see Chapter 5 3 2 1 Image acquisition cell s cell software Manual Chapter 5 Experiment Manager All positions in one display Select this option if you want to have just one online Viewport that always shows the most recently acquired image of the respective image type If a stage loop con tains more than one Image Acquisition command each of them will have its own Viewport Each position Select this option if you prefer to have the online images for each stage position displ
261. ime you start up Confirm operations Drag amp Drop Decide whether you wish or not to receive a query message when you Copy image or Delete image in the image manager using drag amp drop To select these options set the tick marks in the respective small boxes These queries apply only to the dragging amp dropping of im ages You will not receive a query when using the Edit Delete Image and Edit Copy com mands Overlay Decide whether you wish or not to receive a query message by selecting the check box Delete overlay If you select Burn overlay the overlays will be saved together with the image thus it will change the image data Image Manager tabs Select here whether the Image Manager should list all loaded images as a list with name entries as thumbnail gallery or both 13 5 2 The Preferences gt View Tab The settings of this tab apply to the cell4R cell4M Graphical User Interface Overview e Choose the font size for cell4R cell M image overlays and sheets in this tab cell s cell software Manual Chapter 13 The Special Menu and the Window Menu e Decide whether the mouse cursor is to be transformed into the shape of an arrowhead or of cross hairs during interactive measuring e Decide whether the name of a function is to appear along with a brief description of the com mand in the status bar e Indicate whether you re working with a general or individual workspace e Align the image document al
262. in the right field of the status bar M OY Running 00 00 13 694 Frames Acquired 27 Stored 26 Total 100 5 4 2 4 Pausing an experiment uu Pause To interrupt an experiment press the Pause button As a consequence experiment execution will be interrupted immediately If the Pause button is pressed while the system is taking an image the interruption will take place after the process is completed In the paused state the experiment can also be aborted see below To resume the experiment press the Start button O The experiment time will continue to elapse during an interruption meaning that e The total experiment time will increase by the duration of the interruption If the ex periment is interrupted during acquisition of an image sequence Time Lapse command the cycle time of the current loop as well as the total loop time will in crease by the duration of the interruption cell s cell software Manual Chapter 5 Experiment Manager The time information attached to the image data will reflect the interruption There fore the break will be reflected also in post acquisition temporal analysis of the im ages The experiment time display in the status bar continues counting 5 4 2 5 Setting markers during an experiment z Set markers To mark a certain event during the course of an experiment e g the application of a substance to your sample during a time lapse experiment press the Set Marker bu
263. ine the image name automatically set for image acquisition Imaging functions The settings in this group are relevant for image processing operations for altering gray or color values of an image Available image buffers Enter the desired number of image buffers to be available in the image buffer box of the Image Manager into the Available image buffers field You can select between 16 and 200 image buffers Any change in the number of image buffers only becomes effective after you restart cell4R cellAM Circular switch Select the Circular switch check box to activate the circular switch As a conse quence the destination image buffer will automatically be different from the source image buffer i e the active image cannot be overwritten during image operations Destination image buffer and circular switch The image buffer used as destination buffer depends on various settings a the number of Viewports displayed in the image document b whether the image buffer is protected and c the circular switch settings Single view If you only have one Viewport displayed in the image document the destination image buffer will be set to the next highest image buffer after each image operation The highest image buffer available represents the limit of the range of the circular switch At this point the destination image buffer will be set back to image buffer 1 If you set the range of the circular switch at e g 8 the destination image
264. ined in the interpreter mode After that the active C module will be searched Lastly the prede fined symbols in the Exported by Tool range are searched Upper lower case Wild cards When making a search request upper or lower case is of no rele vance Any parts of a symbol s name you don t know can be substituted with an asterisk letters you don t know can be substituted with a question mark cell s cell software Manual Chapter 14 Imaging C 323 14 14 Quick Watch Use this command to determine the value of the selected expression or variable during an error search i e debugging Available This command is available when a text document is open and active You may also use the lt Shift F9 gt keys Before you use this command open the source text of the Imaging C module or macro Position the cursor on the variable whose value you wish to view Use the Toggle Breakpoint command to set a breakpoint within that line Then initiate the function containing the expression or variable in question Imaging C functions of modules and programs are executed by you via a corresponding MAPI function e g use the menu command of the MAPI function Execute macros using the Run Macro command in the Special menu or just press lt F5 gt or using the Single Step command in the Special menu or just press lt F10 gt cell4R cell4M will move into the debug mode When is the debug mode active As far as Im
265. inetics in Time and Z ccccecceecceeeceeeceeeeeeeneeeeeeeeeeeaeeees 207 10 7 DeltaF F AF F Analysis seseiceinecsicdecetcness adidas seas 208 TO General eero r E EN 208 10 7 2 Generating a AF F Sequence ccccceeeecseeeceeeecseeeceeseseeeeesees 208 10 85 Ralo ANAYSIS oree E e EE EA E E E EEEE S 210 109 8 GONCK alist dedecenestetees shes enc nies EEEE N 210 10 8 2 Generating a Ratio SEQUENCE ccccceeeceeeeeeeeeeeeeeeseaeeeeenaeeeeens 211 109 Spectral UnmiIxNg eai a a 212 109 1 APHICIUO Mea a Saale eecia aneesees sce enorme 212 10 92 WING FODDION aseene eE 213 10 9 NG Solulon ense ese eet eh eae e ears 214 10 9 4 How Does it Work cccccceecccescecceeeeceeecceeeecaeeesaeeesceeeeseseesees 215 10 9 5 Spectral Unmixing with cell4R CelIAM cceceeeeeeeseeeeeeeeeeeeens 216 10 96 Cal FAllON ain a a aE aa 217 TUF AOMUMUIAG ean ea ea ee aeS 218 10 9 8 Unmixing of Color Camera Images ccccescceeeeeeeeeeeeeeeeneeeeeens 220 10 10 Phase Color Coding and AnalySiS ccccccccccssseeeeseeeeeeseeeeeenaeeeeens 221 10 101 Phase Color Goding aserrean E ver tisdveara 221 TOTO Z PHASE ANd SIS mieie E a e 222 TOTI COIOCANZ QAUOM kas E a eat 222 10 12 The FRET Software Module ccccsccccssscecseseeeeeeeseeeeeeseessaeeeeees 224 VO312 AIM aAGE ACOUISINION rosin a cde cient elite 224 10 12 2 FRET Image Correction Factors cccccsccecsseeeesseseeeeeeeeessaeeeeens 226 10 1
266. ing into the red the green and the blue channel so that the display remains the same seemingly a combination of cyan yellow and red The contents of the three RGB channels however are not identical to the contents of the three channels resulting in the other case 7 9 5 Invert Use this command to generate a negative of the original image Available This command is available for all types of images It is however only available for false color images if the False Color Images check box has been selected in the Image tab in the Pref erences dialog box in the Special menu cell cell software Manual Chapter 7 Image Data Handling 121 Transfer function If the greatest number for gray value display is G and G stands for a pixel s gray value in the resulting image then this pixel s gray value will have the following value in the resulting image G Ga G What will happen For 8 bit images G_ equals 255 For 16 bit images G equals 65535 In binary images the white areas will become black and vice versa 7 10 Image Information Use this dialog box to change the name of the active image buffer to enter an image comment and or to have image information displayed Image Information x Image Information as a General Dimensions Markers General Dimensions Markers Image name ee Color Channels 2 File name D Marchive LifeScienceDemo 4X560858_Do Image buffer 3 Frame 1 7 6 Time Framez 61
267. ing this MAPI function the corresponding command is not available will appear gray Example Imaging C Code WORD OnInitMenu WORD wMenuID Switch wMenulIlD case TDM SAMPLE return MENU ENABLE return MENU ENABLE Additional Constants To make the availability of MAPI functions controllable using the OnlnitMenu you ll need to put the constant MAPI_IDLECHECK at the 5th position within the definition instruction of the MAPI func tion Example Imaging C Code export UINT SampleFunction lt MAPI IDM SAMPLE Info in status bar Sample amp Function MAPI IDLECHECK gt 305 306 Chapter 14 Imaging C OLYMPUS Auto configuration If you have selected the Auto configuration check box the new C module will contain a source file having the file name extension SFM as well as a C module configuration file SCX This configura tion file contains the definition for a button bar of the same name as the module as well as contain ing the T button The MAPI function Sample function can be activated by clicking on this button When the new C module is closed the button bar will automatically vanish It will reappear at its previous location when the C Module is loaded once again O If you desire to change the name of the MAPI function SampleFunction after the C module has been generated be sure not to forget to change the assignment of the SampleFunction s T button as well String
268. inter nally but not in the text document 13 1 8 Set as Default Macro Use this command to define the active text document as the default macro What will happen Using the Run Macro command will have the active text document executed as a macro Execution is not possible if the image document is active The Set as Default Macro command defines a text document that will then be executed when the command Run Macro is selected This permits the execution of the associated macro even if a non text document is the active document 13 1 9 Define Macros Use this command to add new macros to the configuration The corresponding dialog box opens Define Macros Macros Macro description Macro text Define as macro function Reset interpreter 2 2 Chapter 13 The Special Menu and the Window Menu OLYMPUS All macros that are integrated into the cell4R cell M Graphical User Interface using the Define Macros command must be saved in a user configuration Use the Spe cial gt Configuration gt Save command to save the configuration Macros Name your macro in the Macros field Add Click on the Add button after entering the name of the macro into the Macros field The name will be added to the existing macros list Warning A macro it must be added to the list in order to be defined It is not sufficient to exit the dialog box by clicking on OK Delete To delete a macro from the list select it and t
269. ion and Hardware Control OLYMPUS Shutter The shutter can be opened and closed by pressing the Shutter button to start and stop the fluorescence illumination of the microscope Excitation filter buttons The Illumination System MT20 MT10 has a built in filter wheel with eight positions for different excitation filters To select a specific excitation filter click on the respective button The color of the buttons and their connection with the respective excitation filters and filter positions in the MT20 MT10 is set in the cell R cell M Configuration Software see Chapter 15 cell4R cell M Configuration Intensity The MT20 s MT10 s built in attenuator controls the brightness of the illumination 14 respectively seven levels are available between about 1 and 100 The intensity can be adjusted by moving the Intensity slider Once this is active after a mouse click it can be moved by the scroll wheel of the mouse as well PIFOC If your cell4R cell4M imaging Station is equipped with an objective PIFOC or nosepiece PIFOC that has been configured in the cell R cell M Configuration Software see Chapter 15 cell4R cell M Configuration the Illumination system MT20 MT10 window features the PIFOC field The position of the objective can be adjusted by moving the slider or by typing in a position height value Information field In the lowest part of the window you find information about the Burner type The Burner hours counte
270. ion dialog select the inserted excitation filter from the drop list of the current filter wheel position The list offers many standard excitation filters provided by Olym pus Soft Imaging Solutions If the inserted excitation filter is a special filter not listed here or if you want to use a different name for the filter you may just enter or change the filter name by clicking into the edit field and typing the desired filter name The filter name defined here will appear on the respective switch button of the Illumination system MT20 MT10 dialog in the cell4M cell R Imaging Software see Chapter 4 3 Illumination Control Color The rectangular field adjacent to the filter name box shows the color of the switch button in the Illumination system MT20 MT10 dialog that will move the respective filter into the illumina tion path Each standard excitation filter which can be selected from the drop list has a predefined default color setting You can change define the switch button color by clicking on the color pal ette button to the right of the color field With the three slider bars you can adjust the Hue the Saturation and the Brightness of the switch button color respectively The Hue values can be set in a range between 60 and 280 Following 332 Chapter 15 cell M cell4R Configuration OLYMPUS the conventions 0 corresponds to red 180 to cyan Each value has a corresponding wavelength indicated below the quadratic color fiel
271. ipal im age display parameters The Camera Control window can be moved freely across the screen The cell4R cell4M Imaging Software provides the unique possibility to change all the above men tioned parameters during Live View which makes it very convenient to adjust the acquisition for each experiment The Camera Control window contains the same Snapshot and Live View buttons as the Acquisi tion toolbar described in Chapter 4 1 Simple image Acquisition 22 Chapter 4 Image Acquisition and Hardware Control OLYMPUS a Snapshot W Live View Camera control 22x Bini 10044000 vl BH a Bera tat 028800 _v Exposure time Exposure time J2 a Adjust with binning ms C Show saturation Adjust with binning 5 A Standard Extended Brightness adjustment Automatic Fj Online Histogram Min gt Max Yo Offset Color control subframe T 9 203153 BE 475 Lite Co White Balance Shutter 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
272. is displayed in the upper left cor ner of the overlay as long as the Label fields check box has not been selected Normally one digit after the decimal is displayed If you would like more than one make the follow ing entry in the ANALYSIS INI file in the cell4R cell4M program folder on the hard disk for as many places as desired e g 2 SYSTEM MesGridDigits 2 110 Chapter 7 Image Data Handling OLYMPUS Imagesize 6 60 mm 5 16 mm Gridsize Horizontal 1 mm H Vertical h i an Cancel Options LI Use starting point Label fields Automatic grdsize Hep minimum of 5 E fields Hep Colors Grid Text Options group Use starting point Select the Use starting point check box along with the Interactive button to determine grid frame position in the image If you clear the check box the grid will always start in the upper left corner of the image at the coordinates 0 0 Label fields 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 by the numbers 1 2 3 99 Each frame can be precisely 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 in that case enlarged If you select the Label fields check box the grid size will not be displayed Automatic grid size Select the Automatic gr
273. ith the Experiment Manager any such experiment can be defined in an easy graphical way by setting up an Experiment Plan Once defined an Experiment Plan can be saved as an independent file Each user can thus define a set of Experiment Plans for repeated experimental runs Experiment Plans can be transferred and executed on any other cell4R cellAM imaging station however if the system configurations differ certain changes might be required Also the plans are stored together with the image data in the database after execution of an ex periment Preparing an experiment Depending on the variability of your samples it may be necessary to individually adjust certain parameters of an Experiment Plan for each sample for example it might be necessary to optimize the exposure times according to the quality of the staining Such parameters are variables in an Experiment Plan and can be changed easily Executing an experiment The same Experiment Plan can be executed as often as necessary During execution of an experi ment the acquired images can be displayed live on the monitor allowing online observation of the sample in one or several fluorescence channels User interference during experiment execution is possible via the Experiment Manager s control field The experiment may be paused continued or stopped It is also possible to interactively set markers for example to indicate external events such as the application of an agonist
274. ith the excep tion that for example in ratio images the actual ratio value will be given instead of the scaled dis play intensity which depends on the Scaling Factor used in the ratio calculation see Chapter 10 8 2 Generating a Ratio Sequence cell a cell software Manual Chapter 10 Intensity Analyses 205 10 5 Background Subtraction 10 5 1 General Background intensity is unavoidable in wide field fluorescence spectroscopy Working in a dark room can eliminate environmental light but there will always be a certain amount of excitation light that reaches the camera Causes are imperfect filter sets and the reflection of light at interfaces the cover slip and the specimen Background intensity reduces the contrast of the image and conse quently background subtraction should be a routine operation 10 5 2 Subtracting the Image Background A Background Open the Background dialog window with the Background button or via Process gt Background Subtraction Background Background Subtraction Background Subtraction Select ROIs From Image Constant Constant ee 2 Fura 3 ROIs w Oro L E ORG Select ROI Image FOLI ROLI v Background Subtraction Constant Select Background Image ORO 3 Fura Image 206 Chapter 10 Intensity Analyses OLYMPUS O This function will create a new data set The original source data remain untouched Background Settings tab Constant This
275. ithin the text document 328 Chapter 14 Imaging C OLYMPUS Meaning breakpoint is enabled active disabled not active Delete All Click on the Delete All button to delete all breakpoints from the Breakpoint list and from source texts Deletion is not reversible o o ee Available The following buttons are only available if you have selected a breakpoint Go To Click on the Go To button to go to the breakpoint you ve selected in the dialog box in the source text The text document will be opened if it had been closed The line containing the break point will be colored Condition Click on the Condition button to determine a condition under which a selected breakpoint is valid and when it is to be skipped Condition Stop at breakpoint when following z T Cancel expression is TRUE Po Example Imaging C Code If a breakpoint is e g to only be valid if the value of variable i is greater than 10 and the value of variable j is less than 10 the condition will have to be as follows 1 gt 10 amp amp j lt 10 Disable Click on the Disable button to skip the selected breakpoint when executing the function The breakpoint will be turned off i e disabled in the text document The status of the breakpoint in the Breakpoint list will go from E to D As needed you can reactivate the breakpoint Enable The Enable button activates a deactivated breakpoint The status of the breakpoint in
276. ition the mouse cursor at the origin of the angle and left click 2 Position the mouse cursor onto a point of the angle s first arm A straight line will ap pear in the overlay 3 Left click to fix the first arm It will be drawn into the overlay 4 A second arm will appear starting from the initial point of the first arm upon movement of the mouse Draw the second arm in the same way cell s cell software Manual Chapter 8 Image Processing 155 5 The Rotate window will open again with the interactively defined angle set in the Angle box 6 Continue with setting the Direction and the Options 7 Execute the rotation by clicking OK Direction Chose between a left counterclockwise and right clockwise rotation Interpolation If this option is selected pixel colors may be created by mixing the colors of neighboring original pixels This will give the rotated image a smoother appearance middle image below If the option is not selected the result may make the features look coarse right image However in case of biological samples the interpolation effect will hardly ever be visible It is of greater importance in case of graphics with sharp borders between areas of different colors left image below 8 4 3 Mirror Properties O Horizontal ae O Diagonal Diagonal oy 156 Chapter 8 Image Processing OLYMPUS The function Process gt Image Geometry gt Mirror allows to reflect single imag
277. ity projection are calcu lated analogously The functions can be selected from the pick lists of the Navigate Z and Navi gate Time buttons 97 98 Chapter 6 Image Display and Navigation OLYMPUS z KK K B H fc 1 Ho bH Process Measure Graph Special Window 7 l i i i Intensit j pa a3 Adjust Colors Maximum Intensity Projection Maximum Intensity Projection Minimum Intensity Projection Minimum Intensity Projection Set Thresholds Mean Intensity Projection Mean Intensity Projection Binarize Extended Focus Define Shading Correction Shading Correction Background Subtraction Projection Maximum Intensity T Define Filter j Minimum Intensity T Filter j Mean Intensit T Arithmetic Operations Maximum Intensity Z foecs i Minimum Intensity 2 Mean Intensity 2 RiGB Studica r Extended Focus Image 3D Images b EFI Options O The projection functions in the pick lists of the Navigate Z and Navigate Time buttons do not create a new image The projections are only displayed temporarily In order to generate new projection image data choose the projection commands from the Process gt Projection menu 6 4 2 EFI Extended Focal Imaging The EFI function can be regarded as sharpness projection for Z stacks time sequences are not accepted as source files The algorithm extracts the image features with the sharpest contrast from all layers of the stack and merges them into a single image To execu
278. ive commands in the sub dialog that is opened with the arrow adjacent to the Label box Color ROIs are colored differently by default The color can be changed by selection from the pull down palette prior to drawing To change the color of an existing ROI use the respective command in the sub dialog that is opened with the arrow adjacent to the Label box Active ROIs This list displays the current ROIs Clicking on the check box deactivates individual ROls Such ROls are not displayed Tools These buttons activate different drawing tools and close the dialog box ROIs are drawn with the left mouse button using the first six tools With the right mouse button the two ends of polygon defining lines are connected to generate closed polygons the drawing command is termi nated and the dialog box reopens rq Polygon Polygon Mark the corner points of a polygon straight lines then connect the corners a Interpolating polygon Interpolating polygon Mark the corner points of a polygon and the software automatically gener ates a perimeter curve around it 2 Freehand polygon Freehand polygon Delineate the ROI by marking its boundaries by continuously dragging the mouse cursor Ol Ellipse Ellipse Mark the center of the ellipse and adjust size and form by dragging the mouse Additionally press the Shift key to draw a circle After releasing the left mouse button the position can be changed By clicking anew the shape can be adjusted
279. ively the intensity range between the threshold values defines a phase Objects in gray value images are defined with the Process gt Set Thresholds command see Chapter 8 5 Thresholds and Binarization Up to eight phases can be selected For each phase a color for display is selected Gray value areas not assigned to a phase will remain uncolored 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 have selected image structures accentuated For true color im ages you
280. ized view The cell R cell M Execution Center Ed Minimize and Maximize button The cell R cell M Execution Center accessible via View gt Execution Center lt Ctrl e gt or the Minimize button is a minimized version of the Experiment Manager window that features only the Control Center button bar the Maximize button and the status bar These are the only compo nents that may be of use while an experiment is carried out However they are not available if the entire Experiment Manager is minimized The cell R cell M Execution Center is especially use ful during experiments with online display of images because the Experiment Manager window blocks a large area of the screen Click on the Enlarge button on the right side to re enlarge the view Execution Center ye gt TF te O0 00 00 00 Frames Acquired 0 Stored 0 Total 41 42 Chapter 5 Experiment Manager OLYMPUS 5 3 Setting Up Experiment Plans To set up new experiments you can either change an existing Experiment Plan or start from scratch with an empty editor surface To obtain an empty editor surface use the Experiment Manager menu File gt New If the current Experiment Plan has not yet been saved a dialog will appear asking whether it should be saved Experiment plans are set up in a simple graphical manner without the necessity of any program ming knowledge Icons that symbolize commands or series of commands have to be placed or dered and connecte
281. just the number of frames displayed per second Loops In this dialog box you can choose how often the image stack is to be played Choosing Play you can define how many time s the loop is played Selecting the Auto Repeat button the image stack animation is continuously repeated until stopped 96 Chapter 6 Image Display and Navigation OLYMPUS Direction In this field you can select the direction of the animation Choose here whether the stack is to be animated uni directionally or meandering back and forth Options Frame rate od Framers Loops Play i iis time s Direction i 6 3 5 Z Stacks Navigate Z In Z 8D experiments stacks of images are acquired at different focal planes If a Z stack is in the active Viewport the Navigate Z button is enabled in the Navigation toolbar You can navigate and animate the Z Stack in the same way as described for time lapse experi ments in the previous chapter 6 3 6 Multi dimensional Sequences Image Navigator Navigate Time selected Data sets are not limited to three dimensions in the cell4R cell4M Imaging Software You can acquire image sequences with up to five dimensions the three spatial dimensions plus time and cell s cell software Manual Chapter 6 Image Display and Navigation color The Navigation toolbar allows selecting through which dimension to navigate either time or Z If the Navigate Time button is activated the navigati
282. l amp cell Software Manual Chapter 4 Image Acquisition and Hardware Control 4 1 2 AVI Recorder This function is not active after a standard installation of the cell4M cell4R software Execute Spe cial gt Add In Manger Click Add in the corresponding window and navigate to the cell4M cell4R program folder Open AviRec dlx Close the Add In Manger window and restart the program The Acquisition toolbar now contains the Start Stop Avi recording button iad Start Stop Avi recording Click the Start Stop AVI recording button to have the current live image be acquired as a video During the acquisition the settings in the Acquire AVI Recording Options dialog will be used Click on the Start Stop AVI recording button once more or the Snapshot button to end the acqui sition process The results of the acquisition will be saved on your hard disk in the form of a video file AVI file You can edit the name and storage location in the AVI Recorder Options dialog box In the image document you will still see the live image The video file that you create can become very large Make sure that there is sufficient free space on your hard disk before you begin the acquisition and use a suitable com pressor 4 2 Camera Control aa Camera Control The Camera Control button or Acquisition gt Camera settings opens the window to set the acquisition parameters Exposure time Binning factor and Subframe as well as the princ
283. l adjustment when more than one channel is being displayed The histogram of the selected channel is moved to the foreground Load LUT This is the same command as in the Image Display toolbar see Chapter 6 2 9 False Color Edit Fluorescence Color This is command is described in detail in Chapter 6 2 8 Edit Fluores cence Color Histograms field Here the intensity histograms the distribution of pixel intensities of the cur rently active channels are displayed The scaling is automatically set so that it encompasses only the range between dimmest and brightest pixels If a single channel is being displayed or if one channel has been selected for adjustment with its Adjust Channel button vertical lines indicate the lower and upper thresholds of each channel Min and Max respectively Every value below the Min and above the Max will be displayed with the dimmest most often black or brightest most often the pure spectral color of the channel s palette respectively 201 4095 Min Max For a single active channel the minimum and maximum values are set either by drag ging the vertical lines in the histogram by typing the values in the respective fields or via the Min and Max scroll bars Auto Click here for an automatic adjustment of the active channels The software checks each channel for the minimum and maximum intensities and scales linearly in between under consid eration of the Clip values Clip This gro
284. l the time makes them more difficult to read Macro recorder Named arguments Select this option to record the argument names together with the invoked functions with the macro recorder Image buffer arguments Tick mark this check box to have the macro recorder record the index of image buffers involved absolute image buffer address If the check box is deselected the re corded functions will refer to the current values for the source and destination image buffers rela tive image buffer address In this case the macro recorder will record every interactive image buffer selection i e every click within the Image Manager Issue error message for invalid pointers When this option is selected and you use a pointer variable you will get an error message in case the value it contains is not valid cell s cell software Manual Chapter 13 The Special Menu and the Window Menu 13 5 6 The Preferences gt Graph Tab The settings on this tab apply to the handling of graphs Preferences Image View File Measure Module Graph Database Report Graph functions Available graph butters EBE Drag amp Drop in graph manager C Confirm delete C Confirm copy Background pattern Overview e You can adjust the number of available graph buffers in the image manager e You can determine whether the graphs are displayed in the list view e You can select whether you want to receive a query message when you delete or co
285. lable The List View that lists the names the XY size and the bit depth of the images The Gallery View that shows thumbnails of the images The Graph View that shows thumbnails of loaded graphs The Measurement View that lists the results of area and length measurements in images amp OND Documents Area which always contains Viewport displays the active image or a selection of currently loaded images Graph document the currently active graph if a corresponding analysis has been carried out It is minimized when cell4R is started Additionally the Documents Area may contain Database Documents Data Sheets Text Documents and Macros 3 1 1 The Image Manager In the Image Manager different image types like single color images multi color images or the various image sequences z stack time lapse etc are represented by different symbols The highlighted image frame in the Image Manager field is active and displayed in the Viewport Manager and the Viewport 10 Chapter 3 Brief Introduction and First Steps OLYMPUS single color image multi color image Z stack multi color Z stack vos single color time lapse la multi color time lapse E single color Z stacks in time lapse E multi color Z stacks in time lapse 3 1 2 The Viewport Manager The image in the Viewport Manager in the top left corner of the cell R cell4M window shows a red rectangle It represents the region of the image currently
286. lay tab Horizontal Vertical Shift Use the arrow buttons to shift the selected channel relative to the oth ers Center Use this button to move the active channel back to the original position Save values for other uses If this option is selected the values of the current shift settings will be loaded automatically when the command is executed the next time and can simply be confirmed with OK 157 158 Chapter 8 Image Processing OLYMPUS 8 5 Thresholds and Binarization 8 5 1 Set Thresholds This command 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 O 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 Separate command see Chapter 7 6 Separate 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 im
287. le bar 107 Shading correction 126 Shift correction 157 Chapter 16 Installing the cell M cell R Software 365 Snapshot 12 Software installation 351 Spectral unmixing 212 Calibration 217 Unmixing 218 Status bar 298 Thresholds 158 Viewport 10 76 Viewport Manager 10 297 Window 295 ZDC 57
288. lick on this button to insert the name of the selected symbol into the active text docu ment This name will be written into the text where the cursor is 14 12 Find Symbol Use this command to access the complete name of a symbol in the active text document Available This command is available when a text document is open and active You may also use the lt Ctrl F12 gt keys What s it for This command is for use when you re programming in Imaging C and you don t know the precise name of a symbol or the symbol s name is extremely long What you would do in this case would be to e g enter the first few letters of the symbol name you re looking for and then use the lt Ctrl F12 gt keys A symbol name with these letters at its beginning will be written into the text document If the symbol name written into your text is not the one you were looking for use the above mentioned keys once more lt Ctrl F12 gt Then the next symbol name with these letters at its beginning will be written into the text document If you still haven t found what you re looking for simply continue to use the above mentioned key combination until you do find what 322 Chapter 14 Imaging C OLYMPUS you were seeking You may want to try using other letters if you still cannot locate the desired symbol name Where are symbol names searched for Firstly symbols are searched for among the symbols defined in the interpreter mode After that the
289. licking with the mouse into the frame can entirely mark the first part Then it can be copied and pasted lt Ctl c gt and lt Ctrl v gt Another mouse click places the new frame in the desired position Af terwards only the connection between the TTL Out OFF icon and the new frame has to be drawn and the two image acquisition commands have to be connected as well the connection is lost upon copy and paste The result of this experiment would be two dual color time sequences one resulting from the first loop the other from the second loop The sequences can afterwards be combined into one dual color time sequence with the Combine command using the Combine button or Image gt Combine 5 3 3 8 Experiments with image acquisitions and different stage positions If the cell4R cell4M imaging station contains a motorized stage it is possible to automatically exe cute experiments at different stage positions that have to be defined in a positions list beforehand see Chapter 4 5 1 Defining a Positions List An example would be to acquire z stacks with two different Image Types at each stage position the Stage frame has to be drawn around the Z stack frame and this has to be repeated several times the Time Loop frame has to be the outmost one The Experiment Plan would look like shown below Within the same experiment folder in the database individual dual color 3 D time lapse series will be stored for each stage positio
290. ll s cell software Manual Chapter 6 Image Display and Navigation 95 Animate An additional feature enables the user to animate the acquired image sequence Pressing the Ani mate button opens the Animate Image Stack window Animate Image Stack lalol Reverse Stop and Play Here you find the buttons to start Play the animation to Stop it and to play it in the Reverse mode tea aa o gt poi First Frame Previous Frame Next Frame and Last Frame Alternatively you can navigate frame by frame through the image stack or go directly to the first and the last frame respectively EE Mark In and Mark Out The slider displayed at the bottom of the window indicates the position of the currently displayed frame within the sequence Furthermore the Animation tool gives you the possibility to mark a subset of frames within the image stack to be animated To do so move the slider to the desired starting frame position and click on the Mark In button Then move the slider to a subsequent frame and click on the Mark Out button to set the end frame for the slide show The selected frame scan range will be marked in the scale bar underneath the slider Options The parameters for the animated slide show are defined in the Options of the Animate Image Stack window To open this dialogue press the Options button Here you can set the parameters which define how the image stack is animated Frame rate In the field you can ad
291. ll crosshair or a Large crosshair which covers the entire image Primary Determine the temporary color for example of a ROI while drawing it After terminating the action the color switches to the designated permanent color 283 284 Chapter 13 The Special Menu and the Window Menu OLYMPUS Auxiliary Determine the temporary secondary color for example of the distance to be measured After terminating the action the color switches to the designated permanent color Navigator Determine the color of the frame indicating the current image cutout in the Viewport manager General Show function names amp arguments in status bar If selected function name and arguments of a command will be shown in the status bar lower right corner of the cell4R cell4M window in addi tion to a brief description To view the respective status bar information simply select the com mand of interest To see the information about buttons in the tool bar simply position the mouse cursor on the button of interest Startup with user dependent settings If selected on startup your own individual workspace will be displayed If this check box has already been selected clear the check box and then select it once more to enable startup with your own workspace To load the general workspace for all users clear this check box Allow tiling and cascading of image window If selected the image document will be aligned as well when you have all document
292. lumination system MT20 MT10 and any excitation filter Shutter Image types based on the usage of the illumination system MT20 MT10 automatically result in the opening of the shutter before the image acquisition and its closing immediately after wards The MT Shutter will be set by default in the Shutter field in these cases However if the transmission light path of the microscope is for example equipped with a shutter it has to be se lected from the pick list here cell s cell software Manual Chapter 15 cell4M cell4R Configuration 341 I OBS System Configuration Illumination System Image Type Illumination Shutter Filter Cube Observation Fluorescence General Excitation Filter Emission Filter Color ion Fiker Dapi 403 DAPI s MT Shutter DapiFiteTxRed empty vii 2 ix Burner a E i p To i zi a j Full Control Fitc 492 FITC MT Shutter w DapiFitcTxRed w empty m amp ni Errors ay as rae ea 7 Microscope TxRed 572 TxRed MT shutter v DapiFitcTxRed w empty vi 2 Tl General Z Drive Indo405 Indo MT Shutter w m T Objectives Space Indo465 Indo mr Shutter l Indo485 v es l r Miia tunes TRFGFP free mer iv tiRrere ww llemty a ee TIRFCyS free v TIRF Cy5 w empty Pifoc Stage Trans Transmission Mic Shutter Trar bi empty is empty E T v
293. mand icon Use for interrupting the experiment for a specified time before execution of the next command cell4R cell4M is a real time system and thus the timer elapsed remaining time continues to count during the pause Properties Delay time This parameter sets the delay before the execution of the subsequent command 5 3 2 10 The Autofocus command AF Autofocus command icon The color indicates the Image Type to be used the exposure time is displayed in the little box on the right Use to automatically find the focus during an experiment The Autofocus is a very useful feature in many experiments especially in those where a thermal focus shifts are to be expected over time and b a motorized stage is used to image the sample at different stage positions In the latter case it cannot be expected that the focal position will be the same over the entire sample For general explanations of the Autofocus see Chapter 4 6 Autofocus As explained there the initial Autofocus scan will always be followed by a second finer one Properties Autofocus Z device If more than one Z device is installed select the one to be used for the Autofocus from the shortlist Range Set here the range of Z positions to be scanned during the Autofocus process The range is centered on the current Z position or in case the Experiment Plan features a Stage loop the Z position defined in the positions list The narrower the range t
294. mand when the Status Bar is On 13 6 7 Command Window Use this command to open the Command text window or to activate it You may also simply use the short cut lt Alt F2 gt Application This command will open a text window for writing Imaging C and Macro functions Subsequently these functions can be executed directly Furthermore the window can be used as a calculator Simply preface any mathematical task with a question mark The result will appear in the next line of the Command Window Example Entering 5 0og 43 5 100 will yield the result 178 863804690473 Editing Command Lines Executing Function A greater than gt sign at the left border indicates the beginning of a command line You can only edit the last current command line Any lines pre ceding this last one can no longer be edited Anything you ve got in the clipboard can also only be pasted into the last current command line Command lines are ended with a semicolon Press En ter to have the function of the current line executed cell4R cell4M will execute that function im mediately The result s of any calculation will then be written in the next line of the Command Window You will receive an error message e g if you have made a syntax error writing the Imag ing C function if the function has too few parameters or if the semicolon is missing at the end Pasting Preceding Command Lines Preceding command lines can be neither edited nor exe cuted
295. me If you select the Monochrome check box the polygon will become yellow All changes will be applied to all three colors simultaneously thus enabling you to define a mono chrome gray value LUT File Click on the File button to open the standard dialog box for the opening and loading of files Files will be saved using the LUP format Linear This function sets the polygon of the active color back to linear 0 255 O The LUP format cannot be used by Imaging C functions to alter the lookup table of an image As a consequence you should save the LUP type palettes as LUT files as well see above Additionally only the LUT files but not the LUPs will be listed in the False Color dialog box see above 6 2 10 3 The Formula tab Red Green Blue check box If the check box on the left side is not selected the corresponding LUT will be given a constant value of 0 Otherwise enter the formula which defines the function of the corresponding palette in the fields Red Green and Blue Edit LUT Sheet Polygon Formula Linear Red 255 pow n 3333 pow 255 33 Linit Green nl Limit Blue 255 pow n 3 powf255 3 Lirnit Writing a Formula 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 O and 255 and the result displayed in the dia gram Simultaneously the image is continuously updated on the monitor using this palette Yo
296. ment displays via selection from the pick list any other open document for example the data base or the graph window E E B g i E Show additional components displays via selection from the pick list the Image Manager or Viewport Manager FY ly 6 2 Image Display 6 2 1 General The commands in the Image gt Image Display menu are image processing functions that do not alter the image data Only the appearance of an image on the screen the image s display will be changed while the original image parameters remain untouched in the source image buffer 6 2 2 Adjust Display Display Intensity Use this command to adjust the onscreen display of image sets without changing the data General A 16 bit gray value image consists of gray values ranging from O to 65535 A nx16 bit image sets has potentially the same amount of intensity levels in each and every color channel cell s cell software Manual Chapter 6 Image Display and Navigation 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 Color monitors generate colors by mixing the three existing color channels red green and blue with different intensities Each channel is able to pro vide 256 levels of intensity 8 bit each The result is a 8x8 bit image composed of more than 16 million possible colors This is the so called true color forma
297. ms such as crashes for example Keep file type in file input output dialogs Select this check box to have the file type you last used offered to you the next time you save or open a file This check box affects the standard Win dows dialog box for opening and saving files in the File menu Keep in mind that the file type of fered to you will depend on the document you wish to save or open Examples If you have saved for example your latest new sheet document in the xls format the next time you wish to save a new sheet the x s format will be offered to you Say that the last time you opened a text document you used the All format the next time you wish to open a text document you ll be shown all files of the currently set directory Preselect file type All Formats in Open dialog If this box is checked all files of all formats will be shown in the File Open dialog Otherwise only the files of the most recently chosen format will be shown cell cell software Manual Chapter 13 The Special Menu and the Window Menu 289 Number of entries in recent file list Here you can select how many of the recently used files are displayed in the file list within the File and Database menu 13 5 4 The Preferences gt Measure Tab The settings in this tab are mainly concerned with interactive measurement functions of the Meas urements toolbar Preferences Image Wiew File Measure Module Graph Database Repo
298. n Both the positions list of the stage and the Z stack properties see Chapter 5 2 3 2 The Z Stack Frame contain parameters for the Z positioning of the system In order to avoid conflicts in an experiment like the one above the absolute position information for the top and bottom position in the Z stack properties will be ignored The relevant information is the Height of the Z stack as defined in the Z stack properties The center position of the Z stack of this height to be acquired at each stage position is the Z value set for each position in the positions list 710 50 um 20 x 30 00 min 67 68 Chapter 5 Experiment Manager OLYMPUS A Stage frame can also be drawn around a Time Loop frame In that case a time lapse sequence is first acquired entirely at the first stage position then the stage moves to the second position where another time lapse sequence is acquired and so on It is also possible that a Time Loop frame contains a Stage frame that again contains another inner Time Loop frame For each stage position the result will be a time lapse sequence that contains blocks of equidistant images These blocks result from the inner Time Loop frame and are separated by the time lapse resulting from one loop through all stage positions 5 3 3 9 Experiments with autofocus Long lasting experiments always carry the risk of focal shifts over time It is not uncommon that say after a few hours the specimen is out of focu
299. n Experiments with an Observation Filter Wheel In case an observation filter wheel is used to monitor the two channels a standard dual color im age acquisition Experiment Plan has to be setup see Chapter 5 3 3 4 Time lapse experiments monochromatic or in multiple colors Again it is possible to calculate online a ratio image sequence out of the overlay or a kinetic of in tensity changes see Chapter 5 3 2 6 Online ratio image Chapter 5 3 3 6 Experiments with online analyses and Chapter 10 8 Ratio Analysis An Experiment Plan would look like this for example 50 x 500 00 ms 225 226 Chapter 10 Intensity Analyses OLYMPUS 10 12 2 FRET Image Correction Factors FRET Correction The analysis of FRET image series becomes complicated by signal bleed through or excitation cross talk that is common for typical donor acceptor pairs The excitation characteristics of the fluorophores usually cause a certain direct excitation of the acceptor when the donor excitation filter is used and vice versa This excitation is not caused by energy transfer from one fluorophore to the other and has to be corrected for before a meaningful quantitative analysis is possible Two reference samples are necessary one labeled exclusively with the donor and one exclusively with the acceptor They are used to determine the channel bleed through The practical approach is somewhat similar to that used for spectral unmixing see Chapter 10 9 Spectr
300. n activated Use for the online quantification of fluorescence intensity changes within ROIs over time and their display as a graph See also Chapter 10 6 Intensity Kinetics in Time and Z cell s cell software Manual Chapter 5 Experiment Manager 53 Properties Kinetic Properties Kinetic Store Use ROls From Image Stack layer Stage Position Use ROIs From Image The ROIs have to be drawn into a snapshot or any image of the same XY dimension as the image to be acquired before the start of the experiment Choose this snapshot from the list in this box Z Stack layer In a 3 D Time Lapse series only one Z layer can be chosen for the online calculation of a kinetic Set this layer here Stage Position An online kinetics analysis is possible only for one of the stage positions in an experiment to be carried out at different positions Set this position here Properties Store If the option Store is marked this is indicated by the storage symbol in the top right corner of the icon the kinetics graph that is being calculated online will be stored on the hard disk after the experiment has been finished otherwise it will be trashed O The display of the online kinetics is automatic and does not need to be activated as in case of the online ratio image calculation It is possible to analyze several image sets in one experiment in other words to connect Online Kinetics commands to several Im age Acquisition comm
301. n information Select an entry in the list of add ins to view a brief description of that pro gram It will be displayed in the lower field of the dialog box Close Click on the Close button to close the dialog box and to save any alterations you ve made Add Click on the Add button to insert an add in to the list of available add ins The standard dialog box for loading files will be opened The file types available to you are the following add in formats SXU and DLX Generally you ll only need to insert add ins in two cases either you had deleted existent add ins or an additional add in has been purchased Remove Click on the Remove button to remove an entry from the list of available add ins This button only removes the entry from the dialog box The add in file will not be deleted Any add in you ve removed can thus be re added at any time However you need to know the corresponding sxu or dIx file name 13 3 Define Menu Bar Use this command to alter the configuration of the menu bar or context menu Define Menu Bar E ds onmands OK RACIUTE Cancel Snapshot amp Camera Control Camera Ckonfiguratior AboutModule Acquire Acquirel mage Set amp lnput ubohocus Autofocus Options lidlurnination Settings Show Main Menu Down Description Acquisition AddDicomlntoT oBut Addi nll anager AddT oModule Adjust BBitDisplay AdjustDisplay AdjustDisplayaubo AdjustImnageWi
302. n one sheet Generate statistics of the sheet s This function is currently disabled One sheet per image Select this option if the results of several images are to be combined in one sheet O 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 for details see Chapter 7 1 Calibrate Image Editing sheets Several editing functions are available in the Sheet Context Menu that can be opened by right clicking on a sheet 189 190 Chapter 9 Measurements OLYMPUS Cu Ctrl Copy Ctrl C Delete Delete Edit Column Header Sort Ascending Sort Descending Create Copy Autofilter Create Graph Define Statistics Statistics Define Context Menu 9 4 3 Deleting Measurement Results kos Delete Measurement from Image Use this command to delete individual objects and their measurements interactively 1 Click the Delete Measurement from Image button and select the object to be them in the image overlay 2 Delete another object or terminate the function via right click or lt Esc gt O Selecting them in the measurement display and pressing the lt Del gt key can also delete objects and their measurements x Delete Measurement button Use this command to delete the entire measurement results of the active image cell s cell software Manual Chapter 9 Measurement
303. n seven entries are listed To add a new field it would be necessary to remove another entry first Choose View Use the command Choose View in the Database gt View menu or the context menu the pick list that appears upon a right click on the database window to open the Choose View dialog window Choose View Table View Gallery View Horizontal Gallery View Narrow view C Structure Strip Gallery Strip Six different views are available for the database window By default the database window opens with the Full View gt Gallery View the one shown in the previous chapter Two other alternative displays for the Full View can be selected cell s cell software Manual Chapter 12 Database In the Table View the thumbnails of the Gallery is converted into a table with icons instead of thumbnails Additionally it lists the cryptic default names under which the files are stored and appear in disk management programs see Chapter 12 4 1 General Remarks When the Horizontal Gallery View is selected the gallery is displayed as a scrollable single row of thumbnails Thus more space remains for the Form view In the Narrow View the Form view is removed and the Gallery displayed as a single column The database window is attached to the Image Manager and Viewport Manager windows but can be moved freely at will Full View Narrow View The Full View and Narrow View buttons allow toggling between th
304. n the X di rection cell a cell software Manual Chapter 11 Graph Display and Graph Analysis Zoom Out Click the Zoom Out button to increase the displayed X range i e to compress the graph in the X direction Scale X Click the Scale X button to enlarge any X segment of the displayed graph to full axis size Mark the left and the right margins by mouse click The first click generates a blue vertical line the second a green one Both can be moved via mouse drag While doing so the current X position of the line is continuously shown on the status bar Right click to accept the new scale The two margins remain visible as long as the button remains pressed once the button is depressed they are deleted 11 2 3 2 Y Scale Zoom Up Click the Zoom Up button to decrease the displayed Y range i e to stretch the graph in the Y direction Zoom Down Click the Zoom Down button to increase the displayed Y range i e to compress the graph in the Y direction Scale Y Click the Scale Y button to enlarge any X segment of the displayed graph to full axis size Mark the left and the right margins by mouse click The first click generates a blue vertical line the second a green one Both can be moved via mouse drag While doing so the current X position of the line is continuously shown on the status bar Right click to accept the new scale The two margins remain visible as long as the button remains pressed once the button is dep
305. nd Y coordinate of the top left corner and the width W and the height H However it is much more convenient to define the ROI in the Camera Control dialog box and read it in via Get Current Camera Settings in the Properties gt Image page Properties Display Display If this option is selected an on line image will be displayed and constantly updated on the Viewport during the experiment for example a time lapse or a Z stack acquisition In this case the Image Acquisition icon shows a monitor symbol el Fite Image Acquisition with on line display If this option is deselected no images are displayed on the Viewport during the experiment The following controls are not required in this case and are disabled 45 46 Chapter 5 Experiment Manager OLYMPUS On line display is only possible in time lapse or Z stack experiments If the option is activated in single image experiments an error message is generated prior to the ex periment execution Brightness This mapping function is mainly used to adjust the brightness of the image display on the computer monitor Note that mapping does not change the image data You have two options to define which mapping applies to your online image display e Automap The mapping range min to max values is automatically determined according to the minimal and maximal intensity values of the acquired image e Map fixed from to Predefine the mapping range min to max values manu
306. nd of the calibration structure with the second line cursor and click again 11 Click on OK to confirm the calibration 7 1 4 Z Calibration Calibrate Image ser Calibration Z Calibration Calibration of the plane Scale 0 108 Scale Y 0 108 Calibration of the range Frames 2 2 0ffzet s6000 um 2 5 pacing ois E um Z Offset This parameter refers to the absolute position of the lowest layer Z stack and is of minor importance It is read in automatically when loading cell4R cell M data cell s cell software Manual Chapter 7 Image Data Handling 107 Z Spacing This is the step width between two neighboring layers of a stack It is read in automati cally when loading cell4R cell4M data Unit This function opens the same Set Unit dialog box as in XY Calibration tab 7 2 Scale Bar Image Process Measure Graph Special Window F Image Displa Tu o J a a3 C Set Magnification Scale Bar Properties w Show in viewport Shift F4 Draw into Overlay Overlay Bar Show Markers 7 2 1 General In all scientific reports the calibration of an image must be specified which can be done with this command This tool is useful for making video prints hard copies or exporting images to word processing applications because the inherent calibration is visualized in the image It may be nec essary to choose a different location or a specific length for the scale bar Both can be defined in the S
307. ndaw Adjust mage lt oom Align AplActyate window Apl amp ddRecornd Apt ddMFields AplArchivel ven Aesults AplArchiving AplarrangeF ields AplChangePassword Starts continuous acquisition using active input device C Insert sub menu Command groups All Groups Macros MAFI Configuration Programs w All C Modules W Internal Functions cell cell software Manual Chapter 13 The Special Menu and the Window Menu Any alterations you make to menu definitions via the Define Menu Bar command and integrate into the cell4R cell M Graphical User Interface have to be saved in a user configuration Use the Special gt Configuration gt Save command to save the configuration Explanation Menus are organized hierarchically Main menu entries contain commands and sub menus An arrowhead to the right of an entry denotes a submenu Submenus contain further com mands Three dots behind a command indicate that a dialog box will open Menu The contents in the Menu list can be toggled with the Show Main Menu and Show Sub Menu buttons As soon as you select an entry in this list that entry will appear in the field above the list This entry is the menu element ready for editing You can e g alter a name in the list of all main menu entries Commands can be removed from or submenus can be added to the list of all menu commands Show Sub Menu Click on the Show Sub Menu button to move down a level in the menu hier
308. ne 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 O 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 you close the dia log 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
309. nels 340 nm and 380 nm is set in the respective boxes Value 1 and 212 Chapter 10 Intensity Analyses OLYMPUS Value 2 ROI The typical approach to background subtraction is to mark a ROI in an image area that contains only background intensity and use the average intensity in this ROI as background value This value is determined individually in each image of each channel The last option is to subtract a predetermined background image from each of the images of the data set Kinetic field This field is only active if the Output option Kinetics has been marked Here you have to select from which data set the ROIs shall be taken and for which the analysis shall be performed If the option Highlight selected ROIs is activated those ROIs are shown with bold lines If Sheet is activated in addition to the graph also the corresponding spreadsheet is generated Thresholds field The thresholds for the two bands can be set in the respective boxes For any pixel with an intensity value below the threshold the ratio value will be set to 0 The thresholding is performed to prevent low signal areas in the original channels from resulting in areas of pro nounced noise in the ratio sequence the reason being the division of small values by other small values Output Scaling field Digital images displayed on computer screens are based on integer numbers representing intensities Division of two of such integers generates so called floating point num ber
310. ng systems in this context e MSWindows 2000 requires administrator user rights for cell4R cell4M to function properly e Standard user accounts can be installed in case of MSWindows XP How to run cell R cell4M in a normal user account under MSWindows 2000 1 Log on as Administrator 2 Create a new user using Settings gt Control Panel Users and passwords from the Start menu 3 Select Add to open the assistant for editing a new user Fill the required fields In the Level of Access dialogue you have to select Administrator from the Other pick list 4 Check that the newly created user has permission to write into the Archive directory where the databases are created when working with cell4R cell4M To check the secu rity of a directory file the Security tab in the Properties dialog to be opened via right click has to be made visible To check the security settings of the Archive use the Windows Explorer and do the fol lowing Select the Archive directory Right click and select File gt Properties Select the Security tab Click Add select the group or the name from the list Group or user names and exit with OK Check that the permissions for the user include Modify Read amp Execute List Folder Contents Read and Write ao oO D 358 Chapter 16 Installing the cell M cell4R Software OLYMPUS 5 Check that the Environment variables for the user are set to D Temp by doing the fol lowing
311. ng the Autofocus process in the Autofocus normal mode see below The range is centered on the current Z position The nar rower the range the shorter will be the time required for the Autofocus scan to finish However the focus cannot be found if it is outside the range Thus certain cautiousness is necessary when set ting the range Autofocus Options Range 33 33 Big Step 4 1 Fine Step 1 00 Use entire range L Display d Use ROI Low Signal mode Big Step Set here the step width to be made between neighboring Z positions during the first of the two Autofocus scans Whenever the Range is newly set a default step size is being set in dependence of it Fine Step Set here the step width to be made between neighboring Z positions during the second of the two Autofocus scans Whenever the Range is newly set a default step size is being set in dependence of it 35 36 Chapter 4 Image Acquisition and Hardware Control OLYMPUS Use entire range If this box is NOT checked the following will be done if the focality initially in creases during an Autofocus scan and then worsens again the process will be terminated and the found focality maximum will be set as reference position for the second finer scan If the box is checked however the process will not be terminated after a local focality maximum is determined but the entire range will be examined Obviously this causes the Autofocus scan to last longer but
312. ng the Record Macro command the Recorder button bar will appear e This menu command s function will be changed to Stop Macro Recorder e cell4R cell4M will generally open a new text document at this stage If a text document is al ready open you can append the commands you re recording at the end of this text To do so activate the text document before activating the Execute Macro command e All operations you execute within cell4R cell4M will now be recorded in this text document Each step will result in one or more functions being recorded in the text document If you d like you can have the image buffer selection in the Image Manager recorded this has to be preset in the Special gt Preferences Module tab e Stop the recording with the Stop Macro button Saving macros Use the Save command in the File menu to save the recorded macro Now you will be able to re load modify or add this macro in cell4R cellAM 13 1 3 Executing Macros There are a variety of ways to execute macros e Activate the text document containing the macro Start up the macro with lt F5 gt e Activate the text via the macro Use the Special Recorder gt Set as Default Macro command in the menu see below Now the macro can be executed anytime pressing lt F5 gt even if the text document isn t active e Select the Special gt Define Macros command to integrate the macro into a particular con figuration as bu
313. nged by mouse drag or by clicking into the field the curve will be fitted to reach the point that was clicked Adjust Display Adjust Display Detail Mapping Oooo 4 2 Linear Click here to get back to a linear scaling from Min to Max Reset Click here to get back to the scaling that was active before the Adjust Display window was opened 81 82 Chapter 6 Image Display and Navigation OLYMPUS 6 2 3 Auto Adjust Display Auto Adjust Display This is a quick way to adjust the channel intensities of multi dimensional image sets The entire data set not just the current image is checked for the brightest and the dimmest pixel and their intensity is used to set the minimum and maximum values of the display Additionally the current clipping settings in the Adjust Display dialog are considered 6 2 4 White Balance Many cameras tend to adulterate falsify image colors at acquisition This kind of color displace ment can be corrected retroactively To do so you need to have an image that has an area where you know it should be white Instead it looks for example yellowish This yellowish tinge the color tinge can be corrected throughout the whole image Available This function is only available for single images of the RGB format and not for n x 16 bit images acquired with the Experiment Manager In microscopy it is only useful for transmission images acquired with a color camera O This command d
314. nly exists on screen No new file is written into the database To create an overlaid image as new data set use Edit Copy and Edit Paste lt Ctrl c gt and lt Ctrl p gt and store the new image This image is a 3x8 bit RGB image not a nx16 bit image 100 Chapter 6 Image Display and Navigation OLYMPUS 6 6 Intensity Modulated Display Images sets that are created by pixel by pixel division of source images or color channels most significantly those resulting from Delta F F or Ratio calculations Chapter 10 7 Delta F F AF F Analysis and 10 8 Ratio Analysis have one disadvantage they do not reveal if a certain ratio re sults from an area of originally rather intense or rather dim fluorescence The ratio of two small numbers may after all be similar to that of two large numbers This effect often causes a loss of structural information in the ratio images To give an example the Intensity Modulated Display allows modulating the intensity of a false color ratio image middle image below may be one with a Rainbow palette Chapter 6 2 9 False Color with the brightness information of the original image left image below The resulting in tensity modulated ratio image is on the right and has an appearance that is much more similar to that of the original image than the standard ratio image cell amp cell Software Manual l If the system is configured thoroughly all images will have the correct size
315. nt Manger are automatically displayed in the color defined with the Image Type in the cell R cell M Configuration Software ObsConfig exe see Chapter 15 4 Definition of Image Types Snapshots however are displayed with a gray palette Use the Edit Fluorescence Color command to modify the color of snapshots or of individual color bands of multi color images Edit Fluorescence Color we p Saturation 1000 Brightness 1000 Hue This parameter sets the color tone spectral color Wavelength Instead of changing the Hue you may type in the desired wavelength Saturation Reducing this parameter adds whiteness to the color In the RGB color space this corresponds to linearly increased intensity in the non saturated channels Brightness Reducing this parameter adds black to the color In the RGB color space this corre sponds to linearly decreased intensity in all channels cell s cell software Manual Chapter 6 Image Display and Navigation 85 6 2 9 False Color Al False Color button Use this command or Image gt Image Display Load LUT to select a color palette from the list in the dialog box RGB Lookup Tables of Color Channel lt gt The library of palette files provided with cell4R cell4M is stored in Cell4R M LUT Available This command is available for all image types however the false color palette will only be used if a single color channel of the image is being displayed Fal
316. ntensity Analyses OLYMPUS see a relatively strong but undesired fluorescence of the nucleus upon YFP excitation This phe nomenon Known as bleed through strongly reduces color resolution and constrains scientific conclusions 10 9 3 The Solution cell4R cell4M is the first imaging system to implement Spectral Imaging and Linear Unmixing a technique adapted from satellite imaging into widefield fluorescence microscopy With this tech nique it is possible to separate and resort the contribution of different fluorochromes to the total signal in each color channel Figure 2 Images of a GFP YFP double labeled sample GFP fused with H2B histone protein and YFP with tubulin taken with YFP dichroic and emitter Left GFP exciter right YFP exciter cell a cell software Manual Chapter 10 Intensity Analyses 10 9 4 How Does it Work The principles of the method can easily be explained considering the example shown in Figure 2 To unmix the spectral information of the fluorochromes with strongly overlapping emission spec tra it is necessary to determine the spectral properties of the individual fluorochromes under the same imaging conditions used for the multi labeled samples the system has to be calibrated for each fluorochrome This is performed by taking reference images of single labeled samples with these fluorochromes using the same filter set excitation filters dichroic mirror and emission filter as for the la
317. ny other com mand because they will not be synchronized 59 60 Chapter 5 Experiment Manager OLYMPUS 5 3 3 Types of Experiments 5 3 3 1 Single images monochromatic or in multiple colors Dal Monochromatic single image The Image Manager indicates a single monochromatic image by a black amp white cell symbol The simplest possible Experiment Plan consists of a single Image Acquisition icon Obviously the result would be a single image file taken with a certain excitation filter intensity and exposure time the camera exposure being synchronized with the MT20 MT10 shutter Of course with cell4R cell4M it is easy to acquire images subsequently with different excitation illumination settings exposure times and even different ROIs and binning factors Such an Experi ment Plan would consist of a series of Image Acquisition icons connected with arrows to indicate the order see the example below The result would be three monochromatic image files respec tively named Dapi Fitc and TxRed E 7 3 I Dapi Fitc TxRed g O This simple Experiment Plan of three icons codes for a quite long list of commands and actions They shall be listed here as an example cell R is able to execute some ac tions in parallel to minimize idle times This is not possible in cell M here the actions are executed consecutively The train of commands is based on the usage of a triple band fluorescence filter cube that is already in po
318. ny time So you can be sure to get the true data if you compute a histogram or if you measure the pixel values of your image even though you have overlay elements present Overlays are vector graphics Images are stored in a bitmap like format that contains information about each individual 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 Image and Symbol These are bitmaps added to the overlay 7 5 2 Activating the Overlay Toolbar Activate the toolbar by selection in the pick list that appears when right clicking on any button bar or via Image gt Overlay Toolbar 112 Chapter 7 Image Data Handling OLYMPUS 7 5 3 Creating and Editing Overlays KS Edit Overlay This button allows you to select an existing object in an overlay Upon clicking the button the cur sor automatically moves into the image and is confined to this area until a right mouse click termi nates the Edit function Any object active at this point can be edited with the overlay tools Clicking into an empty area of the overlay while being in the Edit mode deactivates any active object Once active you can change size and shape of objects by mouse drag To select more than one object use the shift button while clicking ir Sele
319. o Image Types with the different filter combinations be defined in the ObsConfig software An example is shown below see also Chapter 15 3 Definition of Image Types and Chapter 15 2 6 Configuration of the Dual View Micro Imager Image Type Illumination Shutter Filter Cube Observation Fluorescence Excitation Filter Emission Filter Color Fdon amp Ffret CFP split 430 CFP y CFP FP Image Splitter ae EP split 500 YFP J CFPYFP Image Splitter w As usually it is possible to calculate online kinetics and ratios of one or both images see Chapter 5 3 2 6 Online ratio image Chapter 5 3 3 6 Experiments with online analyses and Chapter 10 8 Ratio Analysis An Experiment Plan would look like this for example T EJ 4 CFP split 40 i Kinetic YFF split 40 100 x 1 00 s It is not possible to combine two split images with a multi color frame with the intention to generate a four channel image cell cell software Manual Chapter 10 Intensity Analyses The image acquired with the acceptor exciter 500 YFP in the example contains a channel that is without use for the analysis the short wavelength channel with the acceptor fluorescence blue channel of the second image in the example The three relevant channels in the FRET Analysis window have to be selected as follows Fdon the short wavelength channel of the donor exciter image blue channel of the first image in the example Facc
320. o obtain an overview of the symbols defined in Imaging C You may also use the lt Alt F12 gt keys The Browser dialog box is non modal i e you can continue working with other commands with out having to close this dialog box Query Dp Foin O v Range Exported by Tool ka Result Description Type Defined ir _ The Query group is where you enter the symbol you re looking for Select the type of symbol you re looking for from the Type list e g Function Internal Function Object Function etc Enter the name of the symbol you re looking for into the Symbol field You can use wild cards here Select where you are to find the desired symbol from the Range list e g Exported by Loaded Modules This list will then disappear and replaced by the German function names check box if you ve selected the Internal Function type of symbol Query Definition A Symbol can be a function a structure a union an object a variable a dialog or a constant Type The Type list contains various types of symbols Internal Function Function Structure Type Union Object Type Variable Dialog Constant and All Symbol Enter the name of the symbol you re looking for into the Symbol field If you only know a part of the symbol s name you can substitute an asterisk for unknown text and for unknown cell a cell software
321. ocument can be deleted by pressing the Del key 11 3 3 Graph Information 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 How to open the Graph Information dialog box e Double click on any graph buffer within the Image Manager to view information on that graph e lt Alt Return gt keys use these keys to view information on the active graph e Image Manager context menu right click on any graph buffer to open the corresponding con text menu and then click on Graph Information to see the information of this particular graph without making it the active one displaying it e Graph Document context menu right click on the graph window to open the corresponding context menu and then click on Graph Information 11 3 3 1 The General tab This tab contains general Graph information Title displays the title of the graph document The title can be changed It can be up to 39 charac ters long Date displays the date the graph was created Time displays the time the graph was created Comment Use this field to add any comments concerning the graph The comment can be up to 114 characters long 245 246 Chapter 11 Graph Display and Graph Analysis OLYMPUS X axis and Y axis Displays the title legend and the unit of the X and Y axis respectively The parameters can be changed The changes will be displayed in the graph document All
322. ocument to the active module e g another source text What will happen If the document has already been saved as a file the file will be inserted into the list of C module files If not the Save Text as dialog box will be opened where you can enter a name for the file If the file is already in a module a message informing you of this will be displayed 14 5 Save Module Configuration Use Special gt Imaging C Save Module Configuration to save the current configuration of the Graphical User Interface GUI in the active C module Available This command is only available e when aC module is active e if there is a MKU make file for this module and if e the current configuration contains a MAPI function of the active module Before you use this command activate the module in question in the module manager Then you can check and see whether the module has a MKU make file using the Open button in the Open Module dialog box Configuration file The configuration of the Graphical User Interface GUI will be saved in the SCX configuration file of the C module 312 Chapter 14 Imaging C OLYMPUS Save Shared Configuration Keep the lt Shift gt key depressed while you call up the menu This command will then turn into the Save Shared Configuration command The information on which MAPI functions of other modules will be included in the current Graphical User Interface GUI con figuration will be saved in the SCX file of t
323. oe eR ra ete et Pe DRE Ne eee ete 136 IAC cA E E E A EE AT E A EEE TS 136 MEAN seoir r a a a a Ea 137 Vedi aNs na a aa aGaers 137 Pseudo Fe aran a a E aE 138 OG R E E E 139 RODOM S a ens r E O E atieiaoee eS 139 REIME cxsbsinecdovsise doves netavscende a R 140 Ser FO ossessi e tense ates cin Radel cane ts lic snd ren alee a 141 NAN occas tect eases a e E ee emnceencecantae 142 WOW DAS Sein cacesttntonetantae a hee ie renent coe cence sacar 143 EQO ela are g O iuris aa a Were nner metros ten near Ome ihre ter rere tee 143 PRA Kc eee E 144 Chapter 8 Image Processing 125 126 Chapter 8 Image Processing OLYMPUS 8 2 20 DCE Differential Contrast Enhancement c ccceseeceeeeeeeeeees 146 922l Sepalo oon Sadat stes rs tenie aaa 148 8 3 ArithimeliG ODGratlOnS sv ceuctedeececsac deeds ei odeeednceces iadenetectiensdiadiee tase 151 GAs mage GSOMeMy icc esl cnet eee acoseatenniaaensnd ad scaebuoeel teal 152 8 4 1 PRO SIZ pepe e ccatenctauseensencetealiacueenee cette teceacenccanchens couenenes eueatecentes 152 EZ OU AUC wicca ec a cae ec nde a nce eee cdc ts Seance een 154 Bie WNIT OR eana a a a a a a ateans 155 84A Algi E ai 156 8 459 AUtOAlON Z roaie e EEE EEEE TEE EE REE ER 156 840 colli GORTECUGM n a a r r r ceed citrinete 157 8 5 Thresholds and BinariZation cccccceceeeceeeeeeeeceeeceeeveveueueeeeeeeeeenes 158 851 SetThresholds aissei aa EA EEEa 158 85 2 S6LCOlor inresholdSanorne aaa detect eeteeauec
324. oes not affect the image data it only alters the on screen display To apply the white balance a circular image area has to be defined where it is not that the pixels should be white black or gray but at present are tinged Afterwards three correction factors will be calculated based on the pixels within this 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 Using these correction factors the whole image will be corrected This is the procedure 1 Select Image gt Image Display gt White Balance 2 Left click within the image A red circle will appear in the overlay 3 Position this circle with the mouse To resize it keep the left mouse button pressed as you move the mouse cell s cell software Manual Chapter 6 Image Display and Navigation 4 Right click to confirm the circle and apply the white balance 6 2 5 Black Balance This function is similar to the white balance That difference is that here a dark image area can be defined that should be displayed in black All other colors will then be corrected with the same correction value Available This function is only available for single images of the RGB format and not for n x 16 bit images acquired with the Experiment Manager In microscopy it is only useful for transmission images acquired with a color camera O This command does not affect the image data
325. of a sheet document being active a text document is actually active In this case it s sensible to turn this automatic activation off 299 300 Chapter 13 The Special Menu and the Window Menu OLYMPUS cell amp cell Software Manual 14 Imaging C Chapter 14 Imaging C 301 cell4R cell4M has its own macro or programming language Imaging C It is a variation of the ANSI C standard which has been expanded by a number of functions Why program in Imaging C Imaging C offers the possibility of automating processes you use over and over as well as expanding cell R cell M by adding new image processing functions You can program your own macros using the integrated Imaging C Interpreter This allows you to adapt im age processing functions to your particular application needs If you are experienced in using mod ern programming languages especially Standard C programming with Imaging C will not cause you any difficulties This way cell R cell4 M can serve as a development platform for developing prob lem oriented application s The new macros can be integrated into the graphical user interface and used just like any of the other functions in cell R cell4M tat Generaler e a Glad dio EE a O 302 14 2 New Module a snnesnesnneennnennennrrnerrnerrnrennrenrrenrnerrnernnernnrennnennennene 303 T43 Open Module oore o a E EE E E OE EE EE 309 144 Add to MOGUIC 5 cos csiis cates vata teiciebitctea tess ite r
326. of the icon the ratio sequence that is being calculated online will be stored on the hard disk after the experiment has been finished otherwise it will be trashed Properties Display For a description see Chapter 5 3 2 1 Image Acquisition 52 Chapter 5 Experiment Manager OLYMPUS Properties Ratio Calibration Store Display Background Subtraction Subtract a constant Subtract an image Subtract a AOI From Image Allmage 5 For Channel 1 TROI 1 For Channel 2 TROI 1 Thresholds For Channel 1 For Channel 2 Output Scaling Scaling Factor O The calculation of a ratio images and their online display is a time consuming com mand for the PC processor The software estimates the additional CPU usage and changes the minimal cycle in dependence of online analyses However in rather fast and complex experiments it still may happen that the acquisition of image pairs is faster than the calculation of the ratio images by the PC especially for 1x1 binning and full frames with online display In such a case the image acquisition timing has the highest priority and the analysis will remain incomplete That means while certain ratio images will be missing all images will be acquired as designed in the Experiment Plan and stored in the database The ratio calculation has then to be repeated offline 5 3 2 7 Online kinetics Ei E gt Hed Kinetics kinetic command icon with store optio
327. of the line Left click to activate the Rotate mode and re orientate the arrow Left click to activate the Resize mode and drag the mouse cursor with the left button clicked Adjust the length of the pink arrow as desired The width of the red rectangle de termines the range that will be averaged Repeat the rotating and resizing actions until position length and thickness are as re quired Right click to fix this line A graph with the intensity profile will be created Draw another line by continuing with step 2 or terminate the action with the Esc button cell a cell software Manual Chapter 10 Intensity Analyses 201 10 4 Regions of Interest ROIs 10 4 1 General Regions of Interest or ROIs are parts of images that are defined by the user for subsequent analy ses The ROIs do not become part of the images even if being displayed that is they do not change the data and can be removed at will 10 4 2 Drawing ROls Rol Open the Define ROIs dialog window with the ROI button or via Image gt ROI E Define ROIs B x Label Color CO DOES Active Ala Tools Label ira E 1 ROI 2ROI2 rae 3ROl 3 me Pai Label By default i e if no name is typed into the box the ROIs are labeled ROI lt consecutive number gt ROIs with identical names will likewise be numbered consecutively Labels can be de 202 Chapter 10 Intensity Analyses OLYMPUS leted or changed by clicking on the respect
328. on The NxN filter a smoothing filter is most 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 cell cell software Manual Chapter 8 Image Processing Define NxN Define NxN x Iterations 3 see CE Iterations Enter the number of times the filter is to be applied successively to the image Select an entry between 1 and 25 The preset value is 3 The greater the number of iterations the greater is the extent of the averaging Size 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 O The number of iterations and matrix size also determine the time required to complete the filter operation 8 2 16 Lowpass Application A Lowpass filter transmits space frequency in a highly unadulterated form High space frequencies generally image noise or fixed pattern camera noise will be suppressed and strong contrasts will be smoothed Low space frequencies which generally carry the actual image infor mation pass the filter 8 2 17 Edge Enhance This filter is based on the definitions set in the Processing gt Define Filter gt Edge Enhance dialog box Application Edge enhancement 143 144 Chapter 8 Image Processing OLYMPUS The term edge refer
329. on 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 work ing 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 159 160 Chapter 8 Image Processing OLYMPUS 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 1st Deriva tion 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
330. on tools work with respect to the time axis If Navigate z button is activated they work with respect to the Z axis The Select Color Channel button is always active to enable the selection of individual color channels 6 3 7 Parallel Navigation in Multiple Viewports O The data dimensions have to match for this operation For example one cannot navi gate through a Z stack and a time series simultaneously However it is not necessary that the dimensions present in the data sets have the same size in X Y Z and time It thus is possible for example to navigate through a short and a long time series at the same time up to the last frame of the short series If a multiple Viewport arrangement is selected see Chapter 6 1 The Viewport it is possible to navigate through several data sets or different color band selections of the same set in different Viewports simultaneously To do so more than one Viewport has to be active Multiple activation is achieved via lt Shift gt click 6 4 Projections and Extended Focal Imaging 6 4 1 Projections Along the Z and Time Axes Projections are a means to condense the information of an entire Z stack or time series into a sin gle image Most useful probably is the Maximum Intensity projection The algorithm checks each single image of a stack or series for the brightest intensity at any given X Y position and uses this for the projection The Minimum Intensity projection and Average Intens
331. onfig software see Chapter 15 3 Configuring the Microscope Thus the contents of the dialog window may differ from setup to setup The Microscope dialog window renders a permanent status overview of the microscope indicated by the icons of the toggle buttons and by the optical elements listed in the boxes that is updated several times per minute Thus if the user makes changes directly at the microscope the dialog box will display these changes Z drive control A slider control for adjusting the Z position is displayed together with the current position if the microscope frame features a motorized Z drive Once the slider is activated after a mouse click it can be moved with the scroll wheel of the mouse as well This slider does NOT control a piezo electric objective mover or nosepiece mover PIFOC see Chapter 4 3 Illumination Control Objective Select an objective from the pick list the nosepiece moves automatically into the corresponding position 27 28 Chapter 4 Image Acquisition and Hardware Control OLYMPUS Note If a piezo electric objective mover PIFOC is installed the nosepiece might not necessarily use the shortest way to move from one objective to the next in order to avoid the winding up of the PIFOC cables Magnification Changer The default setting is 1 X and has to remain activated if the manually op erated optional magnification changer slide on the right side of the IX81 microscope is not u
332. ong with all other document windows when you use the commands of the Window menu to sort the document windows of the Graphical User Interface GUI Preferences Image Wew File Measure Module Graph Database Report Overlay Sheet Font size 3 El Eont Arial Pen size Auto Font size ho E Decimal places 2 E Mouse cursor Style Frimnary Auusillary Navigator General C Show function names amp arguments in status bar C Startup with user dependent settings Allow tiling and cascading of image window Button Bars Auto Arrange Button Bars Overlay Set the Font size for texts in the overlay and the Pen size line thickness for the overlay drawing tools Sheet Determine sheet font and font size for sheets in the Sheet group Arial is the default font set in the Font list the Font size is automatically set to 10 Select the settings from the pull down lists ac cordingly to change the Font or the Font size The settings apply to the values in the sheets them selves as well as the column headers and the first sheet column containing the row numbers Any alterations you make to the settings here apply to any open sheet document Set the number of Decimal places of the values displayed in the sheets Mouse cursor Style Determine the shape of the mouse cursor This applies only to interactive commands like drawing ROIs measurements and overlay drawings You have three choices an Arrowhead a Sma
333. ons in the Tree view Image data and graphs can also be loaded by drag amp drop into slots of the Viewport Manager or Graph Manager 12 5 2 Inserting Documents Images acquired via the Experiment Manager and the Experiment Plans used for the acquisition are stored automatically in the database Snapshots processed images and analyses however are not stored automatically To store these documents within the database the Insert function has to be used The Save as function leads to data export jt ll Insert Image The active image set can be inserted into the active folder of the database with the Insert Image button Alternatively an image can be inserted by dragging it from the Image Manager into a data base folder More than one image can be selected by using lt Shift gt click or lt Ctrl gt click A dialog box opens where the Record Name and Sample Preparation can be given and a Com ment can be added Upon clicking Insert the image set will be moved from the temporary folder to the database For all other types of data the menu Database gt Insert has to be used Database gt Insert gt Experiment This command creates a new Database folder in the active database cell s cell software Manual Chapter 12 Database 261 Database gt Insert gt Image This has the same function as the Insert Image button Database gt Insert gt Images This command allows inserting a series of images from the Image Manag
334. onvenient 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 241 242 Chapter 11 Graph Display and Graph Analysis OLYMPUS 11 3 The Graph Menu The Graph menu contains special commands for graph processing they do not work on images Theses commands are described in the following 11 3 1 Markers and Labels 11 3 1 1 Display markers Use Graph gt Markers and Labels gt Display Markers to display the markers set during image acquisition in the graph document 11 3 1 2 Set labels Use the Graph gt Markers and labels gt Set Labels command to add and position annotations to the active graph Alternatively right click within a graph document to open the context menu and then click Set Labels Show Markers in Graph Set Labels Graph Information Convert to Sheet Define Context Menu Graph context menu O It is convenient to change the display of the graph so that the interesting region of the graph is completely displayed in the graph document before you use this command Label text 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 th
335. onvert between the three palette editing methods In fact there are only a few ex ceptions where this would make any sense There is however one important exception if you switch from either the Polygon or Formula method to the Sheet tab the palette you have defined in the former will be inserted into the sheet This exception enables you for example to have a whole palette generated using a formula and then to edit individual values in the sheet A switch to the Sheet tab deletes the sheet values of the previous palette You always should save see below any palette you have defined before you switch to another tab O Imaging C functions can only use the LUT format to alter the lookup table of an image Therefore it becomes necessary that all LUTs originally saved in the polygon or for mula format are saved as LUT files as well See below Furthermore only LUT files are listed in the False Color dialog box See above Saving Loading palettes A palette can be saved as a separate file independent of its image This allows reloading the file at a later time and using the palette on the same image or transferring it to another image To do this click on the File button contained by all three tabs 6 2 10 1 The Sheet tab Sheet You can edit sheet values manually Click into the sheet field and select any particular field using the mouse or the cursor keys The input can be terminated by the lt Tab gt key moving yo
336. option subtracts a fixed value from all pixel intensities in all images of all channels The constant has to be typed into the box or can be changed by using the adjacent scroll arrows ROI The most useful way is to mark a ROI in an image area that contains only background inten sity and use the average intensity in this ROI as background value This value is determined indi vidually in each image of each channel Image This option allows subtracting a predetermined background image set from each of the images of the data set The background image has to have the same X Y size and the same num ber of channels as the data set to be corrected It can be either a single image or a time series or Z stack as long as it has the same number of frames respectively layers as the data set of interest Background Background Settings Dimensions Color Channels Furas4o0 Furaz o Z Layers L TE ee esee e TE Time Frames From io E to 61 step Cee Ce Dimensions tab In the fields Color Channels Z Layers and Time Frames you can choose the range of images in the different dimensions for which the analysis shall be performed in case only a subset of the data is of interest By default the entire data set is selected With the option Step in Z Layers and Time Frames you can select every second third fourth image and so on to be analyzed Copy non processed data If this option is NOT selected the newly creat
337. or smoothing purposes The original image consists of a simple rectangle of light shading upon a dark background The image has strong noise amplitude interference In addi tion 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 dis played 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 effect 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 on Original NxN image Iterations a 150 150 S725 10 S S J oE E 100 100 S 50 50 0 0 Mean Median 2 150 i E m 100 amp E 100 S o 50 134 Chapter 8 Image Processing OLYMPUS the parameters set The Median filter reduces noise without broadening the rectangle and elimi nates Shot Noise completely Filter parameter 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 In the following chapters the available filters will be explained in detail Before using the User Filter
338. or you may activate it directly from the command window For example if the macro name is MyMacro you can activate it by entering MyMacro into the command window This check box is only available if your program version includes the C Module menu Reset Interpreter If the Reset Interpreter check box has been selected cell4R cell4M will reset the interpreter before running the macro This means that all definitions made in the interpreter mode e g variable definitions function definitions etc will become invalid This ensures that macro definitions will not conflict with previous definitions This check box is only available if your program version includes the C Module menu cell s cell software Manual Chapter 13 The Special Menu and the Window Menu 273 13 2 Add In Manager Use this command to add load optional extensions to the cell4R cell M standard configuration Add In Manager Available add ins Audio support Black Balance CMA Colocalization Excel Dynamic Data Exchange Experiment Manager Extended Focal Imaging Grains Graph Processing v Restart the application to enable disable the add in 30 Deconvolutian File D cell A decon dix Version 2 5469 Copyright Doft Imaging System GmbH 2005 BD Description Blind Deconvolution Inverse Filter and Deblur Filters for 3 0 Definition The range of functions cell4R cell4M provides can be extended as much as you
339. ossible acquisition speed with most cameras an exception is the VF2T 23 24 Chapter 4 Image Acquisition and Hardware Control OLYMPUS Define It is very convenient to define the sub frame via a region of interest either in snapshots or live images When the button is clicked a rectangle drawing tool appears in the Viewport It can be moved freely and the size can be adjusted via mouse drag A right click defines the ROI and the sub frame settings are set accordingly in the Camera control window Reset This function sets the readout back to full frame it is also possible to double click on the frame selection area Extended tab The availability of the functions on this tab depends on the camera Gain The gain factor in CCD imaging defines how many photon generated electrons of each indi vidual CCD pixel are converted into one intensity count by the A D converter Usually the system gain is set so that with gain factor 1 the full well capacity matches the full range of the converter With a gain factor larger than 1 fewer electrons are converted into one count causing the image brightness to increase and the noise as well Set the value by using the slider or type in a number Offset Images captured by CCD cameras usually have a certain offset that means even pixels of images taken in the absence of light and with the shortest possible exposure time where any pos sible dark current does not come into play have a set intensity l
340. otorized z drives and nosepieces it is possible to automatically correct the individual offset Once done the objective can be switched without loosing focus This function is available in the cell M cell R software not in the configuration software ObsConfig exe 1 Select Acquire gt Parfocality Correction z in the cell4M cell4R software The Par focality Correction z window will open cell cell software Manual Chapter 15 cell4M cell R Configuration 347 T Start the camera Live mode and focus the sample Press the lt lt Read button The current z drive position will be set in the Position box Click the next Objective button in the window Parfocality Correction z The nosepiece will move this objective into position Focus again Press the lt lt Read button The new z drive position will be set in the Position box of the chosen objective Press OK when the correction is completed for all objectives The set differences in parfocality will be considered in experiments involving the switch of objectives 15 9 Configuration of the Dual View Micro Imager This optional device may not be part of your cell4M cell R Imaging Station 15 9 1 Configuring the Emission Filters Open the ObsConfig software and go to page Image Splitter The empty square button gives the default setting for systems without Micro Imager The beam splitting function in the cell4M cell4R software is disabled Ve
341. ou that the burner is still turned on if you want to exit the cell4R cell4M Imaging software Display warning message if user is logging out and burner f lightsource is still switched on cell s cell software Manual Chapter 15 cell4M cell4R Configuration 333 15 1 3 Using MT20 MT10 without the Imaging Software You can operate the Illumination System MT20 MT10 with the dialog box Illumination System gt Full control without using the cell4R cell4M Imaging Software that is you can switch the burner on and off change the excitation filter and the illumination intensity 15 2 Configuring the Microscope In case the microscope is not listed in the OBS System Configuration window right click on the ObsConfig symbol in the upper left corner and select Components in the context menu that opens and then click on the Microscope check box in the Select components window that pops up The microscope configuration is required for the operation of the motorized microscopes IX81 and BX61 via the cell4R cell4M software For the non motorized microscopes it is recommended to configure the objectives here as well Select components 2 Light Source Components Microscope Imagetype Shutter Move C ImageSplitter C Pifoc Minimize Stage C opticrid x Close Alt F4 About ObsConfig 15 2 1 General Configuration Type Select the type of microscope from the shortlist Connectivity In case of a motoriz
342. ouse scrolling 344 Chapter 15 cell M cell R Configuration OLYMPUS 15 6 Configuration of the Motorized Stage cell4R cell4M currently support a number of motorized stages by Marzhauser GmbH and Prior Scientific Instruments Ltd see the screenshot below In order to address a stage via software it needs to be configured by use of the Stage dialog OBS System Configuration Illumination System General Excitation Filters Corvus Maerzhaeuser Scan IM IX Disconnected Burner Full Contral Errors Microscope General 2 Drive Objectives Acceleration Filter Cubes Contrast Inserts Calibration Filters Image Types Shutter Image Splitter Pifoc Position Loop 2 device teat 2 Drive ha Speed velocity Type Select the stage type from the shortlist If the stage is properly connected this will be stated as Connected in the box on the right side Otherwise it will show Disconnected Prior Pro Scan II H 107 Ts Connected Mone Corvus Maerzhaeuser Scan IM IX Corvus Maerzhaeuser Scan 100x80 Bx Corvus Maerzhaeuser Scan 6 Slides Bx a Prior Pro Scan I H 107 Ix rans Prior Pro Scan IL H 101 Bx Prior Pro Scan II H 138 Bx ror s 2 Speed The maximum possible Velocity and Acceleration the selected stage can deliver will be set by default However for certain applications it might be advantageous to decrease these set cell s cell software Manual Chapter 15 cell4M cell4R
343. pearance and functions of the Graphical User Interface GUI A configuration consists of the following elements e User defined menus or an altered pre defined menu using the Special Define Menu Bar command 278 Chapter 13 The Special Menu and the Window Menu OLYMPUS e Macros generated using the Special Macros gt Define Macros command The configuration does not include the settings for the Viewport Manager or the Image Manager Settings of individual dialog boxes are not part of the configuration either Information about the loaded modules is not part of a configuration file This means that after load ing the standard configuration no modules will be removed Modules that are part of a configura tion file are those whose MAPI functions e g are used in the configuration s menus These modules will be automatically loaded along with the respective configuration If cell4R cell4M is being used alternatively by various users or for varying tasks it makes sense to set up individual workspaces The configuration of the relevant workspace used last will be auto matically loaded along with many other settings when you start up cell4R cell M 13 4 1 Reset Use this command to restore the original standard configuration What will happen Once you have modified a menu or a button bar or after you have integrated mac ros and programs into the system selecting Reset returns you to the original standard configuration
344. pen however each of the compo nent files must have been saved at least once already in order for files to be saved automatically they must have an existing file name or path name A source text does not necessarily have to be saved to run a macro however this is obligatory for running a C module Background When a C module is created only the text documents that have already been saved will be compiled Thus to avoid problems we recommend you generally to select this check box The header of the active text document indicates whether that document has been saved or not If you ve made alterations to the active text document and these have not yet been saved an aster isk will appear next to the name of the document Reload on startup Select this option to have all C modules that were loaded when you shut down cell4R cell4M reloaded when you start cell R cell4M again Debug mode Having selected the Debug mode you can use the Assert and Verify Imaging C functions for the development of your own Imaging C modules Both functions can be used for tracking down errors in Imaging C modules 292 Chapter 13 The Special Menu and the Window Menu OLYMPUS Maximum number of modules Enter the maximum number of C modules you wish to have loaded simultaneously The standard value here is 20 You should only increase this number if you really need all those modules in order not to occupy more system capacity than necessary
345. piezo electric objective mover or nosepiece mover PIFOC The Autofocus button will appear in the Acquisition button bar only if any of these devices is configured in the ObsConfig software see Chapter 15 cell R cell M Configuration The Autofocus function automatically finds the mounted specimen by acquiring images at different Z positions and analyzes the focality sharpness of the image contents The time required for the Autofocus to find the focus depends on the Autofocus options settings see below and the dis tance of the focus from the starting position cell amp cell Software Manual Chapter 4 Image Acquisition and Hardware Control The focal position may not be found if the signal to noise ratio is too low in other words the cam era settings Exposure time and Binning are not suitable see Chapter 4 2 Camera Control Also quite obviously the focus cannot be found if it is outside the set range The Autofocus is a two step procedure First an Autofocus scan is performed according to the settings and analyzed The found focal position will be used as reference position for a second Autofocus scan that uses a finer step size to improve the focus The resulting position of best fo cus will be used as focal position 4 6 1 Autofocus options Z device If more than one Z device is installed select the one to be used for the Autofocus from the shortlist Range Set here the range of Z positions to be scanned duri
346. programming language Macros on the other hand are supposed to handle simpler tasks They are mainly used to combine available internal functions with C modules together forming a sort of chain link fence of mutually dependent elements e g linking a frequently used series of functions into one single function Whereas C modules are more loosely integrated into a configuration remaining independent files a macro will fully become part of the configuration itself 13 1 2 Record Macro Recorder x Recorder toolbar Use the Record Macro command left button to record a series of commands in the macro lan guage Imaging C while you are executing the commands These recorded series of commands cell s cell software Manual Chapter 13 The Special Menu and the Window Menu can then be used as the basis for developing your own macros Macros take several steps and integrate them into one macro Application Certain image analysis processes frequently require series of commands that are repeated over and over again The macro recorder enables you to record these commands and subsequently have them all run automatically instead of having them execute one at a time again and again General settings Select Special Preferences or press lt F8 gt The Module tab contains the Macro recorder field The selectable settings have to do with the recording of argument names and image buffers What will happen e After activati
347. pter 7 Basic processing routine like size calibration overlay drawings extrac tion and conversion of image sets 9 3 3 5 4 5 4 1 5 4 2 5 4 3 6 6 1 6 2 6 2 1 6 2 2 6 2 3 6 2 4 6 2 5 6 2 6 6 2 7 6 2 8 6 2 9 6 2 10 6 3 6 3 1 6 3 2 6 3 3 6 3 4 6 3 5 6 3 6 6 3 7 6 4 6 4 1 6 4 2 6 5 6 6 T7 7 1 7 1 1 1 1 2 7 1 3 7 1 4 7 2 7 2 1 7 2 2 7 2 3 7 2 4 7 3 7 4 1 9 OLYMPUS Types of EX DEMING INS assisen peaa EE R Er ENEE E 60 Conducting Experiments Data ACQUISItION ssccceeeeeeeeeeeees 69 Opening a Database nnnoennnsennnernnresrnrrsrnrernrrresnnrrrnnrrrnrrrnrnnene 69 Executing an Experiment cccsccceseesseecseeecseesesecsesseneenseeseneeses 70 eta Ol ae eassteucscareceseene 2 veneeesaceeucmsienetes E 74 Image Display and Navigation cssscsssessscesscesseeseenssenseees 75 IES WIGW DOM oa e a a r 76 image DIS DIY srs E E E E TE ES E OS 78 Gene Fal cisssse vated ia r Gen aee eRe E 78 AQGJUSE DIS DIY errore e a aa aa 78 PAULO AGIUSE DIS DIY orea Sie cSilecoe sil Stee career a ui an 82 WV TITS B QAI Emecan r a 82 BIACK BAANG sescuuncncecccteovaccenenctsaceseuesneneneesceaeesncceacasnccceeteeceunctaecces 83 Gray SCAG danei A ee 83 Fluorescence CS OlOM sci soars ecco octet eae Cie la tice ioc tew edie 84 Edit Fluorescence Color ccccsscccseseeceeeccaeeecseeecceeeceuseesaneessaees 84 FAIS Ol Ole s artes teat ade casa ee erence a eases t ai a Ei 85
348. py graphs from the graph buffer via drag amp drop Graph functions Available graph buffers Enter the desired number of graph buffers to be available in the graph buffer box of the Image Manager You can select values between 12 and 100 graph buffers Any change in the number of image buffers only becomes effective after a cell R cell4M restart Display as list view This check box is not activated by default and the graph buffer box appears like the Gallery buffer box with graph thumbnails If you activate this check box the graph buffer box will look similar to the images list buffer box without thumbnails Drag amp drop in the Graph Manager Confirm delete copy Decide whether you wish or not to receive a query message when you copy or delete graphs in the graph manager These queries apply only to the dragging amp dropping of graphs You will not receive a query when using the Edit Copy command Background pattern When activated the graph window and the graph thumbnails in the graph buffer box of the Image Manager will have a background pattern of diagonal stripes 293 294 Chapter 13 The Special Menu and the Window Menu OLYMPUS 13 5 7 The Preferences gt Database Tab Use this tab to define presets for a database you wish to create See also Chapter 12 1 Directories for Data Storage Preferences Image View File Measure Module Graph Database Report Thumbnail display size Database defaults T
349. r Boundary shape Cancel Execute emaath File js o oie Fine Coarse ja O Bum black a m Bis 4 O Burn white iio General usage procedure The separator is adjusted to special image properties 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 gen erally drawn in Now increase the Fine Coarse parameter until you are close to the correct set ting 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 Boundary shape The separator affects light or dark lines dividing objects of the same color or affects objects dis tinguishable from the background due to their intensity value Dark Select this option if light objects have a dark outline Bright Select this option if dark objects have a light outline cell cell software Manual Chapter 8 Image Processing Step Select this option if the individual objects are distinguishable by their varying intensity values Select this option when you wish to separate particles distinguishable from the background due to their gray value Sigma 0 The image c
350. r 5 3 3 7 Complex experiments with trigger pulses and several time loops If the Experiment Plan contains a Ratio Image command connected to a color frame with two acquisition commands see Chapter 5 3 3 6 Experiments with online analyses with activated Store option the ratio data will be stored as child object of the parent image data one level lower in the data hierarchy see the screen shot below Online Kinetics with activated Store option are stored as child objects of the image set that was being analyzed Additionally the image used to predefine the ROls of the kinetics will be stored Tv1 in the screenshot below G 340Fura 380Fura Ee J Ratio of 340Fura 380Fura Kinetics of Ratio of 340Fura 380Fura ms Leal Tul LifeScienceDemoST AR HeLa cells with Fura cell a cell software Manual Chapter 6 Image Display and Navigation 6 Image Display and Navigation The software interface features a special window the Viewport to display up to 16 images Raw images usually need a little touching up in the display to look appealing contrast brightness and color can be adjusted Images series created in multi dimensional experiment may contain dozens or even hundreds of individual images taken at different points in time different Z positions or with different excitation wavelengths Such data sets require tools for navigation and animation All this is explained in the following sections Gili TINE VI
351. r tells you for how many hours the burner has already been used A bright green circle behind Burner indicates that the burner is on dark green indicates it is off Similarly a bright or dark green circle behind Sample illumination indicates that the shutter is open or closed This information field can be closed to save space on the screen and reopened again by pressing the arrowhead on the top right corner 4 4 Microscope Control Microscope Control The cell4R cell4M Imaging Software features a module for the easy control and efficient usage of the automated microscopes IX81 and BX61 Its use will be described in this chapter The IX and the BX modules are similar any differences between the two will be mentioned in detail Use the Microscope Control button to open the control panel of the microscope The control of the IX81 BX61 is fully electronic via the Microscope dialog window and or via the hand switch keys and frame keys on the microscope The Z drive has a rotation knob on both sides of the microscope that enables you to manually adjust the focus as well cell amp cell Software Manual Chapter 4 Image Acquisition and Hardware Control vz Microscope aH sa Microscope aHa 2 Drive Drive 20056 5 Hri Change limits Change limits Objectives Objectives Fluorescence turret Fluorescence turret Lamp afl 2 Hm i The Microscope dialog window features only those modules that have been config ured with the ObsC
352. r those loaded from an archive are accessible in the Graph page of the Image Manager that automatically opens if a Graph window is activated Image Manager 3 gt 2 1 8 Src fe Dest Src 2 Mask 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 operations with two source graphs for example for the addition of two graphs The Graph page closes again if the Viewport or the List or Gallery pages of the Image Manager are activated Graph windows are located in the document area just like the Viewport They contain special buttons for setting the graph display see Chapter 11 2 3 The Graphs Button Bar cell a cell software Manual Chapter 11 Graph Display and Graph Analysis 11 2 The Graph Window Graphs 1 Kinetics of Ratio Image Fura 2 eel FJ Paw amp EM TE Ovl yellows 1 Kinetics of Ratio Image Fura 2 Ratio 2 0 00 pm Upon start of the cell R cell4M Imaging Software the Graph window is minimized and located in the bottom left corner of the document area If not minimized it might be hidden behind the View port but will be brought to the front if the Graph tab of the Image Manager is activated To display a graph select the corresponding graph buffer If the mouse cursor
353. ral veces es once as eedsaee E E onia 107 Setting the Scale Bar PropertiesS cccssccecesseeeeeeeeeeeseeeeeeneaeees 107 SNOW M VIEW DOME arreire sepsis rE enue a r e E ace 108 Draw into OVESNAY cccccseecccsseeceeseecaesecseeeceusessageessueessaueessaeeess 108 Show Markers Time and Z Information ccceceececeeceeeeceeeeeeeeens 109 Cg S PEE EE mee re AE EA A A A ee Pee ene eee 109 STAY S copi cee hc ce a aia 111 cell amp cell Software Manual Chapter 8 Data changing tools to improve the image qual ity for example by in creasing the contrast or reducing the noise 7 9 5 7 10 7 10 1 7 10 2 8 8 1 8 1 1 8 1 2 8 1 3 8 2 8 2 1 8 2 2 8 2 3 8 2 4 8 2 5 8 2 6 8 2 8 2 8 8 2 9 8 2 10 8 2 11 8 2 12 8 2 13 8 2 14 8 2 15 8 2 16 8 2 17 8 2 18 8 2 19 8 2 20 8 2 21 8 3 8 4 Contents GOON Al misss easier decd ern ec tetes ieee ice tll dade du beaals sevetn ste 111 Activating the Overlay Toolbar cccccssssceesseseeeseeseeeseeseeesaasees 111 Creating and Editing Over layS ccccsssccecsseeeesseseeesseeeeseeeeeenees 112 SEPE ae ee ee ee PPE ore EO eee eT 116 EXE JO pene ene renee CRC pee Re cee neRr er erent cheer er er eer err eee eens ee 116 COMDIN Eiis a a a e EE ee es eee 117 Generala E E 117 Combining Data Sets lt 2 vecssosinecssenavedeieniccciienieeisvediseasicenieciiodinestes 118 Conven MAGS erreren EENE OEN OSEERE CERSA NEENA 119 General ae E E N E E EE O ees ence
354. rating a new image that contains the binarized channels of the source image as well as a channel displaying the colocalization areas 4 Generating a sheet with a statistical analysis of the areas above threshold in the new bi narized image Thresholding of the first color channel Click on the Define Threshold 1st button to open the Set Threshold dialog window Set the bi narization threshold for the first color channel This and thresholding in general is explained in detail in Chapter 8 5 1 Set Thresholds Thresholding of the second color channel Click on the Define Threshold 2nd button to open the Set Threshold dialog window Set the bi narization threshold for the second color channel Binarized image with Colocalization channel Click on the Colocalization button to generate a new image It contains the two color channels now binarized using the two threshold settings as well as the binarized colocalization channel The latter shows all image areas that have intensities above threshold in both color channels of the original Binarization is explained in detail in Chapter 8 5 3 Binarization Statistical analysis Activate the new binarized image and click on the Create Sheet button to generate a sheet that lists a statistical analysis of the image areas that are above threshold in the three channels of the binarized image 223 224 Chapter 10 Intensity Analyses OLYMPUS 10 12 The FRET Software Module FRET Foerster
355. rchy represent the experiments and are usually created automatically by the Experiment Manager They contain the image data ex periment plans graphs and other documents Double click an entry to expand or to reduce the active level Gallery If an experiment folder is the active entry in the Tree view the Gallery field on top right displays thumbnails of the items contained Form The Form view in the lower right part displays a table with information details of the currently active item in case of image data for example all relevant acquisition parameters are listed 12 4 3 Adjusting the Database Window Arrange fields Use the command Arrange fields in the Database gt View menu or the context menu the pick list that appears upon a right click on the database window to open the Arrange fields dialog window Arrange Fields View ype g Info Windows Cg Esperiment E Image Graph Document E Data ef Form view C Experiment amn Graph Document E Data E Table View s Print an Query by Example J Insert Available Audio File Calibration Unit Channel Document Files Size Document Insertion D ate Document Insertion Time Document Modification D Document Modification Ti Experiment Comment Experiment Hame File Name Frames Graph Comment Height ID Image Comment Image Name 4 i Current Record Name Sample Preparation Comment Document Creation D ate Doc
356. rd copy paste from the source text when you re looking for errors in this text The easiest thing to do is to open the Watch Variables dialog box first and add the expression in the Quick Watch dialog box using the Add button to the Watch Variables dialog box cell cell software Manual Chapter 14 Imaging C E Watch Variables Expression ee Expression list This dialog box is non modal meaning it can be kept open during debugging This enables you to track a variable s current value during the debugging Expression Add Expression list The Add button will evaluate the Imaging C expression entered into the Expression field and will add the expression and its value to the Expression list Do not put a semicolon at the end of the expression If a value is undefined the following will appear This will apply e g to the value of a non initialized pointer variable The Add button is only available if the Expression field contains an entry When will values be calculated If the Watch Variables dialog box is not closed and the de bugging process continues the values of the expressions in the Expression list field will be recal culated once execution is halted due to a breakpoint or due to single step execution Delete Clicking on the Delete button will delete the selected entry in the Expression list field This button is only available if the Expression list field contains an entry Delete All
357. re accessible via the But ton bar of the database window The lower border displays the Status bar with information about the active folder or file By default the database window is displayed in Full view subdivided into four areas Preview Tree view Gallery and Form The size of the four areas can be freely ad justed by mouse dragging of the borders The mouse cursor when on top of an area border changes from the typical arrow to a double arrow crossed by two bars to indicate the dragging possibility amp tricolor apl kidney 008 ey tricolor apl H Ca kidney _o02 E kidney _003 Field name Field value E J kidney _004 Experiment Name kidney 008 Date 31 05 2005 Time 14 57 a ee ee Experiment Comment H y kidney 008 Form view H Cg kidney 012 m Pa bidaman 47 E i tricolor 1 kidney 008 cell s cell software Manual Chapter 12 Database Preview This area in the upper left corner shows a thumbnail of the currently active item in the Tree view Tree view The Tree view in the lower left corner resembles the structure Known from file man agement programs like Windows Explorer However only the database folder itself called lt data base name gt apl in the top most level appears as such in a standard file manager program The main entries that is the folders in the second level in the hiera
358. re changed accordingly automatically The parameters bot tom level and top level have the highest priority which means they restrict the parame ters Layers and Step Width PIFOC usage For technical reasons the PIFOCs move in multiples of their inherent minimal step size about 6 1 nm in case of an objective PIFOC The software will automatically adjust the Height according to the best matching Step Width times number of Layers Once the top and bottom positions are defined any change in the number of Layers will automati cally lead to a corresponding change of the Step Width and vice versa so that the Height is main tained In case of an arithmetic mismatch between Step Width number of Layers and Height caused by the step size limits of the selected Z device a fit of the bottom and top positions will be done automatically with minimal possible changes cell s cell software Manual Chapter 5 Experiment Manager 49 This is a convenient way to set the top and bottom position Ww 1 Start a Live View acquisition see Chapter 4 1 Simple Image Acquisition 2 Move the selected Z device either with the PIFOC slider in the Illumination system MT20 MT10 window see Chapter 4 3 Illumination Control or the Z drive slider in the Microscope window see Chapter 4 4 Microscope Control until the desired position is reached 3 Transfer the settings to the Experiment Manager with the Set top or Set bottom button in the Z stack P
359. re comparable to those of the Sobel and the Laplace filters but some what less precise however faster to compute 140 Chapter 8 Image Processing OLYMPUS 8 2 13 Reimer This command is based on the definitions set in the Processing gt Define Filter gt Reimer dialog box What will happen The Reimer filter subdivides the entire image into areas of pixels with the same intensity within a certain tolerance range The borderlines between the areas are displayed in white all the rest in black It results an image that resembles the isobars in a weather chart a so called equidensitometric image of the second order Why the Reimer Filter The Reimer filter is able to subdivide continuous gray value ranges within an image which the naked eye alone has difficulties to distinguish Superimposing the filtered im age and the original helps determine areas of similar intensities Original Define Parameter gt Interval The Interval sets the gray value tolerance range 1 254 for pixels to be grouped into one area The preset interval value is 32 The lower the selected interval the more image details are visible in the filtered image Define Reimer Interval a C Preview cell s cell software Manual Chapter 8 Image Processing 141 8 2 14 User Filter This command is based on the definitions set in the Processing gt Define Filter User Filter dialog box Application A user defined filter provides yo
360. re most noticeable when broad bandwidths and strong enhancement are in use Ramp optimize Select this 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 Overflow Enter the percentage of dark pixels to be set to black and of light pixels to be set to white into the 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 O or 255 respectively for 8 bit images These gray color values are available for spreading the remaining gray intensity values Percentages be 147 148 Chapter 8 Image Processing OLYMPUS tween 0 05and 50 00 can be entered When you select 50 a gray value image will look very simi lar to a binary image colors will be either white or black 8 2 21 Separator This filter is based on the definitions set in the Processing gt Define Filter Separator dialog box Application separating objects such as connected particles or such as various intensity regions Define Separator filter Define Separato
361. re part of the Measure Preferences to be found on the Measure tab in Special Preferences Shortcuts to these options are the F8 key and the Measurement Prop erties button in Measurement Display of the Image Manager Tolerance This parameter decides de degree of similarity to the origin that a pixel must have to be selected The match has to very good in case of low values a small number of pixels will be se lected For high values the similarity can be rather remote and a large area will result Smoothing Depending on the dynamic range and the evenness of labeling within the selected image object the borders of the area selected by the magic wand can be raggedy A smoothing factor larger than 1 will effect the selection of objects with more regular shape Color Space The color space used determines the general criterion for the pixel selection with respect to color and intensity lop RGB Monitor colors and images acquired with color cameras are composed of the three primary colors red green and blue in different intensities Multi color images acquired with cell4R cellAM may contain channels of different colors but are converted into RGB images upon display With the RGB space the similarity criterion has to be fulfilled in all three channels for pixels to be selected This color space is especially useful for multi color fluorescence images Shusi HSI This is a more abstract color space than RGB Hue the color tone is explaine
362. red on the Microscope gt General page see chapter 15 3 1 have to be configured here E ops system Configuration Illumination System General Excitation Filters Burner Full Contral Errors Microscope General 2 Drive Objectives Filter Cubes Contrast Inserts ii Filters TIRF 2 TTL Port 2 OPEN low Image Types up Image Splitter Pifoc Stage Shutter Type Connection Polarity Transmission TIRF 1 TTL Port 1 OPEN low cancel Shutter Type Type the name of the first additional shutter in the first available slot The first shutter slot automatically lists the shutter of the illumination system MT20 MT10 if it is selected on the Illumination System gt General page the first entry row is not accessible If a shutter has been configured on the Microscope gt General page see chapter 15 3 1 it will automatically be listed in the second shutter slot the first entry row is not accessible in that case Connection Select the trigger port on the imaging PC front panel that is connected with the shut ter via a BNC cable Polarity If the shutter is triggered to open with a TTL high pulse select OPEN high otherwise select OPEN low If in doubt check the specifications of the manufacturer Transmission Check here to make the shutter available for image types with Transmission as setting in the Illumination Excitation Filter column in the Image Types page see previous chap ter cell
363. red cell icon in the Image Manager indicates a multi color single image 62 Chapter 5 Experiment Manager OLYMPUS O Once an Image Acquisition command has been placed and defined on the editor sur face outside the Multi Color Frame it cannot be subsequently moved into the frame and connected with another command placed in there It would be necessary to delete the frame connect the icons and redraw the frame Similarly the commands within a multi color frame cannot be connected with commands outside However it is possible to add an Image Acquisition icon to an existing multi color frame or to draw an empty multi color frame into the editor surface and subsequently fill it with Image Acquisition commands 5 3 3 2 Single images transmission Transmission light is usually much less intense and less harmful than fluorescence excitation light For this reason microscopes often do not feature any electronic shutter in the transmission light path In such setups transmission imaging in praxis cannot be combined with fluorescence imaging in one experiment because the transmission illumination causes such a strong background that any fluorescence would be largely or entirely overpowered If however the system is equipped with a transmission shutter and the software is correctly con figured compare Chapter 15 3 Configuring the Microscope and Chapter 15 4 Definition of Image Types the acquisition of a transmission image will be prece
364. requires three sets of calibration ratio data even if the specimen contains only two labels In such a case per form a dummy calibration as a third measurement by using any of the two reference images and setting the first ROI which usually marks a labeled area in the calibration step into a background or auto fluorescence area Unmixing The multi labeled specimen has to be imaged using the same filter set as used for the calibration The unmixing is identical to steps 2 11 in Chapter 10 9 7 Unmixing In the Unmixing dialog win dow step 4 be sure to use the red calibration as input for 1 Fluorochrome green for the 2 Fluorochrome and blue for the 3 Fluorochrome cell cell software Manual Chapter 10 Intensity Analyses 221 10 10 Phase Color Coding and Analysis 10 10 1 Phase Color Coding This command 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 Allow operations on false color images check box within the Special Preferences gt 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 respect
365. ressed they are deleted Max Y Click the Max Y button to autoscale the Y range from the minimum to the maximum Y value of the displayed graph segment 239 240 Chapter 11 Graph Display and Graph Analysis OLYMPUS Auto Max Y This is the dynamic analog of Max Y The scaling is automatically updated when moving the graph in the X direction by using the scroll bar at the bottom of the graph document 11 2 3 3 Further zooming and scaling functions Define Display Area Click the Define Display Area button to zoom up a rectangular area of the current graph docu ment 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 interesting part of the graph Click the right mouse button to return to the Define Display Area dialog box Alterna tively you may enter the absolute X and Y limits of the graph area into the fields of the dialog box Click the OK button to zoom the selected area of the graph to the whole graph window Default Size Click the Default Size button to rescale the graph so that the total range is visible Log X Click the Log X button to change the X axis scale type from linear to logarithmic Log Y Click the Log Y button to change the Y axis scale type from linear to logarithmic 11 2 3 4 Diverse functions Delete All Labels Click the Delete All Labels button to delete all text labels in t
366. rily filters out isolated pixels with gray values which differ greatly from their immediate sur roundings Rather homogeneous gray value areas and edges are retained In contrast to filtering with a median filter the filtered image loses none of its original clarity cell cell software Manual Chapter 8 Image Processing Define Rank Filter Define Rank Filter Rank Order sog a Cancel Execute Clete ie Help Size Enter the radius of the area to be considered You can select radii from 1 to 11 at intervals of 0 5 The preset value is 3 Rank The pixel intensities within the area are ranked Enter the rank in percent with which the central pixel is to be replaced 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 With the preset rank of 50 the central pixel will be set to the median gray value of the rank se quence 8 2 19 Sigma This filter is based on the definitions set in the Processing gt Define Filter gt Sigma dialog box Application statistical noise filtering Assumption Local noise in an image follows a Gaussian distribution The Gaussian curve can be described by its half width 20 A small o indicates a narrow curve with high maximum this is the intensity distribution in a homogeneous object with low noise A large o indicates a flat and wide shape this is the intensity distribution in
367. ritax error 324 Chapter 14 Imaging C OLYMPUS Add Use the Add button to add the expression to the Expression list in the Watch Variables dialog box The Watch Variables dialog box will be opened if it wasn t already This dialog box is non modal meaning that it can remain open during debugging This enables you to track a vari able s current value during the error search Command Use the Command button to compute the results of expressions and the values of variables The Command dialog box will be opened What s it for This dialog box is for reading out expressions and variables of the operator e g for converting a value into hexadecimal format Command a Close Expressions Result Symbol PY Undefined variable i Expression Execute Result The Execute button will execute the Imaging C expression in the Expression field Do not put a semicolon at the end of the expression The value of the expression will be displayed in the Result field Example File types Expression oxff Hex 42 sin PI 2 Image 1 Protect Image 2 Name 14 15 Watch Variables Use this command to track the value of variables and expressions while searching for errors You may also click on the Add button in the Quick Watch dialog box How to add expressions in the dialog box Expressions and variables can be either entered di rectly into the Expression field in the dialog box or added via the clipboa
368. roperties page respectively i l 4 Stop the Live View acquisition by clicking the Snapshot button 5 3 2 4 The Time Loop Frame a Time Loop frame Use for time lapse experiments Execution of this command will cause the following e The commands within the Time Loop Frame are repeated a specified number of times with a specified time interval e The images are arranged into time sequences and stored as one file e Time sequences may consist of single or multi color images or entire z stacks Properties Properties Time Lapse Repeat Cycle time Total loop duration 00 00 25 000 Approximate minimal cycle tine 350 50 Chapter 5 Experiment Manager OLYMPUS A Time Lapse sequence is defined by the number of times the commands will be repeated and the time a single cycle lasts Repeat This determines the number of times the set of commands within the Time Loop Frame has to be executed Cycle time This parameter sets the repetition time and its unit If this time is larger than the sum of the duration of the commands within one cycle the system automatically adds a delay between the end of the last command of one cycle and the first command of the next one The Total loop duration is the cycle time multiplied with the number of repeats and displayed as read only information Approximate minimal cycle time This read only parameter informs you about the minimal possi ble duration of a single cycle The min
369. rsor cell s cell software Manual Chapter 9 Measurements 185 6 Move the mouse and left click at the corner positions of the area to draw a multi segment line Drag the mouse with the left button pressed to draw a freehand line or left click at the corner positions to draw a multi segment line In case corrections are necessary keeping the lt Shift gt key pressed left click to delete previous figure segments one by one from the overlay Right click to automatically draw a line to close the polygon Draw a second polygon or click lt Esc gt to terminate the measurement The result will be displayed in the overlay and listed in the measurement display 9 2 5 7 Interpolated Polygon El Interpolated Polygon Use this command to measure an irregular object with rounded corners Default measurements are area and perimeter Several measurement series can be conducted successively 1 5 Left click on the border of the area A red line will appear in the overlay between the starting point and the moving mouse cursor Mark two more border points relatively close by A red triangle circumscribed with a blue interpolated line appears Continue by marking more border points Right click to automatically draw a line to close the structure Draw a second polygon or click lt Esc gt to terminate the measurement The result will be displayed in the overlay and listed in the measurement display 186 Chapter 9 M
370. rt General Label style C Use alternating colors C Use inverted r axis Inactive color Ache color Show labels k kz None Background ONunbein Measurement result Include name Magic Wand options Decimal paces 2 g o o E Scale Auto Smoothing b E e a0 Color space kd X Label fort C Zoom text with image General Continuous Measurement If this option is not marked the selected measurement tool becomes disabled after each measurement and has to be selected anew for another measurement Other wise several measurements of the same time can be executed one after the other and the function has to be terminated with the lt Esc gt key Use inverted Y axis Conventionally in imaging the origin of the X Y coordinate system is the upper left corner Mark this option to set the origin to the bottom left corner with rising Y axis Show labels None If this option is chosen measurement objects such as lines and x s will not be labeled in the overlay 290 Chapter 13 The Special Menu and the Window Menu OLYMPUS Numbering If selected individual measurements conducted within a series of measurements will be numerated in the overlay When you select this check box the Measurement results option will be cleared you cannot display numbers and measurements simultaneously in the overlay Measurement results Select this option to label each object being measured with the measure
371. rtical split Click this button if the Dual View Micro Imager is aligned relative to the camera so that the image is split into a left and a right half 348 Chapter 15 cell M cell R Configuration OLYMPUS OBS System Configuration Illumination System TYPE General Excitation Filters Burner Full Control Errors Microscope Image Types Shutter Image Splitter ODHE Type OHH Filters Configuration 1 C O iC Configuration 2 Horizontal split Click this button if the Dual View Micro Imager is aligned relative to the camera so that the image is split into an upper and a lower half Type ADH H Filters Configuration 1 moce v PE o 535 YFP w ee Filters Select a filter from the shortlist or type in a filter name and select a color for each image half as described in Chapter 15 1 1 Configuring the Excitation Filters This color will be used for the display of the corresponding color channel in the dual color image resulting from the overlay of the two halves Quatro Split This button is needed to configure a Quad View Micro Imager cell s cell software Manual Chapter 15 cell4M cell4R Configuration 349 15 9 2 Configuring the Image Types 1 Make sure that the necessary excitation filters and the filter cube are defined on the Ex citation Filter and Filter Cube pages respectively 2 Goto page Image Types 3 Create Image Types for the acquisition of im
372. s O Images acquired in experiments via the Experiment Manager unlike snapshots or live images are stored automatically in the active database in a folder carrying the name of the experiment Likewise stored are online analysis results if the store option is se lected For details see Chapter 5 4 3 Data Storage 12 4 1 General Remarks When looking at the database window you will recognize features that you are familiar with but tons thumbnails and last not least icons in a tree structure somewhat similar to the MS Windows Explorer software However you will not find the data files on the computer or the storage medium under the same names as displayed in the database window Internally the data are stored as files with cryptic default names created by the cell4R cell4M software For example an image time series resulting from an experiment called CFP YFP in the Experiment Plan acquired by the system and stored in a database named FRET for example will be found on the hard disk as a file named something like 4455H85C_FO0000021 tif in the folder FRET 4E55H85C_DocumentFiles Easy and intuitive access to the image and analysis data is only possible from within the cell4R cellAM software The cryptic file names are listed if the option Table View for the database window is selected in the Database gt View gt Choose View dialog see Chapter 12 4 3 Adjusting the Data base Window Database folder A database fold
373. s for example because of temperature drifts This can be avoided by inserting an autofocus scan after certain intervals during the experiment For the setup of the Experiment Plan it is convenient that the cell M cell R Experiment manager supports the placement of time loops within time loops The Experiment Plan shown below defines a long time lapse experiment with an image acquisition once a minute and autofocus scans once every hour Experiments that use a motorized microscope stage for image acquisitions at different XY positions would be problematic without the possibility of an autofocus scan at each position In most every case the focal position will be different at each stage position at least if larger stage areas are included 60 x 10 00 min cell s cell software Manual Chapter 5 Experiment Manager 69 The Experiment Plan above describes an experiment with dual color image acquisitions at different stage positions At each position first an autofocus scan will be performed The entire stage loop will be repeated every 10 minutes 5 4 Conducting Experiments Data Acquisition 5 4 1 Opening a Database Before the start of image acquisition with the Experiment Manager it is necessary to open a cell4R cell4 M database There are two possibilities to open an already existing cell4R cell4M database or to create a new one To open a database select Database Open in the Database menu of the main window In t
374. s which cannot be displayed e g 769 845 0 9100591 These numbers have to be multi plied by a Scale Factor with the positions after the decimal point being clipped With a factor of 1000 which is the default the above number would give 910 With this factor a pixel value above 1000 means signal increase and a smaller value a decrease Dimensions tab See the description in Chapter 10 5 Background Subtraction Calibration tab It is possible to calculate the calcium concentration out of FURA 2 ratio images if the imaging sys tem is calibrated following a procedure described in the literature The calibration parameters have to be entered into this Properties page A calibration protocol can be downloaded from the Molecu lar Probes homepage http www probes com media pis mp03008 pdf 10 9 Spectral Unmixing 10 9 1 Application Having the genome of more and more species been elucidated the next challenge of modern biol ogy will be to investigate the functional outcome of the genes the proteins and their role in physiology and disease conditions The development of computerized microscopes capable of acquiring spectral information together with the development of new life cell fluorescence dyes and new color variants of fluorescent pro teins opens new possibilities for the modern cell biologist Multi dimensional fluorescence micros cell s cell software Manual Chapter 10 Intensity Analyses 213 copy offers the possib
375. s 10 Ov lade MAG VIEW DOMswecSewestcciwoeetuivinenwnkiaCacsae incense 10 3 2 Simple Image Acquisition cccccescecseeeeceeeeceeeeecaeeeceeeeesaeeeeaeeeess 12 3 3 Saving Images The Database cccccccecseseeeseeeeeeseeeeeeseeeeeeenaes 13 9 4 jLOACING IMAGES ssassn rasaan raaa nA a A RTAS 14 3 5 Conducting Experiments with the Experiment Managev 14 3 6 Displaying Multi Color IM QES cccccsesceeeseseeeeeeseeeeeeeeeeseeeeesenaes 16 3 7 Displaying SEQUENCES ccccccseeeecseecseeeeceueecseeecaueecseessaneesseeeess 17 7 8 Chapter 3 Brief Introduction and First Steps OLYMPUS cell 4R screenshots are being displayed throughout this manual cell4 M screenshots are identical but for the label in the upper left corner of the main window 3 1 The cell4M cell4R User Interface l cell R cell M Fie Edit Database Acquire Image Process Measure Graph Special Window DAR D 6 Elala Trat SS ERN sad e m nf Tel Hanan Images Hel vja ala ME
376. s 191 9 4 4 Image Link l Image Link Use this command to locate measurement values in the Measurement display by selecting them in the image overlay This command is useful when many measurements have been taken 1 Click the Image Link button located in the Measurement Display The pointer moves to the image window 2 Click on any measurement object in the image overlay 3 The respective measurement value becomes highlighted in gray in the Measurement Display 4 Terminate the function with the lt Esc gt 9 4 5 Show Hide Statistic x Show Hide Statistics Use the Show Hide Statistics toggle button to hide or show the Statistics field in the lower part of the Measurement Display See also Chapter 9 4 7 Define Statistics 9 4 6 Select Measurements For the different types of measurement objects different measurement functions are available By default different measurement functions are selected for each type for example Area and Perime ter for 2D objects rectangles circles polygons etc Length for Lines Use the Select Measure ments function to open the respective dialog window 192 Chapter 9 Measurements OLYMPUS fea Select Measurements MS Select Measurements El All Measurements Type w 2D Object gA Area dee Angle Fi 2m Aspect Ratio Shape K ar parals C 7 Bisector Inner Max Distance Point C Fai Bisector Inner Mas 0 Angle C 7 Bisector Inner Mean Distance File C A Bisector Inner Min Distance
377. s above will result upon Plan Verification see Chapter 5 4 Conducting Experiments Data Acquisition JPI Multi color time sequence The Image Manager indicates a multi color time sequences with a film stripe icon containing a colored cell It is also possible that a Time Loop frame contains another Time Loop frame The result will be a time lapse sequence that contains blocks of equidistant images These blocks result from the in ner Time Loop frame and are separated by the time lapse resulting from the loop duration set for the outer Time Loop frame cell s cell software Manual Chapter 5 Experiment Manager 5 3 3 5 3 D Time lapse experiments acquisition of series of Z stacks In so called 3 D time lapse experiments the acquisition of Z stacks is repeatedly carried out To set this up in an Experiment Plan a Time Loop Frame has to be drawn around a Z Stack Frame The latter may contain a single Image Acquisition icon or several combined with a Multi Color Frame or not For the color management the same principles apply as in the chapters above they will not be repeated here A typical multi color 3 D time lapse Experiment Plan is shown in the example below Monochromatic and multi color 3 D Time lapse sequence The Image Manager indicates monochromatic 3 D time lapse data sets with a monochromatic stack within film stripes For multi color data sets it is a colored stack within film stripes 21 x 0 50 um
378. s 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 Left click on one of the phases in the color bar Keeping the left mouse button pressed you can move both thresholds simultaneously 8 5 3 Binarize The Binarize command is context sensitive It changes to Binarize Color Image if the active image buffer contains a 24 bit color image Multi dimensional images 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 Separate command see Chapter 7 6 Separate The result ing gray scale images can be handled in the same way as any other gray scale images Why binarize images 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 speed 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 or255 white
379. s here to an image area in which gray values either rise or fall sharply This filter can be used as pre procession before particle detection However noise may also be en hanced What will happen The filter examines the gray values of all pixels within an image area comprising NxN pixels and determines if the central pixel of the matrix is within a certain percentage range of either the minimum or the maximum value of its neighboring pixels using the following algorithm I gt lax Percent 100 I 4 Imin gt Ly I lt lna Percent 100 I 4 Inn gt Ly where intensity of the eena pixel ane maximum intensity inside the NxN pixel area oie minimum intensity inside the NxN pixel area Define Edge Enhance Size Enter the dimensions of the NxN pixel area 8 9 The larger the area the less detailed the resulting image will be The preset size is 5 Define Edge Enhance fx se pE Percent as Percent The Percent 1 99 field defines the threshold for the central pixel to the assigned either the minimum or maximum value of the surrounding pixels With a lower percentage darker gray values of the original image will predominate while a higher percentage will result in the predomina tion of lighter gray values The preset percentage is 50 8 2 18 Rank This filter is based on the definitions set in the Processing gt Define Filter gt Rank dialog box Application Image smoothing It prima
380. se WINKOW cccssccsseessececserseeeecseeecseessesenseesaneeas 206 12 4 3 Adjusting the Database WINdOW ccceceeeeeeeeeeeeeeeeeeeeeeeeeeees 257 12 5 Working with the Database ssesnesnnnnnennnennnnnnnnnrnrnnnnrernennnennne 260 12ST kadno Docu menlS sinisas EE EEEa 260 129 2 INSSrung DOCUMENTS siea e ER 260 et QUEN aa S 262 12 5 4 Administration Defining Organizational and Database Fields 263 252 Chapter 12 Database OLYMPUS 12 1 Directories for Data Storage How to specify the directories for data storage 1 Select Special Preferences from the menu bar or alternatively press lt F8 gt 2 Select the tab Database Preferences Image View File Measure Module Graph Database Report Thumbnail display size Small w Database defaults Thumbnail creation size Small wt Save images File type Compression None tit k Force TIF type for documents Locations pasaste Dame JO e aes D Archive T mpStorageDir sa Backup volume capacity eo iS MB Record name Auto Record Name m 3 Specify a default directory to store all database files in the Database files box Make sure to use a large partition of the hard disk for storage the D partition should be used in an Olympus Soft Imaging Solutions imaging PC 4 Specify a directory in the Temporary storage directory box This directory is used as a temporary storage during data storage and archi
381. se color palettes or lookup tables LUTs enable to display gray value images or individual color channels of n 16 bit images in predefined false colors also called pseudo colors This is done in that the palette assigns each gray value of the image a certain color For example in an 8 bit image with the Rainbows palette See below the intensity 23 which is a near black gray in the standard linear black to white gray palette is displayed as blackish blue while 226 otherwise a very light gray is displayed in an orange tone fj RGB Lookup Tables of Color l B False color palettes are often used to increase contrast This is because the human eye distin guishes only about 50 different gray values but many more colors Another application is to high light certain intensity values How palettes alter image display 184 0 255 43 185 0 255 7 186 0 255 30 187 0 255 24 188 0 255 18 189 0 255 12 190 0 255 E 191 0 255 0 192 5 25 0 193 11 255 oj 194 17 25 0 195 23 255 0 136 23 25 0 197 34255 ole 86 Chapter 6 Image Display and Navigation OLYMPUS The intensity values counts of monochrome images pixels comprise the palette s input e g 0 255 for 8 bit images These intensity values are listed according to their respective line number see the example table below Each intensity value is assigned to a certain intensity of the three colors Red Green and Blue The three color intensities put together
382. se cursor will move to the red co ordinate arrows on the left top of the image overlay 2 The arrows color will change to yellow indicating the ability to move them 3 Move the arrows to the desired location and left click to set the new coordinates The function terminates automatically cell s cell software Manual Chapter 9 Measurements 9 4 2 Create Measurement Sheet fz Create Measurement Sheet All executed measurements create an entry in the measurement display These results remain linked to the image after the image has been saved Additionally it is possible to list the measure ment results in a spreadsheet Such a sheet can then be exported in different formats via File gt Save as Click on the Create Measurement Sheet button located in the measurement button bar to open the Create Measurement Sheet dialog box Create Measurement Sheet Select the images whose measurements will be included in the sheet Image 1 Tvl C 2 Tve O 3 T3 Select All Unselect All Help Cancel Show image name in the first column Generate statistics of the sheets L One sheet per image Create separate column for each type of measurement Image All images are listed that contain any measurement objects Select the images the meas urements of which are to be converted into sheets Show image name in the first column This option is useful if the measurement results of several images are to be combined i
383. se of software updates a new installation is necessary Several administrative requirements have to be fulfilled for the system to function without problems 16 1 Updating the cell4M cell4R Software Updating the cell4M cell4R Hardware Control Selecting the Camera cccccseeseeeeseeeeeneeeeees Single User Systems Administrator Users Multi User Systems c ccscccsseeeeseeeeeeeeeeees PC to Controller Network Connection 16 2 16 3 16 4 16 5 16 6 352 Chapter 16 Installing the cell M cell R Software OLYMPUS O Usually cell4M cell R is installed on the hard disk s large D partition where the ar chive for the image data sets is located by default as well In case you want to install a virus scan software on the cell M cell4R imaging PC be sure that tiff format files are not scanned Otherwise the performance of the system will be considerably hampered because the new images generated in an experiment are tiff files and to scan them is very time consuming 16 1 Updating the cell M cell4R Software i It is not necessary to uninstall the older version first However if you decide to un install the software first start the OBS System Configuration software ObsCon fig exe go to the burner page and deactivate the option Display warning message if user is logging out and burner light source is still switched on Otherwise the un installation will not be complet
384. sed If however the changer is used to increase the magnification by 60 the option 1 6 X in the dia log box has to be selected to ensure a correct size calibration of the images Fluorescence turret Select a fluorescence filter cube from the pick list the filter turret moves automatically into the corresponding position ate Condenser for IX81 microscopes only Select an insert of the Phase Contrast Optical Ele ments Turret of the transmission condenser from the pick list A p Lamp on off Use this toggle button to switch the transmission lamp on and off Moving the slider changes the transmission light intensity ai FE samera Ocular for IX81 microscopes only This toggle button directs the image of the specimen either to the ocular or to the camera a ES 44 Transmission shutter open and close This toggle button opens and closes the op tional transmission shutter and complements the function of the Lamp on off button and the in tensity slider 3 E Side port Bottom port This toggle button directs the image of the specimen either to a camera mounted at the side port or at the bottom port Ee freo fen Image Type buttons These buttons set all microscope and illumination mod ules excitation filters filter cubes shutters as defined for the different image types see Chapter 15 3 Definition of Image Types cell amp cell Software Manual Chapter 4 Image Acquisition and Hardware Control 4 5 Motorized Sta
385. see the left cell image below as a typical example What will happen This method allows the user to select three background areas called Hotspots here in the raw image The algorithm determines the average intensities in these areas and temporarily generates a background image with a corresponding regular intensity gradient and uses this for the correction cell s cell software Manual Chapter 8 Image Processing 129 How to use this method 1 Select the Additive mode in the Assumed deterioration field of the Define Shading Correction dialog for this kind of shading correction Select the Interactive zero level option the Define interactive zero level field will ap pear in the lower left corner of the dialog box Define interactive zero level Hotspot radius E Set the size of the Hotspot radius from 1 64 Execute the Processing gt Shading correction command The mouse cursor will carry a red cross surrounded by a red circle of the size set above Click in to a background region The circle will be fixed in the image overlay Repeat this step twice in other background areas After the third Hotspot is set the shading corrected image will be generated automati cally 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 a new 130 Chapter 8 Image Processing OLYMPUS
386. selected Z device Set top This parameter sets the highest position focal plane the selected Z device will reach during the acquisition of the Z stack It can either be typed in or the current position of the selected Z device can be read in by clicking on the Set top button Clicking on the arrow buttons next to the box changes the position in increments of 10 nm Set bottom This parameter sets the starting position of the selected Z device during the acquisi tion of the Z stack it is the lowest focal plane It can be set as above Top layer slider and bottom layer slider These two sliders in the center of the page can be used as well to change the respective positions Height This read only information lists the total height of the Z stack to be acquired 48 Chapter 5 Experiment Manager OLYMPUS Properties 2 Stack Absolute Movement Layers The total number of layers to be acquired can be typed in or changed by clicking on the arrow buttons next to the box Step Width The distance between successive image levels in the stack can be typed in or changed in increments of 10 nm by clicking on the arrow buttons next to the box If the set values are not reasonable for example if the bottom layer has a higher number than the top position the pale green background of the dialog boxes turns into pale red O The above parameters are not independent A subset of them can be defined freely and the remaining parameters a
387. serts Filters ae pene Image Types Shutter Image Splitter 5 Pifoc 6 Stage OptiGrid Magnification Changer None Gis iex 15 2 4 Configuration of the Fluorescence Filter Turret Fluorescence Turret If a motorized fluorescence turret is available select it from the shortlist to be able to use it via the cell4R cell4M software Shutter This button is available only if the IX2_FRFACA is selected It moves the turret to a shutter position and back Filter cubes slider You can use the slider to move any of the mounted fluorescence filter cubes into position without having to start the cell R cell M software Filter cube names Type in a name for each mounted filter cube or select a name from the shortlist 338 Chapter 15 cell M cell4R Configuration OLYMPUS OBS System Configuration Wumination System Fluorescence Turret sanera Turret I 2_FRFACA v Shutter zE Excitation Filters l irer Filter Cubes Full Control DapiFitcTxRed O D 4 Microscope Fura GEP vl General 2 Drive Free Objectives Baciter Cubes Skip Contrast Inserts Filters Skip Image Types Shutter Skip Image Splitter Pifoc Stage 15 2 5 Configuration of the Transmission Contrast Inserts Condenser Check here if a Motorized condenser is available to be able to use it
388. sional i e they consist of image stacks of dif ferent color channels time sequences or z stacks With the commands in the Navigation toolbar you can easily select single images out of an acquired image sequence to be displayed in the Viewport Image Navigator ae se WW R 3 DI bol RR 6 3 2 Multi Color Images Multi color images consist of sequentially acquired images with different illumination settings For example A sample is labeled with three different fluorochromes The resulting multi color im age acquired with three different excitation filters is displayed as an overlay of the three different color bands ai Select Color Channel To view the individual color bands click with the left mouse button on the arrowhead of the Select Color Channel button and select from the shortlist that opens To select additional color bands use lt Shift gt left click 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 hid den 94 Chapter 6 Image Display and Navigation OLYMPUS 6 3 3 Displaying Different Color Bands in the Tile View Mode It is possible to individually display all color bands of a multi color image at the same time in differ ent Viewports To do so a suitable Viewport arrangement has to be chosen with the Arrange View ports button in the Viewport button bar see Chapter 6 1 The Viewport
389. sition If not in a first step the filter tur ret would move the cube into position If three single band filter cubes would be used the movement of the filter turret would be executed automatically as well without any further commands in the Experiment Plan 1 In parallel cell R only filter wheel moves the DAPI filter into position attenuator sets intensity 2 Shutter opens 3 Camera gets exposed and acquires the image 4 Shutter closes cell s cell software Manual Chapter 5 Experiment Manager 61 5 In parallel cell4R only camera reads out the DAPI image data filter wheel moves into FITC filter into position attenuator sets intensity 6 Shutter opens 7 Camera gets exposed and acquires the image 8 Shutter closes 9 In parallel cell4R only camera reads out the FITC image data filter wheel moves into TxRed filter into position attenuator sets intensity 10 Shutter opens 11 Camera gets exposed and acquires the image 12 Shutter closes 13 Camera reads out the TxRed image data Da Multi color single image The Image Manager indicates a single multi color image by a colored cell icon In order to store the images together in one multi color file the icons have to be framed by a Multi color Frame see below In this case the ROI selection and the binning factor have to be identical for all images The other settings that is exposure time and intensity can differ A colo
390. sitions will be examined during an Autofocus scan Consequently the time required for it is unsure to a certain degree as well If the subsequent command in the Experiment Plan is to be executed immediately after the focus is found the exact point in time cannot be told in advance As a result the acquired images will not be equidistant If the option Ensure constant time is checked the Experiment Manager will calculate the maximal time for an Autofocus scan and add a delay if the scan terminates before the maximal time is expired Thus the timing of the experiment is fixed however at the cost of a certain loss in speed Low Signal mode If this option is checked a Mean filter see Chapter 8 2 8 will be applied on each image before evaluation of the sharpness cell s cell software Manual Chapter 5 Experiment Manager 57 5 3 2 11 The Z drift Compensation ZDC command Zk 4 zpc command icon Use to avoid loss of focus due to microscope Z drifts It is possible especially in long experiments that focus is lost for example due to thermal instabili ties The ZDC is an optional IR laser based device for IX81 microscopes that detects the absolute position of interfaces of differing refractive index In case of oil immersion objects this is the cover slip to specimen interface in case of water immersion objectives it is any of the two water to glass interfaces and in case of air objectives it is the air to glass interf
391. spective buttons Lamp The transmission lamp can be switched on here by clicking on the button without having to start the cell4R cell4M software the intensity can be set by moving the slider cell s cell software Manual Chapter 15 cell4M cell4R Configuration 15 2 2 Z Drive Configuration I ops System Configuration Illumination System Drive General Drive Available Limits Full Excitation Filters Range Range Burner rr F Full Contral J F Errors Current Position Microscope General 20086 5 Hri Objectives Filter Cubes Contrast Inserts Filters Limits Shutter Image Splitter Upper Limit 30000 0 Hm Set Current Position Image Types Pifoc Lower Limit ym Set Current Position Stage Full Range hal w ZOC 2DC Available Z Drive available Activate the motorized Z drive by checking the box if the microscope is equipped with one Position box and range sliders The box displays the current Z drive position You can move the Z drive by typing in a new position or by using the sliders The Z drive can be operated within the set limits see below with the Limits range slider The dark gray bars to both sides of the Full range slider indicate the current range If the Full range slider is moved beyond the limits the fol lowing message will appear ObsContig Limits The screenshot shows typical settings for an IX81 The values have to be optimized for each individual setup 335 336 Chapt
392. spective dialog with a right mouse click cell a cell software Manual Chapter 10 Intensity Analyses 197 be HeLa stack g E List ak Gal Measurement Results Mark the pixels whose coordinates and gray values you wish to determine A cross in the overlay will denote each pixel marked The pixel s X Y coordinates and the gray value of each color channel will be listed in the sheet i Sheet Fura Seles s Coord Y Coord Furas40 Furada pri pri O2 10 568 239 364 19 66 10 46 251 393 15 33 15 98 24 335 22 30 26 45 230 352 Am an Cn Ta md aT i Pixel Measurement Settings Select the Show Labels Numbering check box in the Measure tab of the Special Preferences dialog window to numerate each pixel measured This way the pixels in the overlay will clearly correspond to the values in the sheet 10 2 Histogram Use the command Measure gt Histogram to have an image s gray value distribution calculated and plotted in a graph Definition The Histogram is an X Y diagram in which an image s gray value distribution is dis played in a graph i e number of pixels per gray value versus the gray value itself Results The histogram will appear as a graph The graph will be written by default into the first slot of Graph View in the Image Man ager To avoid overwriting an existing graph just click into an empty slot before calcu lating the histogram 198 Chapter 10 Intensity Analyses
393. ss image detail Define DCE OF Bandwidth 20 Cancel Enhancement 0 4 E Execute C Quality Overflow L Ramp optimize g Z File Preview Help Preview Enhancement indicates the factor by which lesser gray value modulations are to be enhanced Enter a whole number between O 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 de pend on the bandwidth selected Employing a narrow bandwidth in conjunction with strong en hancement will yield the greatest differential contrast Image detail will be accentuated whereas larger image structures will take a step back into the background O 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 Quality Select this 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 larger solid color image areas They a
394. sure gt Intensity Profile gt Arbitrary command to determine the gray value intensity along a line of arbitrary orientation How to measure an arbitrary intensity profile 1 Select the Arbitrary command 2 Left click on the starting position of the line to be drawn 3 Move the mouse toward the end position A blue line marks the current length and orien tation of the line being drawn At each end an orthogonal red line is displayed 4 Left click on the end position 199 200 Chapter 10 Intensity Analyses OLYMPUS 5 Continue with drawing another line or terminate the command with a right click and con firm yes O This command works currently only on the active frame not on an entire multi dimensional image set 10 3 4 Average Intensity of Neighboring Pixels Use this command to set a line of arbitrary orientation and determine the mean gray value intensity of the neighboring pixels within a certain range on both sides of it How to measure an intensity profile of a line of arbitrary orientation and thickness 1 Select the Average command A blue double cross with a central rectangle appears In its center is a pink arrow indicating the direction of the line to be drawn Note that the left mouse button toggles between the Resize and the Rotate modes in dicated by the crosshair and the circular arrow respectively in one corner Roughly place the end of the pink arrow to the designated starting point
395. t 6 2 2 1 The Adjust Display tab Adjust Display Adjust Display Detail Mapping E im im All calor channels a P 201 m Ge a te GL oo Using the Adjust Display dialog box you can define which range of intensity values of the data is to be displayed on the monitor for each channel the images consist of using the 256 available brightness values per channel of the monitor What you re actually doing is defining a so called display palette or LUT lookup table This palette correlates intensity values of the data with color intensities on the screen Its range goes from the minimum value the lowest displayable intensity value to the maximum value the highest displayable intensity value Color channels field top This field contains colored Display Channel buttons to select channels to be displayed and narrow Adjust Channel buttons underneath to select channels to be adjusted Display Channel buttons The white button causes the display of all color channels while the col ored toggle buttons select or deselect the corresponding color channel for display To display a single channel in a three color image deselect the two other channels 80 Chapter 6 Image Display and Navigation OLYMPUS Display Channel colored squares Dapi id 5 Adjust Channel bars below Load LUT Edit Fluorescence Color Oo oe m Adjust Channel buttons These buttons select the corresponding channel for individua
396. t Unit Reciprocal 3 Back in the Configure Input dialog window set Magnification to 1 and X calibration and Y calibration to 6 45 4 Click Save to open the Magnification Table 104 Chapter 7 Image Data Handling OLYMPUS Magnification Table Magnification Pinel width Pixel height urn urm 1 00000 6 45000 6 45000 Delete All V pivel width D et 5 Click Add to load the new settings The following message will appear it refers to the default settings Click Yes to accept 2 The magnification 1 00000 is already defined in the table Do you want to replace the existing entry 6 Click OK to confirm the new Magnification Table 7 Click OK to confirm the new XY Calibration settings 7 1 3 XY Calibration Using the Image gt Calibrate Image dialog box you can recalibrate the image of the active image buffer The XY Calibration tab is designed like the corresponding tab of the Configure Input dia log box described in the previous chapter If the exact magnification of the objective is known there is a certain tolerance here caused by the manufacturing process calibration is straightforward Image pixel size is CCD chip pixel size as specified by the manufacturer divided by magnification If the magnification is not known a calibra tion specimen of Known size can be used Example The cameras F View and Hamamatsu Orca ER both have CCD pixels size of 6 45x6 45 microns In case of a 60x objecti
397. t according to real time z position Imagine the combination of several time sequences to one long sequence If the sorting option is not selected the active sequence will be become the first part of the combined sequence and the others will be added in order of the Feasible image ob jects list If the sorting option is selected the software will check the time information of each se quence and combine the sequences chronologically Analogously in case of the combination of Z stacks the Z position will determine the final order if the option is activated cell s cell software Manual Chapter 7 Image Data Handling 119 7 9 Convert Image 7 9 1 General The Image gt Convert command opens a submenu containing the commands for increasing or reducing the number of bits per pixel i e bit depth This might be necessary if you wish to use images in other application programs Images acquired with cell4R cell M are stored and handled in a proprietary nx16 bit format Other application programs often are only able to read a certain bit depth 3x8 bit true color is the standard bit depth for many graphical and image processing software s Here 8 bit of informa tion is stored in each of the three color channels red green and blue The interplay of the three channels generates the colors displayed on a computer screen Another use for reducing the bit depth is to reduce an image s bit depth to save memory When you do this
398. t click to confirm the polygon E Polygon Click on this button to draw a polygon into the overlay Keep the left mouse button pressed and move the mouse to draw freehand Define the polygon point by point by clicking the left mouse button on each point Straight lines then connect points automatically Right click to connect the end points of the drawn line automatically to form the polygon Z Highlighter Click on this button to place a colored transparent foil on an image section Using this option you may effectively emphasize special image features Drawing is done as for Rectangles If you choose Invert instead of a color in the Object Properties dialog for a highlighter the highlighted image section will be inverted 115 116 Chapter 7 Image Data Handling OLYMPUS 7 6 Separate Separate Color Channels Extract Combine r The Image gt Separate commands allow dividing a multi dimensional data set into several inde pendent subsets within one dimension time Z or color Available The commands for dimensions not contained in the data set are disabled like Z Layers in the screenshot above Color Channel This command decomposes a data set into its color bands Z Layers This command decomposes a data set into its Z layers Time This command decomposes a data set into its time points For example a series of Z stacks will be separated into the individual Z stacks 1 7 Extract J23 Extract
399. t that the User paid for the product These limitations on the liability do not influence claims for reasons of product liability 7 Contract duration legal consequences of violating the license 1 The contract is deemed to be in force for an unspecified period The User rights are automatically terminated if one of the conditions of the contracts has been violated 2 In case of a contract violation the User has to return the original storage devices and all copies thereof including all modified copies all printed and written documentation and the software protection key to OLYMPUS SOFT IMAGING SOLUTIONS or the User has to destroy these items OLYMPUS 3 In addition OLYMPUS SOFT IMAGING SOLUTIONS reserves the right to file a lawsuit to claim reparations for damages non compliance or removal of the software in case of license violations The following laws and or conditions are in effect the conditions of this contract copyright laws and the laws of the civil code cell amp cell Software Manual Contents Chapter 1 Imaging stations for life science experiments Chapter 2 A system chart and a list of all components Chapter 3 Getting you started a quick guide through the main features of the imaging stations and how to use them Chapter 4 Simple ways to take images how to control the hardware modules and which parameters to set Chapter 5 Beyond snapshots setting up simple and comple
400. tatus box Not calibrated turns green and states Calibrated Set Limits This command opens the Stage limits window If no limits have been set previously the four limit position buttons will be marked by a blue question mark Stage limits To set limits move the stage with the joystick for example to the leftmost position of your sample and click X1 The blue question mark will disappear 34 Chapter 4 Image Acquisition and Hardware Control OLYMPUS Once all four limits are set a green cross appears in the center of the four buttons A red dot indi cates the current stage position Any position in the positions list outside the limits will be labeled with a red diamond symbol A reference position outside the limits will be labeled with a red R 3 45875 37290 6264 31 Boo e a a eee os 47974 35775 5729 58 In order to widen the limits so that all positions in the list remain in bounds right click the positions list to open the positions list context menu and select Adapt Corner Limits O The limits do not affect the function of the joystick However the Experiment Manager checks if all positions in the selected list are within the limits Otherwise a warning message is generated 4 6 Autofocus E oF 26 6 w Autofocus normal Autofocus Fine Autofocus very fine Autofocus options Autofocus O The Autofocus function is only available if the system contains either a motorized mi croscope Z drive or
401. te the command select Processing gt Extended Focus Projection O EFI works well for transmission images and deconvolved fluorescence data However the out of focus blur in fluorescence widefield images often impedes useful results cell s cell software Manual Chapter 6 Image Display and Navigation 99 6 5 Fluorescence and Transmission Image Overlay The selective fluorescence labeling of sub cellular structures causes consequently that often a cell is not visible entirely To see where the fluorescent structures are located in the cell it is possi ble to overlay fluorescence and transmission images possibly taken with contrast enhancing methods in the display This requires identical X Y dimensions of both images Multi dimensional image sets can be overlaid either with single images Snapshots or with multi dimensional image sets that have the identical number of time points and Z layers The procedure is straightforward 1 Activate the fluorescence image it can be monochromatic or multi colored a 2 Open the short list of the Select Color Channel button and choose Select Transmis sion A dialog box opens that lists the images that would fit for an overlay 3 Choose the desired image and click OK The Viewport shows the resulting overlay im age 4 Deselect the option Overlay Transmission in the shortlist mentioned above to remove the overlay O The overlaid image is not a new data set it o
402. tegrated Imaging C Module and Macro Recorder gives the opportunity for advanced users to customize applications and automate functions This manual is the complete documentation necessary for using the Olympus imaging station cell4M cell4R correctly and efficiently Special care has been undertaken for this manual to guarantee correct and accurate information although this is subject to changes due to further development of the imaging station cell4M cell4R Thus the manufacturer cannot assume liability for any possible errors We would appreciate reports of any mistakes as well as suggestions or criticism If you find any information missing in this manual or you need additional support please contact your local Olympus dealer cell a cell software Manual Chapter 2 System Overview 3 2 System Overview The following chapter gives you a short overview of the basic cell M cell R system components without additional peripherals The system consists of different hardware and software devices which are full integrated 4 Chapter 2 System Overview OLYMPUS 2 1 System Chart The system chart shown exemplifies the different components of a standard cell4R system and how they are interconnected rear view Computer front view Microscope top view cell a cell software Manual Chapter 2 System Overview 5 2 2 Hardware The hardware devices of the cell4M cell4R imaging station are listed below e Olymp
403. ter 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 Figure 3 and the eYFP Figure 4 samples The ratio of the average intensities of labeled structures ROIs measured at the two excitation wavelengths after background correction gives a constant that is specific for each fluorochrome under the given experimental conditions These two ratios are the prerequisite for the unmixing of the images of double labeled samples YFP oxaae dichroic GFP exciter emitter observed e emission Y k YFP eGFP exci exciter tation i _ eGFP N emission 450 500 550 nm Figure 3 left spectral match between eGFP fluorescence and optical filters HQ480 40x HQ500 20x Q515LP and HQ535m right reference images of an eGFP sample O The reference images have to be taken only once for each fluorochrome in a given ex perimental set up e g filter set labeling method cell type 215 216 Chapter 10 Intensity Analyses OLYMPUS Now the system is set to examine samples labeled with both fluorochromes The intensity meas ured at 450 490 nm excitation is composed of fluorescence due to eGFP plus fluorescence due to eYFP YFP exciter dichroic
404. the amp sign along with a letter e g amp N This means you ll be able to open the menu using lt Alt n gt Select the Insert sub menu check box Click on the Add button The new menu will be appended to the Menu list The new menu entry will be selected in the Menu list Click on the Up button to move the new menu into the menu bar position desired Click on the Show Sub Menu button Select the first command for the new menu Click on the Add button Repeat the above two steps until your menu is complete Click OK cell s cell software Manual Chapter 13 The Special Menu and the Window Menu 277 e Create a sub menu 1 8 Select the Define Menu Bar command in the Special menu Double click on the name of the menu for which you wish to create a submenu Enter the name of the submenu into the Menu field Select the Insert sub menu check box Click on the Add button Double click on the name of the new submenu in the Menu list Select the commands desired for the new menu and click each time on the Add button Click OK e Create a command 1 13 4 Select the Define Menu Bar command in the Special menu Double click on a menu Add a new command or delete an existing command Click on OK GUI Configuration Select Special Configuration to open a sub menu containing the three commands Reset Load and Save Definition The term Configuration refers to the ap
405. the long wavelength channel of the acceptor exciter image yellow channel of the second image in the example Ffret the long wavelength channel of the donor exciter image yellow channel of the first image in the example The other steps of the analysis 1 2 4 and 5 are the same as described for the observation filter wheel images in the previous chapter 10 12 3 5 FRETN Normalized Quantification According to Gordon This method is based on the Youvan method described above It involves the same correction factors and requires the same image types The difference is an additional normalization of the image resulting from the Youvan algorithm for the concentrations of donor and acceptor FRETN MicroFRET Fdon Facc Select the Method FRETn in the FRET Analysis window and continue as described for the Youvan method 10 12 3 6 Normalized Quantification According to Xia This method is similar to the Gordon method but normalizes with the geometric mean of the donor and acceptor images Nfret MicroFRET sart Fdon Facc Select the Method N fret in the FRET Analysis window and continue as described for the Youvan method 233 234 Chapter 10 Intensity Analyses OLYMPUS cell cell software Manual Chapter 11 Graph Display and Graph Analysis 11 Graph Display and Graph Analysis The previous chapter lined out how changes in intensity for example in time lapse experiments can be quantified and plotted in
406. the magnification to corre spond 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 7 1 2 Calibrating the Camera Channel It is very convenient to calibrate the channel with a magnification of 1 Thus if the magnification of the different objectives is set correctly in the OBS Configuration software see Chapter 15 3 3 Con figuration of the Objectives all images acquired will automatically have the correct XY calibration ignoring certain slight variations in the magnification of individual objectives due to the manufacturing process In order to do the calibration the camera CCD chip pixel size has to be known Both for the F View T and the Orca ER camera the pixel size is 6 45 microns cell s cell software Manual Chapter 7 Image Data Handling 103 1 Open the XY Calibration tab in the Acquisition Camera Configuration dialog window Configure Input Display vv Calibration Magnification Image Intensity Magnification calibration um t calibration umn ee ratio 1 Fixed Calibration Snapshot Arbitrary Horizontal O Vertical Calibration length 1 um 2 Click the Unit button to open the Set Unit dialog window and select m as Basic unit and u as Scale and confirm with OK Se
407. 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 pressed 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 Right click on any point of a polygon to delete it lt Shift gt and lt Ctrl gt keys If it is necessary to define a point of a polygon with greater precision keep the lt Shift gt key pressed while positioning the mouse you will only be able to move the cur sor vertically to be able to move it only horizontally keep the lt Ctrl gt key pressed while positioning the mouse Color Select the color you want to change here The polygon representing this color will be placed in the foreground not covered by the other polygons Count and Define Define here the number of points your polygon will contain First you enter the number into the Count field Then click on the Define button All polygon points will be plotted equidistantly with respect to the x axis Up to 32 points can be defined for a single polygon cell s cell software Manual Chapter 6 Image Display and Navigation Monochro
408. this command to measure a rectangular object of arbitrary orientation Default measurements are area and perimeter Several measurement series can be conducted successively As soon as the button is clicked a red rectangle will appear in the image overlay The left mouse button toggles between the Move and the Rotate modes indicated by the crosshair and the circu lar arrow respectively in one corner Dragging without a mouse button clicked performs the mov ing and rotating In either mode a left button drag changes size and shape The best way to draw is the following 1 Open a rectangle at any position and switch to Rotate with a single click on the corner of the rectangle 2 Rotate the rectangle so that the edges are parallel to the desired position 3 Change to Move with another single click on the corner of the rectangle and position the corner opposite to the crosshair to its final position 4 Finally adjust the size of the rectangle via left button mouse drag 5 To exit the measurement press lt ESC gt The result will be displayed in the overlay and listed in the measurement display cell s cell software Manual Chapter 9 Measurements 183 9 2 5 3 3 Points Circle ie 3 Points Circle Use this command to measure a circular object by selecting 3 points on the arc of the circle De fault measurements are area and perimeter Several measurement series can be conducted suc cessively 1 Mark a point at the arc
409. this option to have black object outlines superimposed over the original image Black pixels within the original image are reassigned 1 as intensity value Burn white Select this option to have white object outlines superimposed on the original image White pixels within the original image are reassigned 254 as intensity value Edit Click this button to be able to edit the resulting image interactively after separation This opens the Edit Lines dialog box Edit Lines As soon as you click the Edit button of the Define Separator dialog box the separator will be ap plied to the image according to its current parameters The Define Separator 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 O When editing be sure to use a zoom factor of 100 OK Click here to confirm the current overlay and to execute separation using the settings in the Define Separator Result group Cancel Click this 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 Erase All Click
410. this rectangle is displayed in the Viewport Thus you can use this rectangle to zoom into an area of interest Application You can turn off both Viewport Manager and Image Manager to temporarily enlarge the documents area This will give you e g more space for displaying images within the Image Document What will happen A tick will appear next to this command when the Viewport Manager is on 13 6 5 Image Manager Use this command to turn the Image Manager On or Off You may also use the short cut lt Alt 2 gt What is the Image Manager The Image Manager contains the Operands Box with the source and destination image buffers and the image buffer box with the List Gallery and Graph views Application You can turn off both Viewport Manager and Image Manager to temporarily enlarge the documents area This will give you e g more space for displaying images within the Image Document 297 298 Chapter 13 The Special Menu and the Window Menu OLYMPUS What will happen A tick will appear next to this command when the Image Manager is On 13 6 6 Status Bar Use this command to turn the Status Bar On or Off What is the Status Bar The Status Bar is usually displayed at the bottom of the Graphical User Interface GUI Among other things it displays a brief description of each cell4R cell4M com mand the name of the active input and position and size of the frame What will happen A tick will appear next to this com
411. 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 The Auto Settings tabs Sets the parameters used to automatically calculate thresholds Set Thresholds Manual Suto Settings Ho of phases Background F Preview Set ROI ENE Olh Onee gero Hi togram limita Dynamic Fixed Lower limit 453 El Upper limit 2520 Undertow o alal Overflouy ol The diagram in this dialog box displays a histogram of the image Histogram calculation is based on the portion of the image you have defined via the Set ROI button When you first call up this tab histogram calculation will be based on the whole image 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 Decide whether image structures are to be light or dark in the Background group Select the Low option if image structures are light and the background dark The 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 valu
412. tistical noise e reduction of acquisition errors e g CCD cameras sometimes provide erroneous gray values for individual pixels e restoration of poorly focused images e suppression or accentuation of minute image detail e accentuation of basic structures such as gray value edges Convolution filters Many of the filters in this menu are convolution filters A convolution filter is defined using a filter matrix A matrix can be described as a geometrical array of positive or nega tive whole numbers that serve as a mask placed over individual pixels Any pixel that is to be re calculated 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 ma trix will be multiplied by the matrix element above it by the so called weight factor For the filter ing of the whole image the filter matrix will be shifted across the original image and the gray value of each pixel will be recalculated Image Matrix What will happen W W are the weight factors of a 3x3 filter matrix I are the original intensities of an image area comprising 3x3 pixels The intensity of the central pixel after filter ing is calculated as follows 132 Chapter 8 Image Processing OLYMPUS nad Wich W l W Ig isei ee normalization factor nee Normalization In order for the filter operation not to
413. to the sample Data storage The images acquired during an experiment are automatically sorted arranged and transferred to a structured database from where they can be easily retrieved cell s cell software Manual Chapter 5 Experiment Manager 39 5 2 Concept of Usage R Experiment Manager Open the Experiment Manager with the Experiment Manager button in the toolbar or via the menu Acquisition gt Experiment Manager Close the Experiment Manager in the same way or with the Close button or via the menu File Close in the Experiment Manager window The Experiment Manager always opens in the state and configuration in which it has been closed the last time 5 2 1 Experiment Manager Components lt lt Experiment Manager File Edit View Options Help a Ws EL led fF XT ee gt 1 oO ote A Experiment Muli color Time lapse Duration 00 00 25 000 Total memory 117 PEER ERP RRP ERRERE ERE Eee F 7 m z5x 00s 00 00 00 00 Frames Acquired 0 Stored 0 Total 40 Chapter 5 Experiment Manager OLYMPUS The Experiment Manager consists of different components used to configure the different stages of an experiment planning preparing executing In the default configuration of the Experiment Man ager these components are arranged in the following way The Experiment Manager window in the default settings consists of the following elements a The Men
414. ttern 20 40 20 View port lirik 3x3 e 1600 Factor 0 0 0 0 12 Chapter 3 Brief Introduction and First Steps OLYMPUS 3 2 Simple Image Acquisition ZE ae TR Live View iol Snapshot Camera Control Illumination Settings The most important tools for simple image acquisition are the Live View and Snapshot buttons and the control boxes that are opened by the Camera Control and Illumination Settings buttons all can be found in the Acquisition toolbar In the following a typical procedure for the acquisition of a fluorescence image will briefly be de scribed For detailed explanations see Chapter 4 Image Acquisition and Illumination Control Make sure to select the correct objective and fluorescence filter and place your sample on the stage The usage of the microscope will not be explained here 3 1 il if 2 3 w 4 5 gt 6 j 7 Click Illumination Settings to open the Illumination system MT20 MT10 control box Click Main switch Burner ON to ignite the burner For a stable light output wait about 10 min Click on one of the Excitation filter buttons to select an illumination color Click Camera Control to open the corresponding window Reasonable starting settings are Binning 2x2 Exposure time 50 ms Brightness adjustment automatic These settings have to be adjusted in the course of the working session Direct the light path of the microscope to the
415. tton menu command or key combination This is explained below e The macro can then be integrated into an Imaging C module and will thus be available as a button function To do this you can use e g the settings shown in the Special C Mod ule New Module dialog box Set a name and click on OK to generate a sfm document that has a sample program in it already Simply substitute your macro text for the following line the third last line in the document 267 268 Chapter 13 The Special Menu and the Window Menu OLYMPUS dLooutpun Hello Word and then compile your module using the Build Module command in the Special C Module menu see Chapter 14 7 Build Module A new button that allows to execute the macro module will appear in the main user interface to Editing macros If you wish to alter a macro be aware of the subsequent points Opening dialog boxes The macro recorder records many commands so that linked dialog boxes will not be opened when you execute the macro To change this all you need to do is append a dollar sign to the names of the functions within the recorded macro Example Use the Image gt Filter gt Define Filter gt NxN command to open the corresponding dia log box The macro recorder will record the following entry e g DefineNxN Iterations 3 Size 10 If you accept the command as recorded the dialog box will not be opened and selected parame ters will always remain the same To
416. tton in the Control Center field You can mark several events during an experiment A time trace of all markers numbers will be stored in the experiment folder of the database The markers can be displayed in all offline analyses if the Image Analysis gt Display Markers and the Graph Analysis gt Markers and La bels gt Display Markers options are selected O Setting a marker does not interrupt the experiment 5 4 2 6 Aborting an experiment H Stop To abort an experiment use the Abort button After abortion all image data acquired during the experiment as well as the Experiment Plan and event markers are automatically transferred to the database and will not be lost O Even in the worst case that a hardware malfunction error occurs during experiment execution all the data acquired so far will be stored and not lost 13 74 Chapter 5 Experiment Manager OLYMPUS 5 4 3 Data Storage Images acquired in experiments via the Experiment Manager unlike snapshots or live images are stored automatically in the active database in a folder that is generated automatically and car ries the name of the experiment The folder will contain at least two objects afterwards the Experi ment Plan and a set of images The number of image sets depends on the experiment set up For example an Experiment Plan with two time loops with each containing a color loop with several acquisition commands will result in two image sets see Chapte
417. u can use all expressions allowed in Imaging C Note the following guidelines e Incase all constants in the formula are whole numbers without decimal point all results will be treated as integers instead of floating point numbers i e the digits after the decimal point will be ignored The result may be steps instead of smooth curves Thus all constants should be Chapter 6 Image Display and Navigation OLYMPUS provided with a point e g 100 or 100 0 to enable calculations with the precision of floating decimal point operations e Multiplication and division operations will be performed before addition and subtraction If nec essary use brackets e nmay have a value of 0 Thus you should avoid using n as a denominator You could divide by n 1 instead e Take note of any error messages that appear in the status bar of the cell4R cell4AM window after you ve completed inputting your formula Messages appearing while writing the formula can be ignored e You can also use Imaging C expressions such as the operators amp and e The pixel value n normally an integer long format can be converted into a decimal number double format by typing double n Examples Power n to the power of three pow n 3 Square root sqrt n Logarithm log n Use the following examples for applying the amp and operators to create some typical curves e n amp 64 4 rectangular signal in bin
418. u bar contains functions for file handling e g Save and Load Experiment Plans edit ing and configuration of the Experiment Manager b The Standard Commands toolbar left the Analysis Commands toolbar second from left and the Additional Commands toolbar middle contain the command icons for setting up Ex periment Plans Further commands can be found on the MT20 commands Microscope com mands and CCD Cameras Commands toolbars to be made visible via selection in the View menu c The Control Center toolbar right contains the icons to verify the experiment plan to prepare the hardware and to start pause continue or abort an experiment d On the graphical editing surface the diverse command icons and frames are arranged and inter connected to define the Experiment Plan e The Properties page is a context sensitive page where the parameters of the active commands are set f At the bottom there is the Status bar with information about the progress of a running experi ment 5 2 2 Arrangement and Customization 5 2 2 1 Opening and closing components The two Command toolbars the Properties page and the Control Center field can be closed via the View menu independently from each other The open state is indicated by a tick mark in the list of components Clicking on its x button in the top right corner also closes the properties page 5 2 2 2 Detaching and repositioning components It is possible to detach the toolbars and th
419. u of the graph document Label text In the Label text field enter the new text for the annotations The amount of text is lim ited to 116 signs Click the Set button to position the text frame in the graph document Modify Labels Labels Delete All Label text Anchor text Centered text Relative position isIE Anchor text Centered text and the commands within the Relative position field have the same function as described above for Set Labels see chapter 11 3 1 2 11 3 1 4 Copy Use Graph gt Markers and Labels Copy command to copy the labels of the active graph docu ment into the subsequent graph document or the graph set as dest buffer in the Operands Box of the Image Manager This tool is very useful to set labels in a series of graphs derived from one experiment at the same position O The labels can only be copied if the graph in the dest buffer has the same X axis scal ing as the graph of the src buffer cell a cell software Manual Chapter 11 Graph Display and Graph Analysis O This command is deactivated for graphs resulting from a Kinetics calculation 11 3 2 Protecting and Deleting a Graph Use Graph Analysis gt Protect Graph or click on the Protect Graph button in the Standard toolbar to protect the graph from being deleted or overwritten unintentionally Use Graph Analysis Delete Graph to delete the currently displayed graph document Alterna tively an active graph d
420. u on to the next horizontal field or by the lt Enter gt key which will move you to the next row Click within the left column with the cross shaped cursor if you wish to select one or more rows You can copy the contents of the rows using lt Ctrl c gt and lt Ctrl v gt Use the clipboard to copy these contents into other 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 im mediately File Click on the File button to open the standard dialog box to open and load files Files will be saved using the LUT format Linear When you click on the Linear button the values of the linear standard LUT will be inserted into the sheet cell amp cell Software Manual Chapter 6 Image Display and Navigation Edit LUT sheet Polygon Formula Enter values for lookup table E Linear Interval 0 Interval When you click this button the Set Thresholds dialog box will open Here s 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 Then you have to set the color for the selected rows manually with the Red
421. u 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 add ing the weight factors of the individual factors Another application is modification of existing filters This is done by increasing the mean image brightness by adding an offset What will happen If you apply the user filter the following operation will be applied to each image pixel W 1l 4 W l W 1 tte ZN Offset Normalization factor where I intensity of the central pixel in the original image l l intensity of neighboring pixels in the original image W W matrix elements or weight factors of the user filter Define Parameters Size To determine the size of the editable matrix field select one of the following options 3x3 5x5 7x7 or 9x9 For hexagonal matrices the numbers define the number of fields in the middle matrix line Filter matrix You can enter the individual weight factors via keyboard into the fields in the middle of the dialog box Positive or negative whole numbers are permissible 142 Chapter 8 Image Processing OLYMPUS Sum field The sum adds up all matrix current 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 The value in the Normalization field determines the range of gray values of the r
422. ug any connection ca bles cell s cell software Manual Chapter 16 Installing the cell4M cell4R Software 355 Warning The firmware update process will take a Few minutes This process must not be interrupted otherwise the Firmware might be corrupted Close all other applications Do not use the computer any of its peripherals or unplug any connection cable until the update process is Finished CTR_M PC1o4 CTR_M PIC MT10 1 j All connected devices are up to date Switch off the MT10 MT20 illumination system after the update Switch it on again af ter about 5 seconds This is necessary for a complete re initialization of the electronics The illumination system will not be fully functional otherwise 16 3 Selecting the Camera i This is not necessary after an update to the new software version 1 Press lt F6 gt or select Acquisition Select Camera to open the Set Input dialog win dow E 2 Click the New Channel button to open the Select Channel dialog window 356 Chapter 16 Installing the cell4M cell4R Software OLYMPUS Select Device Set Input Available devices E ay OB SGenenc C 2 OBS Dummy En OBS Dummy EW OBS Megaview OBSGeneric Em OBS Orca Hi Em OBS CC12 Ep OBS Andor Em OBS DP30 Em OBS DPFO Ep OBS Roper C Use calibration of active channel 3 Incase of a F View l T camera select OBS MegaView In case of an Orca ER or DBH1 camera select OBS Orca
423. ulations will be enhanced horizontally while they become filtered out vertically Image noise should be rather limited as this filter is susceptible to image interference Matrix Comparison If you wish to extract all edges of an object then select a Laplace filter 8 2 5 Differentiate Y Application Extraction of gray value edges in the X dimension Matrix 136 Chapter 8 Image Processing OLYMPUS Comparison This filter is analogous to the Differentiate X filter but enhances edges parallel to the X axis 8 2 6 Laplace Application Extraction of gray value edges The Laplace I derivative filter accentuates edges and smaller image structures in both the X and Y dimension It enhances however noise as well as Matrix Comparison If you wish to have image edges accentuated only along one axis then select the either the Differentiate X or the Differentiate Y filter Compare the Sobel filter as well 8 2 7 Laplace Il Application Extraction of gray value edges Matrix Comparison This filter is similar to the Laplace filter but has a more pronounced effect cell a cell software Manual Chapter 8 Image Processing 137 8 2 8 Mean Application Smoothing The Mean filter replaces every pixel with the arithmetical mean of that pixel and its eight neighbor ing pixels This results in noise reduction Abrupt gray value transitions will also be smoothed over however thus appearing blurry after avera
424. ule can be activated without actually having to load it To do this simply use the lt Ctrl Shift gt keys while clicking on the Open button in the Open Module dialog box File name The Module Formats file format is preset This list contains the formats of the make files MKU and of the executable files SXU Adding module to module search path If you ve selected the Add module to module search path check box the directory name of the module will be added to the directories used for the administration of the module manager We recommend selecting this check box if you re working with a particular module frequently Info Click on the Info button to view information on creation date which version and a brief description of the module The Module Info dialog box will be opened cell a cell software Manual Chapter 14 Imaging 311 14 4 Add to Module Use Special C Module gt Add to Module to add the active text document to the active module Available This command is only available e when aC module is active e if there is a MKU make file for this module and if e atext document is open and active Before you use this command Activate the module in question in the module manager Then you can check and see whether the module has a MKU make file using the Open button in the Open Module dialog box Then activate the text document that you wish to add What s it for This command is for adding a text d
425. ument Creation Time Image Dimension Color Channels LaVers Time Frames Size Y 5ize Bits per Piel Binning Exposure Time Z 5tep Width Markers Regions OF Interest AUls i A UK Cancel Help Set to all of this type Get Default The dialog allows to add to remove or to change the entries listed in 2957 258 Chapter 12 Database OLYMPUS e the information table of the Form area e the Info Window e the optional Table View display of the database window e the Query by Example dialog box e the Insert dialog box in the currently active database View gt Type lists all available views and data types Current lists all possible parameters for the active view or data types and Available lists all further parameters that are not yet in the Current list To add a new entry to the Current list select it from the Available list and then press the Add gt gt button To remove an entry from the Current list select it and press lt lt Remove The order in which the entries are displayed can easily be changed To do so select the entry that is to be moved and click the Up or Down arrow button Press Get Default to use the default settings for the different views and data types To use the same settings for a specific data type for all views press Set to all of this type O In the Info Window the number of data fields is limited to seven Therefore the Add gt gt button becomes disabled whe
426. up is available if a single channel is active To make automatic scaling more effective when noise or bright impurities are present you can define how many pixels as a percentage of the total number of pixels will be displayed with maximum or minimum brightness The intensity of the remaining range of pixels will then be scaled linearly according to the selected color palette Percentage values can be anywhere between 0 and 50 0 1 is usually a good value to start with a slight increase in contrast and brightness results cell s cell software Manual Chapter 6 Image Display and Navigation va a ya Brightness Contrast and Gamma Correction These bars become active if one channel is activated The values can be increased or decreased by increments of 0 1 by respectively clicking into the bar above or below the sliders Brightness Values above 50 increase the overall brightness by reducing both the Min and the Max value values below 50 cause the opposite Contrast Values above 50 increase the contrast by reducing the span between Min and Max values below 50 cause the opposite The Gamma Correction allows a non linear adjustment of the display Values above 1 0 increase the middle intensities while values below 1 0 decrease them 6 2 2 2 Detail Mapping Use this function for non linear intensity adjustments The field shows the histogram of the selected channel and its intensity scaling curve The shape of the curve can be cha
427. ure ment display cell s cell software Manual Chapter 9 Measurements 179 How to define a vertical distance A horizontal measurement is done using the same steps as for the vertical distance 9 2 3 3 Arbitrary line al Arbitrary line Use this command to measure distances between two parallel lines of arbitrary orientation Measurement Results The distance to be measured will be denoted by a straight line labeled with the length by default This arbitrary distance will be listed in the measurement display How to define a vertical distance The measurement is done using the same steps as for the Horizontal Distance command 9 2 3 4 Polyline Polyline This command allows determining the length of irregular structures of an image The distance to be measured will be denoted by a polygonal line made up of straight line segments in the overlay labeled with the length by default 1 Position the mouse cursor at the starting point of the distance to be measured and left click A red line will appear in the overlay between the starting point and the moving mouse cursor 2 Move the mouse and left click at the corner positions to draw a multi segment line 180 Chapter 9 Measurements OLYMPUS 3 In case corrections are necessary keeping the lt Shift gt key pressed left click to delete previous figure segments one by one from the overlay 4 Right click to finish the line Its length will be displayed in the
428. us microscope of the IX or BX series according to your specification e CCD camera SIS F View T Hamamatsu ORCA2 AG or others General features are high reso lution CCD cameras mostly 1 3 Megapixels on a 2 3 chip latest generation interline transfer chip with high quantum efficiency 12 bit data output via IEEE 1394 FireWire interface selec tion of sub frames and on chip binning e Illumination System MT10 MT20 bearing the arc burner 150W Xe or 150W Xe Hg the motor ized filter wheel 8 positions attenuator 14 levels and shutter e cell4M System Coordinator cell R Real Time Controller for multi task acquisition bears an I O panel with three BNC plugs to trigger peripherals and RS 232 sockets to integrate external de vices such as motorized microscope controllers UCB piezo objective movers PIFOC e Computer latest generation PC with all standard features modified for hardware control and peripherals integration monitor e System specific accessories filter sets etc 2 2 1 Motorized Microscope Modules Both cell4M and cell4R support the following motorized IX2 and BX2 microscope components e z drive e fluorescence filter turret e transmission condenser e ocular to camera switch e bottom to side port switch e observation filter wheel e transmission shutter 6 Chapter 2 System Overview OLYMPUS 2 3 Software cell4M cell4R Configuration Software to configure the Illumination System MT
429. user names e Check that the permissions for the user include Modify Read amp Execute List Folder Contents Read and Write ao oO 6 Check that the user has the permission to ncrease scheduling priority 7 Click Run in the MSWindows Start menu In the command line of the window that ap pears type mmc c windows system32 secpol msc and click OK cell s cell software Manual Chapter 16 Installing the cell4M cell4R Software 359 8 Select Local Policies User Rights Assignment 9 Select the item Increase scheduling priority on the list on the right side 10 Select Action gt Properties 11 Click Add user or group write the name of the new user under Enter the object names to select and exit with OK and confirm the changes with Apply 16 6 PC to Controller Network Connection Communication between the MSWindows imaging PC and the cell M System Coordinator cell4R Real time Controller is realized through an ordinary albeit internal network connection via a net work card The MSWindows status bar features an icon showing two little monitors If the system works prop erly you will see the mouse over message shown in the screenshot below CTR_R Speed 10 0 Mbps Status Connected EN SP sy 11 10 When double clicking on the icon the CTR Status window will open The Activity field counts the data Packets being Sent and Received It is important that the configuration of the PC to controller network
430. ve an image pixel size of 6 45 60 0 1075 microns in X and Y results cell cell software Manual Chapter 7 Image Data Handling How to calibrate the active image with a fixed X Y ratio and unknown pixel size 1 Select the image 2 Execute the Image gt Calibrate Image command to open the corresponding window Calibrate Image 8Y Calibration 2 Calibration Magnification 1 amp calibration 6 45 urn i Y calibration 6 45 pm F T ratio 1 H Fixed Calibration Arbitrary wl Horizontal Vettical Calibration length 3 Set the magnification in the Magnification box if known 4 Click on Unit and select an appropriate Basic unit usually meter and Scale usually micro in the Set Unit dialog box Set Unit Reciprocal 5 Select the Fixed check box and enter the X to Y axis ratio in the X Y ratio field most always it will be 1 105 106 Chapter 7 Image Data Handling OLYMPUS 6 If the calculated value CCD pixel size magnification is sufficiently accurate type it into the X calibration box The Y calibration value will be automatically set Exit with Ok 7 Otherwise enter the Calibration length for the length of the calibration object or struc ture 8 Check the box Horizontal or Vertical or Arbitrary 9 Start the calibration with the Calibrate button 10 Select the beginning of the calibration structure with the line cursor and click select the e
431. ve phase will be displayed in the color 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 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 ena bling you to look at both gray value range and image structure at the same time allowing you to define thresholds with even greater precision cell cell software Manual Chapter 8 Image Processing 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 already defined and saved Click the Auto button to use automatically calculated thresholds Parameters used for the auto matic calculation of thresholds are taken from the settings in the Auto Settings tab To set automatic
432. via the cell4R cell4M software Contrast inserts slider You can use the slider to move any of the mounted contrast inserts into position without having to start the cell4R cell4M software Contrast insert names Type in a name for each mounted contrast insert or select a name from the shortlist cell s cell software Manual Chapter 15 cell4M cell4R Configuration 339 OBS System Configuration Illumination System Condenser General Motorized Condenser Available Excitation Filters Burner Full Contral ry Errors Beene Microscope General 2 Drive Objectives Filter Cubes Pacontrast Inserts Filters Image Types Shutter Image Splitter Pifoc Stage Contrast Inserts 15 2 6 Configuration of the Filters of a Filter Wheel Filterwheel Select here if an additional filter wheel is available to be able to use it via the cell R cell4M software The illumination system MT20 MT10 features an integrated filter wheel and no additional filter wheel is necessary in the Reflected light path If a transmission light filter wheel not to be confused with the contrast inserts turret is mounted select Transmitted from the shortlist If an emission filter wheel is mounted select Observation from the shortlist Filters slider You can use the slider to move any of the mounted filters into position without having to start the cell4R cell4M software Filters names Type in a name for each mounted filter or sel
433. view the image on the monitor is displayed in gray scale according to the current Brightness adjustment You have the possibility to change the brightness of the displayed image manually with the Min and Max sliders or to use the Automatic adjust ment For further optimization you may adapt the intensity clipping values Min and Max respec tively representing the percentage of the darkest and brightest pixels which are set to black value of 0 or white value of 255 and are not scaled linearly by the automatic adjustment The effect of the brightness adjustment can be viewed directly in the Live View mode O If an image appears either too dim or too bright make sure to control the minimum and maximum intensities in the Min and Max field before judging that the exposure time is respectively too short or too long The appearance might be due to improper settings if the Automatic option is deselected Online histogram This option opens the Histogram window that allows judgment of the intensity distribution in live images at a glance Subframe cell4R cell4M offers the possibility to readout only a sub frame instead of the entire image cap tured by the camera around 1376x1024 pixels in standard CCD cameras The size of readout frame can be customized by moving the slider at each side of the frame that represents the field of view of the camera The advantage of sub frame readout is a reduced amount of data and an in crease in p
434. ving of the database and should like wise be on a large partition of the hard disk 5 Check the preset Backup volume capacity When archiving the database on a CD adjust backup volume capacity in accordance to the size of the volume e g 600 cell s cell software Manual Chapter 12 Database 253 When the system administrator stores the network database the value for the backup volume capacity should be set to 0 On principle it is possible to use network databases or write directly to CD However this is strongly not recommended problems might occur at least in case of large and complex experiments 12 2 Open Database cell4R cell4M offers a number of possibilities for opening an already existing database Select Open Database in the File Acquisition or Database menus or in the pull down pick list of the Open button of the standard toolbar to open the Open Database dialog Navigate to the desired database file apl and press Open A recently used database can also be selected from the file list at the end of the Database menu Open Database Look in CQ FRET O f ea 9 4I5v887F_DocumentFiles J FRET apl File name Files of type Database api w Exclusive Mark the Exclusive control box to set or change for example the password for the database to change the database settings to execute a SQL statement or to delete a database Furthermore the commands Delete Databas
435. wavelength with a film stripe icon containing a black amp white cell 63 64 Chapter 5 Experiment Manager OLYMPUS In a multi color time sequence the cell4R cell4M system will perform the following Images will be taken subsequently first with DAPI illumination then with FITC and finally with TxRed illumination if desired with different exposure times illumination intensities binning factors and sub frame readout regions Each image capture is synchronized with the opening and closing of the shutter to minimize photo bleaching This series of acquisitions will be repeated over and over again as often as defined in the Time Loop Properties and at exactly specified points in time Let s imagine the capture and readout of the three images and the movement of the filter wheel from the TxRed posi tion back to the DAPI position to be ready for the next cycle take a total of 500 ms and the Cycle Time is set to 2000 ms In this case the system will be idle for 1500 ms before the next cycle starts with a DAPI image acquisition In most cases it will be convenient to store the images of different color within one multi color data set In order to do this same as for multi color Z stacks a Multi Color Frame has to be drawn around the Image Acquisition icons Similar to the Z stack acquisition described in the previous chapter the Multi Color Frame must be placed inside the Time Loop Frame not vice versa or the same error message a
436. well as wavelength WL in nm exposure time during acquisition Exp in ms and the binning factors in X and Y Markers In case markers were set during the execution of an experiment they are listed here with Start and End times in ms and the given comments 124 Chapter 7 Image Data Handling OLYMPUS cell amp cell Software Manual 8 The following sections explain how images can be improved by data manipulation This is in differ ence to the display adjustments explained in the previous chapter The shading correction corrects for regular intensity gradients A host of arithmetical filters is available to reduce noise enhance contrast or sharpen object edges Deblurring algorithms filter out of focus haze 8 1 8 1 1 8 1 2 8 1 3 8 2 8 2 1 8 2 2 8 2 3 8 2 4 8 2 5 8 2 6 8 2 7 8 2 8 8 2 9 8 2 10 8 2 11 8 2 12 8 2 13 8 2 14 8 2 15 8 2 16 8 2 17 8 2 18 8 2 19 Image Processing Shading COPrectiOn cccsesscesessceceesseecsesececeeseceeeaseceeseeeeesaseressags 126 General beresna a A ol ace A E E 126 Define Shading COrrection cscccccccseseeceseseeeeseeeeeseeseeeeeeseeeseaes 126 Executing a Shading Correction cccccceceeeeeeeeeeeeeeeeeeeeaeeeees 130 FON EPE E A T AEN O ANT A E EE TT 131 GENea eRe tee ene a ene one eR en A 131 MARDEN leararen a 134 SIUE OLE 0 E ier E E AETA EE ee nee AE eee ee 135 Diferente X oracneir e a 135 IT OKCN ASV cia as athasiac n a EE 135 EIDA OI e a a ie r
437. windows in the Graphical User Interface GUI aligned This set ting applies to the Cascade Tile Vertical and Tile Horizontal commands of the Window menu 13 5 3 The Preferences gt File Tab The settings of this tab apply to various options for saving images and sheets Overview On this tab you decide e the default method of compression for TIFF images and quality of JPEG images e whether the active configuration is automatically saved when you shut down cell4R cell4M or whether you are to receive a query message e whether an automatic back up of the previous text file is made when saving the current version of the respective text document e the default file type for saving and opening images and documents and e how many entries are to be listed in the recent file list Save image files Here you can set defaults for saving images in the TIFF JPEG JPEG200 and LEAD formats Tagged Image Format tif The TIFF format is the standard image format in cell4R cell4M Select another image format only if you wish to export images to another application program which cannot read the TIFF format The TIFF format enables you to save additional information cell s cell software Manual Chapter 13 The Special Menu and the Window Menu along with the image itself In cell4R cell M this non image information refers to e g calibration data image comments and overlays Preferences Image View Fie Measure Module Gr
438. with Z stacks of only moderate size Tiling allows the application to subdivide the large datasets into a size the processing computer s RAM can handle The advantage of larger subdivisions of data being processed is the increase in processing speed and therefore a decrease in the amount of time it takes to deconvolve a dataset Sub Volume Overlap Pixels Tiling often results in clearly visible rectangular artifacts at the bor ders of the sub volumes Increasing the Overlap of the sub volumes can reduce this side effect This however increases the processing time The sub volume overlap setting determines the number of pixels that the montaged sub volumes will overlap The possible values are integers cell cell software Manual Chapter 8 Image Processing from 0 to N 2 where N is the width or height of the XY field in pixels whichever is smaller An over lap of 10 or 25 pixels usually works best If the result of the deconvolution with a value of 10 con tains artifacts having rigid lines edges or an obvious grid structure start with a value of 10 then increase this number Overlapping regions are deconvolved twice so making this number too large e g 100 will increase the deconvolution time 8 6 6 3D Blind Deconvolution O This feature is part of the deconvolution module SW_CELL_3DB_WF of the cell M cell4R software and not available in the basic software packages 3D Deconvolution Filters Projection Filter Selection
439. x experiments in an intuitive graphical way 2 2 5 3 1 5 3 2 Contents MATE OCGUGTION iiaa eaa a aT aa 1 System OvervieW sssssssssnunnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn mnnn 3 SVEMO Na a tcciiete eter reni e ai ea EREE 4 HAWANE asr ia a a 9 Motorized Microscope MOdules ccccccceeececeseceseceeeeeeeeeeeseeenees 5 SOMWA Chie aye oi aa Sl ee oe 6 Brief Introduction to the Software and First Steps 7 The cell4M cell R User Interface cccccccseeecseeeeceseeceseesseeeseaees 8 iine lmage Manage isan as vetnea cece aces teece a etasinlereces neater E 9 The Viewport Manager sccccsccssecccseesseeesneccseessnsecsessneenserseees 10 NG VIEW DOP enano ere ice taavese cate Giccict vaccine nies ada neds Weed eee 10 Simple Image ACQUISITION cccccccccseeeeceeeeceeseecaeeeseeeesaueeesaeeesaaees 12 Saving Images The Database cccccccsssscecseseeeeseeseeesenseeeseaeees 13 Loading MAGES aiias anaa aa aa a a E a RRi 14 Conducting Experiments with the Experiment Manager 14 Displaying Multi Color Images ccccccssseeeeseeeeeeseeeeeeseeeessaeeeeenaes 16 Displaying SEQUENCES ccccsseccceseeceeeeecaueecseeecaueessaeeesaueesseeeesees 17 Image Acquisition and Hardware Control csssessseenseeees 19 Simple Image ACQUISITION ccccccccsseecceeeeceeseecaesecseeeceueessaeeessaees 20 Snapshot and Live View cccccsse
440. y value image on a 600 dpi printer at a 10 cm width even at a quality value of 25 you will hardly be able to distinguish the compressed image print out from a print out of the original But keep in mind the effects compression has on various image processing functions cell cell software Manual Chapter 13 The Special Menu and the Window Menu Sheet format Decide which file format the system will offer by default for your use when you save This format is a pre set file type i e you are free to alter the actual format of a sheet anytime you like To make an alteration select the desired file format in the Files of type list in the File gt Save As dialog box The above mentioned pre set format in this tab is only relevant for new sheets i e those sheets for which there is as yet no existing file These pre sets will only be relevant for the file less sheets if you ve cleared the Keep file type in file input output dialogs check box in the General group When to use which sheet format The standard format must be used if you later wish to be able to read in and continue work on sheet data in cell4R cell4M Use other formats if you wish to export data into other application programs Standard Select the Standard option to save sheets using the cell4R cell4M sheet format whose file name extension is SFS Any sheets you later wish to load and continue work on in cellAR cell4M must be saved using this format Mi
441. ys tem has to check whether all interconnections of the commands are consistent the parameter settings reasonable etc If the experiment plan passes the check the System Ready button changes to the enabled state Once validated an opened Experiment Plan may be repeated several times without additional checking O Loading an Experiment Plan from the database requires validation before execution even if the plan had been used before and consequently was valid 5 4 2 2 System Ready EY System Ready Press the System Ready button to arm the system This downloads the Experiment Plan to the cell4R Real Time Controller cell M System Coordinator and sets the parameter for the camera Also the memory required for image acquisition is allocated on the hard disk The arming of the system may take several seconds depending on the amount of data to be acquired Once the sys tem is ready the Start button will be enabled If no cell4R cell4M database is open the following dialog window will appear Experiment Manager YD There is no open database or the open database cannot be used Please open or create a cell Family database Open or create a cell4R cell4 M database See above Once this is done the System Ready command will be continued O If the Experiment Plan includes online display of images the Viewport is automatically rearranged at this point The images will be arranged one above the other by
442. ystem MT20 MT10 15 1 1 Configuring the Excitation Filters The excitation filters are mounted in the filter wheel of the Illumination System MT20 MT10 which is accessible via the filter flap on the right side of the illumination system housing The filter wheel has 8 slots for different excitation filters and can be rotated so that each filter slot is accessible via the filter flap Filter exchange and configuration of the excitation filters with the OBS System Configuration soft ware is described in detail in Chapter 7 3 3 Filter Exchange of the Hardware Manual Part B mn fas ay ObsConfig exe I ops System Configuration g Illumination System MT Filterwheel General Excitation Filter g Excitation Filters Burner Full Control EEE Errors 432 FITE Microscope Image Types Shutter Image Splitter Pifoc Stage 403 DAPI L 572TxRed 340 Fura 380 Fura Move to handling position le le le le le le le cell s cell software Manual Chapter 15 cell4M cell4R Configuration In brief start the cell4M cell4R configuration software by clicking the ObsConfig exe button and go to the Illumination System gt Excitation filter page A gray arrow in the list of program pages on the left hand side indicates the selection With the vertical slider bar shown in the dialog below you can rotate the filter wheel so that any of the 8 filter wheel positions of the MT20 MT10 lig
443. ystem of your computer If you deactivate this option you may choose between dot and comma for the Decimal symbol and between comma and semicolon for List separator O Certain programs may not read your data properly if you choose not to use the regional settings General Save configuration on exit without confirmation A configuration file contains the structure and content of menus and button bars as well as the assignment of accelerator combinations key code short cuts Select the Save configuration on exit without confirmation check box to have any 287 288 Chapter 13 The Special Menu and the Window Menu OLYMPUS user defined cell4R cell4M configuration saved automatically upon shut down A configuration will only be saved if you ve made alterations to it The current configuration will be saved in the corre sponding configuration file thus overwriting the previous version of the configuration Clear this check box to receive a query message each time you ve made alterations to a user defined con figuration and shut down cell4R cell4M You ll be asked whether you wish to save the current configuration Selecting Yes will open up the Save Configuration dialog box This dialog box is structured like the standard Windows dialog box for saving files The file type at the top of the list is the one for configuration files scy Now you can create a new configuration file or overwrite an existing configuration file

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