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Chapter One: Introduction

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1. n To capture the microscope features type values for the Numerical Aperture of the microscope objective the Refractive Index of the Leica FW4000 User Manual 41 6 4 42 immersion fluid and any information that you will find useful into the Properties of the Point Spread Function area Type the wavelength of light for the probes that are set for the current experiment into the Frequency area of the dialog You can take the wavelength value from the probes for the current experiment or from the list of available Fluorochromes Note You must specify the X Y and Z numerical fields in Microns Note The PSF will be different for different wavelengths Type the dimensions of the physical image size into the Image Dimensions area of the dialog The number of planes is given The distance in microns between adjacent pixels is needed as Is the Separation in microns between image planes Note Each plane of the image stack will be square with its width in pixels specified When you have defined all of these parameters the software creates a new PSF to simulate the appearance of a fine bead viewed under those conditions To allow the PSF to mimic the behaviour of the Correction Collar set the Objective Collar Adjustment to an appropriate value Note This reshapes the PSF to obtain better levels of detail from the image Therefore it is possible to produce related PSFs by varying
2. Leica FW4000 User Manual 275 Setting Up the Print Image Environment 1 To print results 3 4 Click Print Image in the Publish Results dialog Pa main menu This displays the Print Page dialog Print E Hame HitogreenERARedTi Drag an image into the desired position on the page Click on the images to detine print options Click on the margins to define print margins 2 Drag the image into the Print Page dialog grid and drop the image in the appropriate grid square X Note To perform this drag and drop exercise you may need to move the Print dialog to one side of your screen to give you access to the image you 276 Leica FW4000 User Manual wish to drag and drop 3 4 Repeat steps 2 and 3 until you have dragged and dropped all the images you wish to print At any time during the drag and drop activity oy Sigs you can change the print image grid by clicking a the appropriate button If you change the image grid from four images to either two or one image then the images in the right bottom frames are removed as appropriate for the new grid layout To set the page margins double click the page outside the images in the Print Page dialog This displays the Set Margins dialog Margins Lett sae Fight aT LOR 25 o Bottom 29 mm Cancel To specify the size of the margin click in the appropriate text box and type the value in millimetres A typical value would be in
3. Cancel Aw Refer to the Leica Server Documentation for more information on this dialog Mo Leica FW4000 User Manual Viewing Optics Data 1 To view the optics data 3 1 Access the menu by performing one of Right click to display the drop down menu or Openthe View menu from the main toolbar 2 Select Show Microscope from the displayed menu and click Data Petter My ote at ore aat nae a co se Pe SSS ray ara ET EE TT wit z Por ICE NT pce TAT T E iew Setup a e Reviey View 5 ss i fititirre nthe gt Double click on the Objectives shown on the screen to see the Leica Objective dialog You may change a particular attribute of the microscope from the new screen displayed gt To display Leica Optics Data click Optics Data from the Hardware Setup menu in the main toolbar Leica FW4000 User Manual 45 Leica Optics Data x Lens Data AutoFocus Data Calibration Magnification Changer Description Aperture Magnification Lens 2 AQ x fe Lens 3 ee ae Lens 4 e ae Lens 5 a ee Lens 6 8 ee Lens 7 E ee TTT i Cancel Apply Help Refer to the Leica Server Documentation for more information on this dialog To view the optics filter right click to display the drop down menu or open the VIEW menu from the main toolbar select Show Microscope from the displayed menu and click Filter Leica FW4000 User Manual Viewing the Stage Plan n To view the
4. gt Note If you make a mistake in selecting a movie the movie may be easily removed by selecting the movie and clicking the Remove button Leica Deblur and 3D Visualise will remove the highlighted movie from the Movie List For now leave both movies in the Movie List 5 Click the OK button and Leica Deblur and 3D Visualise generates a new movie that comprises both movie files This concatenated movie can then be saved and concatenated again with another movie if desired 6 Click the Loop Mode button on the Movie player bar and then click the Play button Leica Deblur and 3D Visualise displays the frames of the movies in a continuous Loop pattern 7 Click Stop 8 Close all movies before continuing on to the next section Leica FW4000 User Manual 251 Tutorials for Start Deconvolution Start 3D Blind Deconvolution Blind Deconvolution is considered to be the most accurate algorithm available with Leica Deblur and 3D Visualise It does not require the calibration and measurement of the Point Spread Function PSF 3D Blind Deconvolution is an iterative and constrained algorithm It is iterative in the sense that it repeats the same computational Operations many times while converging to the enhanced image solution It is constrained in the sense that it only accepts deconvolved images that have the correct mathematical properties of non negativities that is it does not allow the tracer concentration to have negative values a
5. What do you want to do f Load an existing Point Spread Function f Create a Point Spread Function Create a new synthetic point spread function Based on Probe name FIT FITC1_PSF FITC1_PSF FITC2_PSF FITC2_PSF Requires no image input Generates 4 set of PSF images based on data such as wavelength numerical aperture etc given by the user Images are created and an esperiment made fram them S35 Note The description of the PSF function selected is displayed in the dialog when you have selected your activity 2 Choose the task you wish to perform 3 Choose the probe you wish to use 4 Click Continue to display the dialog shown below or click Cancel to cancel the operation 40 Leica FW4000 User Manual Properties of the Point Spread Function Name Texas Red Description pees Point Spread Functiord Numerical Aperture fi A Objective Eau Objective Collar Adjustment EE zl Refractive Index foi 1 515 1 515 m Frequency Texas Hed Light Wavelength rnm 620 0 nm Select Probe All Probes in current Exp m Image Dimensions width pixels 256 YY Spacing he Depth slices a Z Spacing o7 Create Cancel 2 n To define a new synthetic PSF type the physical parameters that define the image that will be computed into the Properties of the Point Spread Function area
6. eae es ee ATERRAR sai aes PrrrrrpPI 2 Click Continue to continue the time sequence run or STOP to stop the image capture acquisition 3 Ifrequired add a note in the Log This text box 4 If required click Skip Step to move the experiment forward to the next time sequence gt Click Close to close the image capture dialog Click Try Again to rerun the time sequence Note When you rerun the time sequence complex time sequence programs capture large numbers of images and thus require high values of filestore to accommodate those captured images gt To view the images you have captured click Image Viewer Image Viewer Leica FW4000 User Manual 103 When you click Capture again you see the FW4000 Capture dialog Check the box appropriate to the action desire and then click OK Experiment Default Experment Images already exist tor this experiment what do vou want to do Delete existing images and continue Keep existing images and continue Create a new experiment Default E sperment Cancel AS Refer to Chapter 9 Enhancing your Image for a detailed description of how to view the images and manipulate them to maximise your analysis of the results AS Refer to Chapter 2 Setting General Preferences for a description on how to set which image is shown in the Image Viewer 104 Leica FW4000 User Manual Chapter Six Reviewing Images Chapter Overview
7. To deselect an available measurement parameter click the required parameter Click the Deselect button to move It to the list of available parameters m T0 select all available measurement parameters click ae the Select all button to move all available parameters to the list of selected parameters m T0 deselect all available measurement parameters click ee the Deselect all button to move all selected parameters to the list of available parameters Leica FW4000 User Manual 157 158 To change the order of parameters in the selected list select a parameter you wish to move and click either of the up or down arrows as appropriate Click OK to close the dialog saving the changes or Cancel to close the dialog without saving any changes Leica FW4000 User Manual Changing the Colour and Thickness of Lines 1 To change the colour and thickness of lines 3 1 Click on the Setup button in the Measurements dialog to display the Set Up Measurements dialog eSBs Alternatively click Settings in the Measurement dialog and choose Setup 2 Click on the Regions tab F ip amp Regions Features Parameters Limits Classify General Excel Regions List Curent Region Region name Region Class Remove J Thick lines Ok Cancel 3 Click the Highlight Colour button to display the Colour dialog 4 Select the required colour 5 Click OK to clos
8. To select all the captured images click the image gallery Gallery menu and click Select All Images or press the Ctrl A keyboard keys or click the select all images icon see the icon to the right All displayed images will have Select checked To clear the selection of all the captured images click the image gallery Gallery menu and click Clear image selection or press the Ctrl C keyboard keys or click the clear images icon see the icon to the right All displayed images will have Select unchecked ao ites To select or deselect the current image click the image gallery Gallery menu and select an image Click Select Deselect Current Image or press the Ctrl I keyboard keys To delete selected images click the image gallery Gallery menu and select the images to be deleted Click Delete Selected Images or press the Ctrl D keyboard keys or click the Delete icon Click Yes or No in the Delete Conformation dialog that is displayed Leica FW4000 User Manual n To display images for only one probe anh select the Display Probes Composites radio button and select the desired probe Display All Selected Images To show images for all the probes select the Display All Selected Images radio button Leica FW4000 User Manual 109 Displaying and Comparing Sequential Images The images viewed here are the same images that you captured earlier They have not been manipulated in any way to produce a composite ima
9. 220 Leica FW4000 User Manual Slice Viewer S This function generates slices through the image optically and then displays the face of each slice in the Slice Viewer The thickness of each slice corresponds to the Z spacing or step size Viewing in the Slice Viewer is a good way of examining the degree of optical sectioning in the data To control which slice is currently displayed move the slider at the bottom of the view window The current slice number is displayed to the left of the slider Tip You are advised to examine your data in a Slice Viewer because it allows you to see subtle and not so subtle changes from slice to slice To use the slice viewer 1 Make the XY Max Projection of your data set active by clicking within its view 2 Select Slice Viewer from the VISUALIZATIONS menu to generate an optical slices projection of the data set from the XY perspective Note When a projection operation is selected the view formed depends upon the currently active view For example in the previous step the XY Max projection was the active view Thus when the Slice Viewer menu item was selected optical slices were created from an XY perspective 3 To step through the optical slices move the slider at the bottom of the view You can do this by clicking the slider and dragging it to the left or right or by pressing either the left or right arrow key The current slice number is displayed to the left of the AUTO
10. 30 45 60 90 or 180 degrees Generate a movie by setting your own Start Point Mid Point End Point and Step Angles To create a movie 1 Select Movie Maker from the Movie menu The 3D Previewer dialog appears Note Images in the 3D Previewer will open in the XY view only Note This tutorial continues by using the Quick Movies option 2 Within the 3D Previewer dialog click on the Movie menu and select Quick Movies Click on Rotate Y Axis and select the 60 Degree option This will generate a movie that rotates about the Y axis by 60 degrees in the Rock mode To stop the Movie at any time click on the Movie menu again and select Stop Leica FW4000 User Manual D 1 To generate a movie by selecting your own Start Point End Point 3 and Step Angle 1 7 Movie To play the movie again you may click on Play Movie from the Movie menu You may play the movie in Loop mode by selecting Loop mode from the Movie menu and then choosing Play Movie If you wish at any time to play the movie backwards you may do so by selecting Opposite Path from the Movie menu and then clicking on Play Movie To restore your original image select Original View from the Quick Movies option from the Movie menu of the 3D Previewer dialog Restore your original view by selecting Original View from the Quick Movies options Close all views before continuing on to the next section Choose your Start Point by either starting with the or
11. Cancel Share Dio not share A 12 Leica FW4000 User Manual Automatic Calibration The FW4000 can run in combination with an automated Leica microscope or with a manual microscope For either combination the system can calibrate itself using information that is read automatically or entered by the user The following information is required for automatic calibration CCD pixel size This is entered automatically by the software if a Leica DC DFC or camera is connected C mount magnification This is entered by the user Magnification data This is defined by the objective and magnification changer which can either be read automatically from an automated microscope or entered manually by the user Both of these are set at the time the experiment is performed 1 To use automatic calibration 3 4 From the hardware setup menu select Optics Data 2 Click onthe Lens Data tab 3 Either The objectives listed in the Lens Data tab will be filled in automatically if you are connected to a DM microscope Manually complete the Lens Data tab Aw Refer to Leica FW4000 Release Notes tor more information on lens data 6 Click on the Calibration tab 7 Check the Automatic calibration check box The software will take the CCD pixel size and the C mount magnification to calculate the distance between two pixels for each objective in the list WS Note The calibration factors displayed will change depending on the m
12. Capture gt Review gt Process gt Publish gt Archive When you have logged on and chosen an existing experiment or created a new experiment the Leica FW4000 software displays the six stage Experimental Process Buttons You use these buttons to move through an experiment lifecycle You can pause at any stage in the process Leica FW4000 automatically saves the data setup and captured images in the laboratory workbook for your experiment To continue with the experiment open the paused experiment when you next log In and continue from the appropriate stage in the process flow Setting up an Experiment 04 Clicking Setup displays controls for the five physical components you can set up The check box where applicable must be checked before you can set up that aspect of the experiment The XY dialog opens automatically when you click Setup This allows you to change camera and microscope settings to create optimal images You can change the order of the five physical component icons by right clicking on an icon and then dragging and dropping It in the desired position in the sequence In previous versions of the software these buttons contained text This classic look is available as an option in the preferences dialog Leica FW4000 User Manual In order the above buttons allow you to define Fluorochromes selected from the fluorochrome database and define the settings to be used for each fluorochrome s
13. Click OK to accept the new attributes or click Cancel to cancel the operation Leica FW4000 User Manual Preparing Selected Images for Processing To process your images in the Image Viewer double click on one of the selected images in the gallery If you want to process the images in Leica Deblur and 3D Visualise if you have purchased that option you must specifically select them and export them to Image processing To prepare images for processing 1 1 23 Open the Leica Image Gallery 2 Select the individual images you want to process 3 Click the Image Gallery Gallery menu and click Export Selected Images to Image Processing 4 fe a e ae Bee ee HEr peers z ed peer a po beets Oo pect z E a ca aie ae se _ S i ie ee a Reet Pr peas REE SS Close the Leica Image Gallery eee Alternatively to open the image viewer you can double click the required image in the gallery gt 123 Leica FW4000 User Manual Preparing Selected Images for Publishing Before you can publish your images for a montage report you must specifically select them and export them to the publishing area SS Note If you just want a report showing a composite image and its component probe images you can generate this by selecting the Publish icon without going through the gallery 35 Note Before you export a set of images for publishing ensure that you have se
14. For Deconvolution Methods to verify the setting It is set to on Adaptive Blind by default Optics Settings 230 The Optics Settings will prompt you to supply data for the Numerical Aperture of the objective lens and the Refractive Index of the immersion media for the objective lens oil glycerol water or air which was used during data collection Optics Settings also require you to indicate the type of microscope Modality you wish to use Examples include Fluorescence Widefield Brightfield Transmitted Light Laser Scanning Confocal Spinning Disk Scanning Confocal or Multithe Photon Fluorescence Leica FW4000 User Manual On the Image Dimensions tab notice the following Value Pixels Microns Width 90 6 3 Height 120 8 4 Depth 50 7 5 sp T0 change the Spacings microns type the following values Field Value X Spacing field 0 07 Y Spacing field 0 07 Z Spacing field 0 15 On the Point Spread Function PSF Dimensions tab The PSF Dimensions fields are disabled To enable these fields the Non blind method must be selected under the Deconvolution Methods section For this tutorial we have selected the Adaptive Blind method 1 To set the standard settings by using the Optics method Select the Microscope frame Ensure that the Lens NA value is 1 4 Ensure that the Refractive Index field value is Oil 1 515 Ensure that the is selected in the Modality box Select the Emissive Wavelength frame to display the wa
15. Processing Images The Image Viewer is a full display of the component and composite images which you have selected 08 File Edit View Image Database Window Settings 0 OSA 2 PRE GG Ame WH GF s00 The options supplied with the viewer allow you to process images by 4 gt gt gt Defining and selecting regions of interest Performing measurements on your experiment Mixing the channels to enhance your images Rescaling your images Enhancing images by removing background noise smoothing sharpening correcting misalignment and converting images to brightfield Performing no neighbours and nearest neighbours deconvolution Leica FW4000 User Manual Refer to the following chapters for information on how to use the image processing tools 4 4 4 Chapter 7 Processing Images and Deconvolution Chapter amp Performing Measurements Chapter 9 Enhancing your Image Chapter 10 Advanced Deconvolution Leica FW4000 User Manual 59 Publishing Results The methods available for publishing results via the Publishing Results dialog are p 60 4 4 4 4 Print a selection of images Print a report with images embedded Montage images for Z stack or time lapse experiments Make avi movie files from collections of images 8 Print Image Print Report Montage Web Page Web Montage Movie Maker ke A EAE arta poo I 1mm 21 BriohtField Bake atlas
16. Use ROI view RUI Maszk View ROI Outline Setup ROI Shows the objects detected once you have defined the background threshold level using the Remove Background dialog 150 Leica FW4000 User Manual View ROI Shows the current region of interest ROI mask This mask Mask may be applied to limit image processing operations to only those areas of the image under the mask View ROI Displays only the edges of the current ROI Outline gt Set Full Object Detection by selecting Image from the main menu and clicking Background Removal Alternatively click Apply Threshold in the Select ROI dialog Aw Refer to The section Removing the Background in Chapter 9 for information on removing the background from an image XS Note You are able to Load and Save regions by using the File menu in the Measurements dialog Leica FW4000 User Manual 151 152 Leica FW4000 User Manual Chapter Eight Performing measurements Chapter Overview This chapter contains the following topics Using the Measurements Dialog Specifying Measurement Parameters Changing the Colour and Thickness of Lines Changing the Number Format Making Measurements Using the Feature Layer m Changing the Name of a Region m Changing the Colour of a Region Clearing Regions of Interest m Saving a Region Deleting a Region Synchronising the Set of Results with Excel Making a Measurement Manually Measuring Probe Intensity Calibrating an Image Annotating your
17. Alternatively click the ROI icon in the image toolbar PEOI g Alternatively from the Measurement dialog click the por Define Fir Define Ftr Layer 1 button Layer i 3 Select the image to which the ROI will be referenced from the drop down list This image will be displayed during the ROI definition procedure For the editing modes which limit the ROI to the feature boundary this also defines the image that is used as the feature mask 4 Inthe Action area of the dialog specify whether you are adding or removing regions from the ROI Select Regions Adds the regions you defined to the ROI mask Reject Regions Removes defined regions from the ROI mask Select All Add the entire image to the ROI mask Clear Removes all regions from the ROI mask Apply Threshold Identifies the areas of an image which are objects of interest or background but does not actually remove the background Refer to Chapter 9 Removing the Background gives details on removing the background tools 5 Select the appropriate tool for defining the required region of interest Draw a freehand filled region Draw aline Leica FW4000 User Manual Draw a filled square 6 Select the Clip Mode Specify the full area as drawn S Clip the drawing to the area of overlap with the feature mask that Is include only the s feature s specified by the ROI mask not the background Clip the drawing to the en
18. Producing a Report You can select the results for the probes that you want to publish in your report gt Click the appropriate radio g Raw j Processed button and decide whether to mages Images include Raw Images or Processed Images or Both in the published images Make Sure Experiment is selected in the Image Sources options f Both 1 To produce a report 3 1 Select the experiment radio button 2 Expand the image stack for the experiment 3 Select the desired probes at the Z plane level Print Image Montage Web Montage Movie Maker irage Sawee Z1 DAPI Z1 FITC Z1 TRITC r Exported f Experiment Images Image Folders o f fe Please select required image stack ToO Composite Delete Exported Images Zh 280 Leica FW4000 User Manual TA Note Ensure that you highlight the lowest level of selection that is Z1 Z2 Z3 etc to display the full set of publishing buttons in the Publishing Gallery 4 Click Print Report in the Publish Gallery dialog and display the Create Report dialog Report Template Browse C Program Files LercaS SMO emplates Leica4 MDW S dot Limit length of test to E characters Preview Print 5 Select the appropriate report template for the number of probes you have used in your experiment by clicking one of the format buttons in the centre of the Create Report dialog gt Alternatively to select the report format click Browse to display
19. Reduce background fluorescence intensity Increase signal intensity Automatically optimise the image contrast range Increase the sharpness of details and edges Remove unwanted debris and artefacts Correct for image misalignment using shifting gt gt DO These and other image processing options provide flexibility in further enhancing the appearance of images even after you have captured and stored them Leica FW4000 User Manual Starting the Process Environment lo process images click Process in the experimental setup The Process Images dialog Is displayed send selection for processing swnchrornige lab book with processed data show selected in viewer wa a n an en pn ata m n a n m n mn ea m n m n m m m a aa an mn oa a n aha aa oa an oa ats m n m a m n mn oe an mn a n n e m a ota mn an at aa m n ata mn an a tet rere State Sith n n a n a n a a aer PoP T ee Peer Bese n n a n a n a n a n write eee m n a n a n n a a nn pate pocorn eae E EEEE ae pees ee EEEE EnS Seinge ae P EEEE m n n n n n n n n n n an ee ee nunnan nnana nnana ee ee ee nunnan nnn nnn nnana ee ee nnu nnmnnn nn nnn nnan Click View in the Process Images dialog to open the Image Viewer X Note Before you can process your images in the Image Viewer you must already have specifically selected them and exported them for image processing Aw Refer to Chapter 6 Preparing Selected Images for Processing X Note This dialog is
20. The Leica FW4000 Installation dialog appears FW4000 V1 1 12 01 04 Leica FW4000 MICROSYSTEMS W Leica Fvv4000 Leica Fluorescence Workstation Ww Leica Deblur Flease selectthe tems that you wish to install and then press the Install button XS Note The options available in this dialog may vary depending on your licence agreement 4 Ensure that the Leica FW4000 checkbox is ticked 5 Check the Leica Deblur checkbox if you want to install this component and you are licensed to use Leica Deblur and 3D Visualise This allows you to perform Blind Deconvolution and 3D Visualise operations You need the correct dongle for this component to be available It does not work in demo mode 6 When the installation has finished click OK to close the Leica FW4000 User Manual 5 Installation dialog 7 Restart your computer 8 Launch the Leica FW4000 application either by clicking on the desktop icon or by selecting it from the START menu P Leica F400 Leica FW4000 Ha LeicaFw4oo0 mT Leica Leica FW4000 User Manual Help Use the standard customised Leica FW4000 Help by clicking HELP in the main menu Leica FW4000 User Manual 7 Using Leica F W4000 Leica FW4000 has an easy to use and intuitive interface The Microsoft Windows environment allows you to perform tasks quickly and easily The Quick Start option in Chapter 3 helps you to familiarise yourself with the structure operation
21. be edited to suit the requirements of the individual laboratory Printing images with a choice of 1 2 or 4 images per page Printing a selection of images from a Z stack or Time lapse experiment This is called Montage Generating a report as a web page document Making a movie from a set of images All options except the second one require Microsoft Word Leica FW4000 provides a bespoke Microsoft Word template for you to use In compiling your reports Leica FW4000 User Manual Setting up the Publishing Environment Jo publish the images you have captured click Publish in the Experimental Setup process flow The Publish Results dialog is displayed Leste A gt Revien gt roses ED scriee D fae m Mii te H a Web Page i a i z Print Image Print Report Montage web Montage Movie Maker 21 DAFI 21 FITC 2 Texas Red Experiment Composite 1 To produce a Montage report 3 1 Select a set of images in the Leica Image Gallery 2 Open the Gallery menu and click Export Selected images to Publishing Close the Image Gallery 3 Open the Publish Results dialog to view all the images you selected for publishing gt To show all the images used in your experiment and produce a report of composite and component images click Experiment Leica FW4000 User Manual 213 Printing Images Printing images Is a two stage process Select the images
22. 4 gt gt gt o Oo o Leica FW4000 User Manual Introduction to the Leica FW4000 Desktop Administration Tools m Supervisor s Tools for Managing Users m Changing Passwords Changing Users Details m User Password Management Setting Preferences Setting General Preferences Mapping Keyboard Functions Setting Hardware Preferences m Changing the Database Location Opening the Lab Book Changing the Microscope s Current Configuration Viewing Optics Data Viewing the Stage Plan Setting up the Acquisition Device Relative Focus Correction Introduction to the Leica FW4000 Desktop After you have logged on or closed any of the stages in the Experimental Process flow the Leica Desktop is displayed You may resize the Leica Desktop to fit your Windows layout From this Desktop you can perform a number of discrete management actions without needing to enter into the Experimental Process flow For example you can Manage the logon supervisor only Manage users supervisor only Add new users supervisor only the tabs in the dialog are not available when a level 1 user is logged on Logon as a different user Open an existing experiment without logging on again Create a new experiment without logging on again Note Users ID level is level 1 and the supervisor s is level 2 Leica FW4000 User Manual Creica waooo CST cl Sse ear File view Setup Capture Review Process Publish Archive
23. Add new users Delete existing users Reinstate deleted users Set a user s status to logged off particularly when it was erroneously left as logged on because of a previous system failure gt gt gt Leica FW4000 User Manual 21 1 Changing a user s ID level 3 1 Click the Users tab in the Edit Users dialog User Name Level ID Deleted Logged On Machine DefaultUser 1 False a DefaultSupervisar ls FE et eae rane Reinstate s3 e SPA ADEE Delete Cancel 2 Change the Level ID for a user by clicking in the Level ID field for the relevant user and choosing the new ID level from the list of those available 3 Click OK to accept all the changes you have made in the Edit User dialog or click Cancel to cancel the operations 32 Note Users ID level is level 1 and the supervisor s is level 2 1 To add a new user 3 1 Adda new user by clicking Add 2 Type the name of the new user into the New User dialog field Enter the name of the new user User 3 Click OK to add the new user or click Cancel to cancel the operation 4 Click OK to accept all the changes you have made in the Edit 22 Leica FW4000 User Manual User dialog or click Cancel to cancel the operations Current User List ID User Name Password Level 1 Default ser Password Users Defaults upervisor Ai User 2 Add Delete OF Cancel 1 To delete a user 3 1 Hig
24. Cinepak Codec by A adius Microsoft ALE Configure Intel IndeolA 1 Video Als About Indeo video 5 04 ddad Data Rate i EB sec 9 Move the slider to set the Compression Quality 10 Set appropriate values for KB sec and Data Rate 11 Click Configure and set the configuration settings as required for your movie 35 Note The movie settings you can configure depend on the video compressor type you selected at step 6 Leica FW4000 User Manual 291 292 12 Click OK or click Cancel to cancel the operation 13 When you click OK you display the AVIFile Constructor dialog and the real time movie previewer 14 You can watch a preview of the images in the movie sequence from start to finish in the AVIFile Constructor dialog abAawlmagessimage TOOT SOOT 001 COT1 t abAawlmagessimage TOOT SOOT 001 CO tit abAawlmagessimage TOOT SOOT 001 COS aA abl manestlmane CONT SON 00A C1 tit Leica FW4000 User Manual 15 You can see the Video Clip Player dialog OpenFile Aubo Size Window Show Controls Repeat Show Border TimeF ormat Frames illiseconds Volume Update Set width Set Height 16 Toggle the arrow button to turn the Movie Viewer tools on and off Noe iit OpenFile _Openfie a 17 Click OpenFile to select the video file from the ezVid File dialog 18 Browse and choose the movie to show and click OK ez id File Dialog File name aStack
25. Comment field and type the text you require x v2 J Comment oe POY 15 332 03 Position n To exit from this stage in the Environmental Setup process click the OK button in the top right corner of the Visits Sites dialog Leica FW4000 User Manual 87 Defining Time Sequencing Routines Re 88 Note The Time Sequencer is a Leica FW4000 product option that is only available if you have purchased the FW4000T or FW4000TZ modules A time sequence Is a list of instructions that controls the acquisition of images during an experiment The time sequence is defined by using the Time Sequence Builder The sequence Is built from a list of elements that are accessible from control buttons available via the Time Sequence Builder dialog An automatic image capture tool allows you to set up and store all the parameters required to acquire a number of images automatically Once the sequencing routine has been set up the capture and processing of images Is a fully automatic process Each time a routine is run any or all of the steps can be Included For example skipping one time interval when images are defined for a given frequency of capture to another frequency of capture You amend a sequence by inserting or deleting steps or amending step parameters Each element in the list may be edited by the controls that will appear on the left side of the form when the element is selected by cursor keys or by being touched by the m
26. DefaultUser BPAE cells 20s El DefaultUser BRAE Fluoresc El DefaultUser BFAE fluoresce El DefaultUser BPAE Fluoresc El DefautUser BFPAE Fluoresc E DefaultUser BPAE Fluoresc El DefaultUser checking the in El DefaultUser Default E perim El DefaultUser iim 1 El DefaultUser Jims Experiment El DefaultUser Jims Experiment El DefaultUser Jims Experiment El DefaultUser mouse kidnep i i Ag Z gt To sort records in an ascending or descending order 70 at highlight the records to be sorted and click the appropriate button gt To exit from this stage in the Environmental Setup process click the x button in the top right corner of the Archive Experiments dialog Leica FW4000 User Manual 297 Selecting Experiments in Archive Using Properties The Properties dialog allows you to select data and allow experiments to be found in the Lab Book by using experimental properties n To identify and set up the property profile for experiments to be archived click on the Properties button The Properties dialog is displayed Details Filter What felda do you wish to see Available Properties Selected Properties Cancel 298 Leica FW4000 User Manual Details Tab The Details tab allows you to select experimental properties that will appear in the archive window 1 To build up the property details profile for an experiment to be 3 archived 1 Select the Details tab from
27. Experiment Cee 1 pA fE E ed Rawlmages Rae Sper Both CAPD SEER SNL A TES EB PII RTEA INLA NISL SLI LEAS EINE AE RARE ESS RS Please select required image stack El Experiment Dont Touch105218 Raw Images H 1 BrightField Z2 Z3 Composite To publish results 1 oe ie Select the publish arrow to allow the current experiment to be processed for publishing as a document Click Print Image or Print Report from the Publish Results dialog Select the report format either 1 2 or 4 images per page Drag and drop the images you select into the print image frames Click Print Refer to Chapter 11 Publishing Experiments Leica FW4000 User Manual Archiving Experiments A BY XO 7470 3 IN ame WE by Owner WE fall Results f Archive Retrieve A CefaultSupervisor DefaultUser El BRAE cells 20x magn fe BRAE cells 20x magn fe BRAE Fluorescent ce El BPAE Fluorescent ce El BPAE Fluorescent ce H checking the install lt El Default Experiment Al Jims Experiment Dont El Jime Experiment Dont El Jims Experiment Dont El mouse kidney section Owner El DefaultSupery El DefaultSupery El Default ser El Default ser El DefaultUser El Default ser Hl Default ser El Default ser El DefaultUser El DefaultUser El DefaultUser El DefaultU ser El DefaultLiser El DefauktU ser iim Jims E sperment Jims Experiment Jims Experim
28. General Excel Features List Current Feature Feature Mame Feature Class Colours Show feature names Cancel 3 sSetthe fields as appropriate for the measurement you wish to make 4 Click OK to close the dialog and save the changes or Cancel to close the dialog without saving any changes 162 Leica FW4000 User Manual gt Check Show feature names to display the measurements for all the features defined as ROIs in the image 1 To select and load a Feature Layer 3 41 Click File in the Measurement dialog 2 Select Load Feature Layer n to display the Load Feature Layer dialog E ip Feature layer name OK Cancel 3 Select the feature name required 4 Click OK to close the dialog and load the selected ROIs or Cancel to close the dialog without loading any ROIs Leica FW4000 User Manual 163 Changing the Name of a Region 1 To change the name of a Region 3 4 Click the Setup button in the Measurements dialog to display the Set Up Measurements dialog SOU gt Alternatively highlight the name of the region and type the new name 2 Click onthe Regions tab 1 ip amp Regions List Current Region Region name Region Class Remove Ii Thick lines Show region names OF Cancel Click on the Region name in the dialog The Regions List contains a list of all the regions in the order that they were created Typ
29. Intensity Measure Objects oor Pan Add Annotation Display Annotation Clear Annotation Load Annotation Save Annotation gt Alternatively click the Measurements icon r od a fea fie Edt Setings 2228 MM 2 Sf ee ie te Po Apply M F New Save Prit Copy Setup kcal T Apply Measure Frame eo UT EEE TE E L as O OC D O 6h lea Meas R Clear All Measure Measure poe lt lt RR ae P aros Profile Ftri Ftr2 Point To change region name select row and enter new name a 166 Leica FW4000 User Manual 2 Click Settings inthe main menu for Setting the dialog 3 Click Clear Regions Setup Paint Small Square Medium Square Circle Ellipse Line Area Measure Objects Delete Selected Region Clear Regions Saving a Region To save a set of measurements click Save in the main toolbar in the Measurements dialog Deleting a Region To delete a region select the required SAP Settings region in the Measurements dialog open the Edit menu and select Delete poy als Copy Selection Delete Reset Grid Columna Leica FW4000 User Manual 167 Synchronising the Set of Results with Excel 168 The structure of the measurements being taken is compatible with your Excel spreadsheet To change the Excel template Click the Setup button in the Measurements Dialog to open the Set Up Measure
30. Leica FW4000 User Manual We Experiment My latest Experiment f Raw Images mageF alder C Program Files Leica S MDW Leica S MDW Defaults upervisorhy latest f Processed Images f Both Load Options Filter f All images in folder f Ever n th image H My latest Experiment ImageSet p Raw Images I H T Probe Z Insert Reorder OF Cancel Apply 2 Click the appropriate Raw Images or Processed Images radio button 3 Define the Load Options by clicking the appropriate radio button and typing the P Eve nih image e required number of images 4 Define the experiment parameter order you want to use Refer to Chapter 6 Displaying and Comparing Sequential Images for a description on reordering the parameters 5 Define the filter required by building up a filter profile Use the Filter dialog 6 Check the Limit images to those where box Leica FW4000 User Manual 115 7 Selecta filter by clicking Probe in the left filter window Highlight the required filter from the right menu 8 Click Insert 116 Leica FW4000 User Manual 9 Select Z stack Position Number in the left filter window in the following figure 10 Select the function BETWEEN from the centre window 11 Type values for upper and lower limits in the two fields provided 12 Click one of the AND or OR radio buttons 13 Click Insert Filter Number of stack positions 3 stack Upper 5
31. LeicaFW4000 NetworkAssistant exe w Network Assistant _ O x Remote Database on off E Server Change SOL Server Password Share or Unshare Expernments Leica FW4000 User Manual 11 3 Define where your data Is to be stored by Checking the Remote Database on off box to hold the database for the workstation on a remote workstation for example one of the workstations in the network will be treated as the server or Unchecking the Remote Database on off box to hold the database for the workstation on the workstation for example the workstation will act as the server 4 Type the name of the database server in the Server text field for the remote workstations only W Note Available server names are shown in the drop down list This is usually the name of the workstation as It appears to the others on the network but may be something different XS Note The workstation assigned as the server must be switched on and fully operational with the SQL server running for the network set up to work 5 To share experiments which were created when the system was not networked but which are now on a mapped network drive Click Share or Unshare experiments and type the mapped path of the relevant experiments Click Share w Share or Do not Share Experiments e _ O x Location of espernments to be shared C Program Files s Leicar 400040 ata de Path name with mapped network drive
32. Preferences EE E EEE e m EA aan sera ares eee ingots Sample Experiments joe User Interface Language Autocontrast Images during Capture Show Image Viewer after Capture Generate Composites after each capture Generate MIP s after each capture Show Image Gallery after Capture Show Live Image during Capture jf Show Grey Levels as percentages Save colour with images 8 bit Open Gallery after Loading Experiments Show Image Viewer after Capture in Live Image Setup If Use loons for 42 N T Buttons Cancel 26 Leica FW4000 User Manual Setting General Preferences gt Click the General tab in the Preferences dialog User Interface Language English Autocontrast Images during Capture Auto Contrast each Stack Show Image Viewer after Capture None Generate Composites after each capture Generate MIP s after each capture Show mage Galler after Capture Show Live Image during Capture A Ee Ee tee Show Grey Levels as percentages x Save colour with images 6 bit Open Gallery atter Loading Experiments E Show mage Viewer after Capture in Live Image Setup Use Icons for serrar MT Buttons x Cancel gt Choose the User Interface Language from the selection menu gt To generate a composite image using the probes selected after each capture check the Generate Composites after each capture box Leica FW4000 User Manual 21 nb 28 To generate th
33. Solutions 3 In the Tool style section of the dialog select Alternative This changes the style of the toolbar adding the Measure Angle button 1 To measure the angle between two lines A 3 1 Select the Measure Angle button domme 2 Draw the angle on the image by clicking at the start of the first line 3 Click at the vertex 4 Click at the end of the second line 5 The angle will be displayed on the image as shown on the right side Leica FW4000 User Manual 183 Printing a Set of Results To print a set of results click the Print button in the main toolbar in the Measurements dialog S Frint Image m 184 Leica FW4000 User Manual Chapter Nine Enhancing your Image Chapter Overview This chapter contains the following topics Opening the Image Viewer Removing the Background Maximum Projection Channel Mixing Sharpening an Image Smoothing an Image Enhancing the Contrast of an Image Image Equalisation Aligning the Component Images Converting an Image to Brightfield Oe Oo Oo OM Sd Leica FW4000 User Manual 185 Opening the Image Viewer 4 yy 186 Before you start to process images in the Image Viewer you must select and export a component image as required from the Image Gallery You can open the Image Viewer by either Double clicking on the image in question in the gallery Click Process in the Experimental Setup Note Before you export a set
34. This chapter contains the following topics Introduction Viewing Images Displaying and Selecting Images Displaying and Comparing Sequential Images Previewing Composite Images Building a Profile for the Images to be Selected Saving Selected Images Defining the Image Gallery Settings Preparing Selected Images for Processing Preparing Selected Images for Publishing Replacing Images Oae el e e a OU Hh OH FH SO Leica FW4000 User Manual 105 Introduction 106 This chapter describes how to view the images captured during the image capture stage of the Environmental Setup process flow This review only allows you to view the images from your currently loaded experiment or from the experiment you have just run However in the Lab Book you can view the images from a chosen experiment in the gallery by selecting an experiment and clicking the Gallery button Refer to Chapter 7 Processing mages for information on measuring enhancing and deconvolving your images The software allows you to view images in two distinct ways Display the images in the sequence in which they were captured Z or T or by fluorochrome probe Display the images as a composite image of the output of all probes I N T and Z but based on one that you select The images can be manipulated to produce a composite image by combining the effects of different probes Leica FW4000 User Manual Viewing Images m yo review the images you have
35. To change the size of a custom defined image area click the mouse and redraw the required new image area Setup Multi Focus Use same Exposure for all Probes Change Experiment n To ensure that you can view the whole image in the image window right click the mouse in the image area and click Fit to Window n To display an indication of whether the displayed image is over exposed blue is added or under exposed red is added right click the mouse in the image area and click Show Saturation Leica FW4000 User Manual 69 To change the colour of the image frame right click the mouse in the image area and click Frame Colour To show only that portion of the image that appears within the frame right click the mouse in the image area and click Show Framed Image See the Live Image Setup dialog that follows N O i AA 1 Add pee AKARE b Replace BREE One Probe Show Histogram SetupCamera To set the exposure for one probe and then use this setting for all other probes right click the mouse in the image area and click Use same Exposure for all Probes When you select No Save from the capture and save options drop down menus any images you capture are not saved To save the images in these situations right click the mouse in the image area and click Save in Lab Book or click the Save icon To start a
36. User Manual 269 S 210 Note If you were to paste this file into a text editor you would be able to view the actual settings for the deconvolution Paste PSF File Settings This feature allows you to apply the PSF parameters from one file into one or more files in the Batch This feature is generally used when similar files are collected using the same microscope setup To paste PSF settings 1 2 3 4 Select the Pollen_psf deb file from the Batch1 queue Select Copy PSF File Settings by right clicking the mouse and choosing from the menu displayed Click on the TOSpollen_psf deb file to make it active Select Paste PSF File Settings by right clicking the mouse and choosing from the menu displayed This automatically Pastes the Pollen_psf deb PSF parameters into the TOSPollen_psf deb file Close all views before going on to the next tutorial Leica FW4000 User Manual Chapter Eleven Publishing Results Chapter Overview This chapter contains the following topics Introduction Setting up the Publishing Environment Printing Images Selecting the Images to be Published Setting Up the Print Image Environment Producing a Report Montaging Images Producing a Web Page Producing a Web Montage Making Movies Of Leica FW4000 User Manual 271 Introduction 212 You can publish the results of your experiment by Printing reports using the supplied Leica template that can also
37. an image for the restoration of features at a sub pixel resolution level You may process multi frame time series image sets individual colour channels or intensity images The 2D algorithm is able to suppress noise while retaining quantitative accuracy total number of photons in the image thus allowing you the ability to make valid quantitative measurements The new high speed deconvolution algorithm in the 2D Deconvolution decreases processing time by a factor of five or more Leica FW4000 User Manual Setting Up a 2D Deconvolution Select a Region of Interest 1 To select a region of interest 3 1 Click and hold down the left mouse button while dragging out a Region of Interest ROI similar to the one shown below O aloe Lil ott MAE Pry 2 Select 2D Blind Deconvolution from the Deconvolution menu The 2D Deconvolution dialog appears with the Standard tab as the active tab Leica FW4000 User Manual 255 256 a PRESTO TO TOTO A i ak hea honena Ponen a Pona etl a a D Dew euin eo E e w SSE ENN AEE N kandar Erpat 33 Fares late F dehh i erg r red CE Cuert Wize Mutple Frame if lark mj lezin of hiver o while Coo DPetorytlhon Pars nzter mage Irla Be aeda f E Iraze C Reoz o E f oe PHF lua aliri E ae I FCI C Bie Wilts d Inlet En B ars Frei Pav nuli fj Los C Medun Hih Obra Yella 2 4 Set the parameters to th
38. and choose Slice by Slice Cropping The Cropping Mode Selection dialog appears Press the Apply Region button An unchecked check box appears to the left of the number 1 in the list box This represents the first region of interest selected Click on the check box next to the number 1 to select it A highlighted overlay appears in the selected region Note You may now choose the Crop function or the Remove Region function Crop function Crops out the previously selected region of interest and will display the cropped data as a new data set Leica FW4000 User Manual Remove Region Removes the region of interest from the data set and will display the removed region as black within the data set This function is useful in removing artefacts from data sets You may select from one of three following options Apply to current slice only allows you to select a region of interest on the current slice Apply to all slices takes the selected region of interest on the current slice and applies it to all of the slices in the data set A new volume is created from the cropped area of interest Apply to given slice range takes the selected region of interest and allows you to apply it to a specified range of slices The newly created and cropped volume will only contain the slices you specified 1 To crop a region of interest 3 1 For this tutorial select Apply to given slice range option and type18 for the From slice and type 50
39. and principles of the program Aw Refer to Chapter 3for information on running Quick Start 8 Leica FW4000 User Manual Preparing Your Hardware and Samples 13 To prepare your hardware 3 i 1 Ensure that your microscope and fluorescent lamp power supply is switched on If it is an automated microscope you will need to wait until it is initialised before starting Leica FW4000 2 Turn on your workstation and start Leica FW4000 3 Turn onthe camera power supply if applicable 4 Ensure that the fluorescent lamp is working properly and is correctly centred and working at maximum fluorescent capability 5 Check the appropriate filter cubes that visualise each individual fluorochrome are fitted 6 Check that the connection between your workstation and your microscope Is working correctly 1 Preparing a sample 1 Take a sample and visualise it with a high power oil immersion objective The type of sample depends on the type of analysis you are performing When you have a suitable specimen pull out the light path bar to direct all the light to the camera and then close the shutter Leica FW4000 User Manual 9 Using Leica FW4000 in a Network Environment You can network a number of Leica FW4000 workstations in order to access experiments captured on one station over the network on an additional workstation Checks have been added to the software to handle the following scenarios Access of the same experime
40. before deciding whether or not to retain your changes 3 Click Apply or Apply to Selected in the Image Viewer to save your enhanced processed images The Saving Images dialog Leica FW4000 User Manual 207 allows you to define the processed image folder or the defined image set as the location for saving your Images 4 Click OK to save your processed image or stack or click Cancel to close the dialog without saving any changes F Where do you want to sawe the mages 7 f To an existing Image Set Processed Images B To anew Image Set P AS Refer to Chapter 7 Using a Processed Image Setfor further information This option can be used on fluorescent images that have a dark background and a light specimen You can convert the image to brightfield by performing an inversion resulting in a dark specimen on a light background The original image is shown below on the left and the image after brightfield has been applied is shown on the right gt X DAPI E x DAPI 208 Leica FW4000 User Manual Chapter Ten Advanced Deconvolution Chapter Overview This chapter contains the following topics Exporting Images from Leica FW4000 to Leica Deblur and 3D Visualise Tutorials for the Deblur View Menu Items m Single View m Triple View 3D Viewer Enhancement utorials for Projections Menu Items Maximum Projection Minimum Projection Sum Projection Voxel Slice Viewer Alpha Blending Best Focus u
41. button 4 Press the AUTO button to the left of the slider to cause the Slice Viewer to cycle through all the slices automatically AutoPlay displays the slices sequentially 5 Once you have viewed all the slices pressing the STOP button to halt AutoPlay Note It is important you review any data set you would like to enhance by deconvolution You are looking for a smooth transition of data between slices A data set that is a good candidate for deconvolution will show the Leica FW4000 User Manual 221 slices slowly going into focus and then out of focus 6 Close the Slice Viewer dialog clicking the close x in the upper right corner of the Slice Viewer dialog Alpha Blending This function is available only in the 3D Viewer menu and it simulates the effect of transparency translucency and opacity in the data set The alpha blending factor percentage defines by how much the intensity value of the reflected ray is modified after the ray reaches the first threshold value Voxel and passes through the sample on to the next threshold value Voxel The Alpha Blending factor determines how the layers of the volume are displayed To apply Alpha Blending 1 Make the Max Projection of your data set the active view 2 Open the 3D Viewer from the View menu 3 From the Projections menu select Alpha Blending The Alpha Blending projection will be displayed in the window Place the 2 views alongside each other to compare the views 4 Cl
42. can set maximum and minimum pixel intensity for an entire sequence of selected images Equalisation of the image intensities may be applied by clicking on the Contrast icon in the Image Viewer toolbar To set the values for a stack select only the stack in the Image Gallery and then use Apply to Selected n To set the values for a single image click Apply Leica FW4000 User Manual 203 204 To set the values for a sequence of images that you have selected in the Image Gallery set Apply to Selected To take maximum and minimum values from a captured stack that is to take the maximum intensity pixel in the whole stack and the minimum pixel intensity in the whole stack and to apply these to every image in the stack click Get Min Max from Stack followed by Apply to Selected Leica FW4000 User Manual Aligning the Component Images XS Note This operation can only be performed on component images However you can select this option even when the composite image Is selected You do not have to choose a component image before opening the dialog 1 To align the component image 3 1 Open the Image Viewer 2 Select the Settings menu and click Alignment Correction to display the Image Alignment Correction dialog n Alternatively right click on the image window to display the Right Click Menu and choose Alignment Correction l Image to Align l Set Alignmert Texas Red T Reference FITC od Show C C
43. captured click Review in the Environmental Setup process flow The Leica Image Gallery dialog appears SS coure gt Sea PK Puoisn gt gt H BRAE fluorescent cell 1 cab m When you select Display Probes Composites and reorder the probes sequence the Reorder button appears When you click Reorder the radio button Display All Images is selected automatically gt To exit from this stage in the Environmental Setup process flow click in the top right corner of the Leica Image Gallery dialog Tip You can automatically launch the gallery with the images for your experiment displayed after the experiment has finished capturing This option is available by opening the FILE menu and choosing the General tab from Preferences Leica FW4000 User Manual 107 Displaying and Selecting Images 108 The image Gallery menu and the image radio buttons allow you to display and select all the images you have captured or only a subset of them aallery as i ae ee ae ace ae Bands ASE SA T Se Een sis sbirro E TET r ste aim ote aa x seh ate x pasean i3 ae sre ath oth tote ora ats th th ata ot a i k es r EEE yea afk ath F HEEE EEF Re EEEE BE FAAEE EEEE EEEE yr PA EA EEEH a ee oe n a aa J 4 te m m i j a a ae be ana re bet a la al an 3 a x n ma a 4 n 3 a i a a He ae aie a 3 te Past To display all the captured images click the image gallery Gallery menu and click Display All images
44. cases where speed is important A typical processing time is under two minutes with a 256 X 256 X 32 data set Pentium III 450 MHz 1 To use inverse filter deconvolution 3 1 Select Deconvolution Settings from the Settings menu and choose Standard Settings Double click within the image to display the Standard Settings dialog 2 Check the following dimensions for the Image Dimensions tab Dimensions Pixels Microns Width 90 32 124 Leica FW4000 User Manual 259 260 Height 80 28 56 Depth 110 44 3 Change the Spacings Microns by typing the new values Dimensions Microns X 0 3636 Y 0 357 Z 0 4 4 Inthe Microscope frame check the values are Dimensions Selection Numerical Aperture 0 7 Refractive Index Oil 1 515 Modality box Fluorescence Emissive Wavelength frame Probe left blank Channel 1 wavelength 540nm Channel 2 Blank Channel 3 Blank 5 Click OK on the Standard Settings tab 6 Select Inverse Filter from the Launch menu The Inverse Filter Parameters dialog appears 7 The Image Dimensions frame contains the data set s Width Height Depth and Spacings Check that they are the same as listed above The Phase Content Expected should be used if the Specimen Is a Transmitted Light Brightfield data set and exhibits significant phase characteristics for example areas of the Specimen appear brighter than the background For this data leave the Phase Content Expected dialog unchecked Leica FW400
45. data Leica FW4000 User Manual i New Save Print Copy Setup _ Ha V Apply Measure Frame SSS Se aes T a O C D L Gu Bee He Renee Canser ciel Mest EA tar sae Fir Fire iG Circle drag left mouse to determine size To change region name select row and enter new name Click the Enlarge button to enlarge the Measurements dialog The dialog will enlarge to contain the full set of a data gt Leica FW4000 User Manual 155 Specifying Measurement Parameters 1 To specify the parameters you want to measure 3 4 Click the Setup button in the Measurements dialog to display the Set Up Measurements dialog SEUR Alternatively click Settings in the Settings Measurement dialog and choose Setup Setup Point Small Square Medium Square Circle Ellipse Line Area Measure Objects Delete Selected Region Clear Regions 2 Selectthe Parameters tab 156 Leica FW4000 User Manual F p k Available Parameters Selected Parameters Length width Intensity Sum OF Cancel The Parameters tab allows you to specify the parameters that you want to measure The Available Parameters list displays a list of parameters that are currently not in use The Selected Parameters list displays the parameters that are currently in use n To select an available measurement parameter click the required parameter Click the Select button to move it to the list of selected parameters
46. deb deconvolution parameters into the PollenPA deb file Leica FW4000 User Manual Change Settings This feature allows you to change the setup and deconvolution parameters for a file in the Batch 1 To change the setup and deconvolution parameters for a file in the 3 Batch 1 Select the file from the Batch that you would like to change For example the PollenPA deb file 2 Select Change Settings by right clicking the mouse and choosing from the menu displayed The Launch 3D Deconvolution dialog appears Change the Save Interval setting to 20 and click Update Batch This will update the settings in the Batch for the PollenPA deb file Change PSF File This feature allows you to change the PSF file used in a Non blind deconvolution 1 To change a PSF file 3 1 Select the PollenPA_psf deb file in the Batch file 2 Select Change PSF File by right clicking the mouse and choosing from the menu displayed The Open dialog appears Navigate to the Batch folder and select Pollen_psf deb Click the Open button The file will automatically replace the PollenPA_psf deb Copy PSF File Settings This feature allows you to copy the PSF File parameters from one of the files in the Batch 1 To copy PSF file settings 3 1 Select the TOSPollen_psf deb in the Batch by clicking on it 2 Select Copy PSF File Settings by right clicking the mouse and choosing from the menu displayed This feature copies the file s parameters Leica FW4000
47. dialog Leica FW4000 User Manual 303 Retrieving Experiments from Archive 1 To retrieve an experiment from archive 3 4 Click the Retrieve radio button The Retrieve Data from Archive dialog is displayed Woe A my OX DD 78 48 mancen ke eee Undo ae P Archive f Retrieve Browse Overvrite existing experimentis ME Owner Archive Databa Archive Volume Archive Date ay amp DefaultUser El DefaultUser ZTM Pwd S00 Wol 14 01 2004 ZTN F400 Voll 14 01 2004 HitogreenERRedT 1 p F00 wol 14 01 2004 DefaultUser Default E penmenti d Tempe Ft g0 wol 13 01 2004 4 results 2 If the experiment you wish to retrieve is displayed in the list highlight the experiment you wish to retrieve 3 Click the Retrieve from Archive button The Retrieve Experiments dialog is displayed A 304 Leica FW4000 User Manual Do you wish to retrieve the following results to the main database file ArchiveDB_VC Default ser c Program File 4 Click OK to retrieve the experiment or click Cancel to cancel the operation 5 If the experiment you want is not in the list select the Browse button to browse to the location of the experiment Leica FW4000 User Manual 305 306 SS d F400 a Eds A E3 FwArchive FITC2_PSF zip T Mydrchive FITC1_PSF Pe l or SLOP rete al 6 Select the desired experiment s and press Retrieve 7 Close the d
48. flagged for deletion but not deleted immediately When you close the Archive form you must confirm that you still want to delete the experiments gt Click the Undo icon to undo the most recent set of deletes 310 Leica FW4000 User Manual
49. for the To slice 2 Press the Crop button A new Untitled data set of the cropped region appears in a Slice Viewer projection 3 Repeat the steps above and choose Remove Region see step 7 to remove a region of interest Tip If you desire to Crop out a different shape on each slice or only ona few slices you should select the Apply to current slice only choice The Apply Region button must be pressed to have the current selected region take part in the Crop or Remove Region When a region is selected for cropping and the check box is checked the first highlight will be light blue Select another slice using the scroll bar at the bottom of the Slice Viewer and repeat the steps of selecting a region pressing the Apply Region button and checking the box left to the number in the list box XS Note The Clear Selected Regions button will remove the crop overlay from the regions that are currently selected 4 When you have chosen all of the desired cropping regions press the Crop button A cropped volume will be displayed with Leica FW4000 User Manual 245 Ro only the foreground image data from the highlighted regions all else will be setto background Note Do not close Neuron deb Continue on to the next section Extend Slices 246 An image can be extended have additional optical slices added to it along its Z axis This function allows you to generate additional image slices based on linear interpolation and app
50. in the experiment This is the default image set used when the Saving Images dialog appears Aw Refer to Chapter 7 Using a Processed Image Setfor further information Leica FW4000 User Manual 145 Defining a Region of Interest 146 All image enhancement operations apart from background removal can be applied either to the whole image or to a Region of Interest ROI You can define the ROI to encompass any areas or features in the component image in which you are particularly interested There are two methods of defining an ROI By using the manual image processing tools through the ROI option in the Settings main menu or the Image Toolbar The types of region you are recommended to set manually include Straight rough outline or a small number of regions You may only enhance the images in the ROI defined using this method Features defined using the Define Ftr button on the bottom of the Measurement dialog You are recommended to use a feature to define complex regions or regions comprising multiple objects You may measure and enhance ROI when using the Define Ftr button The Define Ftr button allows you to assign colours line thickness and object names to regions The purpose of accessing the ROI dialog from within the measurements area is to use the areas defined for feature measurement as opposed to image processing Note ROI can only be defined on component images However if you choose Use ROI from the Sett
51. is acquired the brightness and contrast are determined by a number of factors Specimen signal intensity Microscope setup Camera settings and integration time Video levels In some cases even careful attention to these parameters does not provide a sufficiently high contrast image In these cases you can use the image enhancement capabilities to adjust the contrast and brightness of an image that has been acquired To enhance the contrast of an image 1 Open the Leica Image Viewer dialog 2 Select the Image menu and click Contrast to display the Image Contrast dialog Alternatively click Contrast in the Image toolbar to display the Image Contrast dialog d Leica FW4000 User Manual File Edit View Image Database Window Settings Contrast Settings Gamma Contrast Brightness Apply Apply to Selected f Change Image Data Get Min hax from Stack Raw Images Image_TO01_S001_2001_COMP tr 844x649 aii n To recover the original image when you have checked Change Display Only click Reset gt Use the Gamma slider to make the darker areas in the image brighter or the lighter areas darker DS Note Gamma correction is a logarithmic transfer function Gamma values less than 1 tend to brighten the darker parts of the image more than the brighter parts Gamma values greater than 1 cause the darker parts to be darkened more than the brighter parts Avval
52. is blank Verify that the Use Recommended Expert Settings is enabled Click OK Total Iterations 232 The Total Iteration field under Deconvolution Settings allows you to choose the number of iterations performed during a deconvolution The higher this number the more times the data will be processed Lower iterative numbers will execute in less time whereas the higher iterative numbers will take more time and generally provide improved resolution Beyond a certain number depending upon the imaging modality widefield or confocal the deconvolution may provide unstable results Leica FW4000 User Manual When you wish to use either Widefield Data or Confocal Data the following guidelines will help you choose the appropriate number of Total Iterations and the deconvolution performance setting For Widefield data you are recommended to use Best High Moderate for the Deconvolution Performance setting and start with a value of 80 for the number of Total Iterations Consider the following criteria when selecting a number of Total Iterations Values Results 1 80 Minimum use if speed is the most important consideration 81 100 Recommended as initial range of values 101 350 Maximum recommended under normal conditions use if resolution is the most important consideration 351 500 The higher iteration settings may provide superior resolution but require a very low noise level lf you choose Faster Processing by ticking Perf
53. measured with a position gauge for example Heidenhain or Mitutoyo Data Background For a Darkfield sample the Data Background should be set to Dark and for a transmitted light brightfield sample the Data Background should be set to Bright Deconvolution Settings Before you are able to deconvolve a data set you must set the Standard Settings and the Expert Settings The objective of this tutorial is to give you a feel for setting both the Standard and the Expert Settings gt Note This tutorial contains many explanations and guidelines that are important to the understanding of Standard and Expert settings It is recommended that you read over the explanations and guidelines to assist you in specifying the Standard and Expert settings Leica FW4000 User Manual 229 Standard Settings The Standard Settings allow you to specify parameters needed for deconvolution There are three sections Deconvolution methods Optics settings Deconvolution settings Inputting these parameters is step one in the deconvolution process To input the standard settings 1 Create an XZ Sum Projection of a suitable data set 2 Select Deconvolution Settings from the DECONVOLUTION menu and click Standard Settings The Standard Settings dialog appears 3 You can now set up the standard settings Deconvolution Methods The Deconvolution Method for Standard Settings allows you to select between Adaptive Blind and Non Blind
54. new experiment or to delete the images in this experiment and start again right click the mouse in the image area and click Change Experiment 70 Leica FW4000 User Manual n To calibrate an image when you capture it right click the mouse in the image area and click Calibrate Aw Refer to Chapter eight Calibrating an Image for further details on calibrating your image Multi focus Acquire Multi focus acquire is available in the T Z and TZ versions of the application but automatic control of the microscope during a multi focus acquire capture will only be possible with the Z and TZ configurations which include Z control of an automated microscope n To use the Multi focus acquire feature click on the probe you wish to acquire in multi focus mode and right mouse click in the image window Select Set up Multi Focus from the menu to display the Multi focus Acquisition Setup dialog ra oe one Image Size Image Viewer Settings LabBook Calibra Set multi focus acquisition parameters Number of steps above focus Number of steps below focus Focus spacing um Manual focus adjustment Total travel 0 0 um Leica FW4000 User Manual 71 3 1 To acquire the multi focus image 34 2 Check on the Enable Multi focus Acquisition box Enter the number of steps below and above your current focal position that you wish to capture images Enter the focus spacing If you are work
55. stage plan select View E ARANA eCup from the main menu and click Stage permenant Plan This displays the Leica X Y Stage Plan dialog Leica SFY Stage Plan X 0 500000 0 0 C Pattern 30000 0 Limit Alarms C 6 D C Define Move Leica FW4000 User Manual Ma m n a n n n n n nm n n mm nnn EIren ejer Er tiTTe SEE 4 Setting up the Acquisition Device gt To change the camera you are Hardware Setup Help connected to select Hardware Setup Microscope Setup from the main menu and click Setup Acquisition Device Setup Acquisition Device dialog Optics Data Initialise Focus Initialise Stage Display Properties Relative Focus Correction This displays the Setup Acquisition Leica Image Driver Select Ea Leica Image Driver Select Ea Devi ce d ia O g Select the Image Driver that you require About Settings _Defauts x Help Cancel a S Refer to Chapter 4 Defining Live Image Acquisition for more information 48 Leica FW4000 User Manual Relative Focus Correction You can measure and correct the Z axis shift between filter cubes By using the Setup Focus Shift Correction dialog you can compensate for any relative focus shifts between filters during the automatic acquisition of component images 1 To enable focus correction 3 1 Click Hardware Setup in the main menu and click Relative Focus Correction This displays the Setup Focu
56. that may leak into the embedding medium among other causes You can enable Minimum Image Intensity Removal by setting the tick box that is found on the Expert Settings form that is a sub menu of Deconvolution Settings Use Recommended Expert Settings You are recommended to use the Use Recommended Expert Settings option WARNING As a general rule DO NOT MODIFY THE EXPERT SETTINGS until you have attained expertise in using the deconvolution application Do not adjust the Expert settings from their original default settings except In rare cases Do not change them casually All adjustments should be tested on a small sub field of the data set for example 64 x 64 x 32 If you disable the Use Recommended Expert Settings uncheck the check box the Go to Expert Settings button becomes active Expert Settings With a suitable data set loaded from the Settings menu select Deconvolution Settings and click on Expert Settings The Expert Settings dialog appears with two sections the Object section and the PSF section Leica FW4000 User Manual 235 i ys Expert Settings i x Subyolume R te Montage Sub volume overlap Fisela 5 Z Montage XY Guardband Pixels 25 H W Dynamic Subvolumes Quardband Fiels e Pre processing Intensity Correction w Accelerated 5 4 Detection W Minimum Intensity Removal Object First Guess Smoathed Raw Image Super Resolution Activate Sub pitel Proce
57. the images you are montaging 3 Choose a page orientation of either portrait or landscape 4 Click the ellipse and choose a template to use for laying out the montaged images 5 Check the Image Details Required radio buttons as required to identify the image details to be published with each image 6 Click OK to print the montage or click Cancel to cancel the operation Leica FW4000 User Manual 283 Producing a Web Page You can select the experiment and the results for the probes that you want to publish in your web page m Click the appropriate radio g Raw Processed a button and decide whether Images Images l Both to include Raw Images or Processed Images or Both In the published images 1 To produce a web page 3 1 Select the experiment radio button 2 Expand the image stack for the selected experiment 3 Select the desired probes at the Z plane level tc Print Image Montage Web Montage Movie Maker Image Sources 21 DAPI ee heli by 1 TRITC a Exported f Experiment Images Image Folders fe i Please select required image stack ST O B ae Composite Delete Exported Images ee ke 284 Leica FW4000 User Manual TA Note Ensure that you highlight the lowest level of selection that is Z1 Z2 or Z3 to display the full set of publishing buttons in the Publishing Gallery 4 Click Web Page in the Publish Galler
58. the Select Template dialog Select the appropriate template and click Open Browse Leica FW4000 User Manual 281 Select Template Look ir Templates do E BA LeicaF Ww 4000 2 dot PA LeicaF w4000 3 dot My Recent MEEA a Documents APF LeicaFW 4000 6 dot PA Leica W4000 8 dot BF MosaicFW4000 dot Desktop Ma D ocumernts a My Computer _ 4 a File name LeicaFw 4000 4 dot My Network Files of type Templates Cancel P aces Open as read only 6 To preview your report click Preview 3 Preview 7 Click Print to print your report or click Close to close the Create Report dialog without printing your report 282 Leica FW4000 User Manual Montaging Images The Montaging feature allows you to select Z and T series images from different image sets and publish them as a single set X Note To use this feature you must have exported the required images from the gallery and the Exported Images option must be selected in the Image Sources options 1 To montage your selected images 3 1 Click Montage from within the Publish Results dialog This displays the Settings for Montage Montage Print dialog i W x Columns s Orientation Portrait Temolate c Program Files Lerca4 SMDW T amplates Leicad SMD 2 dot i Image Details Required W Capture Time J Stage Position 2 Choose the number of columns to give the best layout for
59. you wish to print Setup the print parameters Selecting the Images to be Published 274 You must select and prepare the composite or component images you wish to publish To prepare images for publishing 1 Open the Image Gallery dialog 2 Select the composite and component images to be published Refer to Chapter 6 Preparing Selected Images for Publishing for more information 3 Open the Gallery menu and click Export selected Images to Publishing You can select the precise images you want to publish by expanding your experiments image stack and then dragging and dropping those images into the print image frame Click the appropriate radio g Raw Processed button and choose whether to Images ss mages include Processed Images or Raw Images in the set of published images or to use Both C Both Leica FW4000 User Manual 1 To select the images you wish to publish 3 1 Expand the image stack for the selected experiment tia EE me EJ Print Image Montage Web Montage Movie Maker Image Sources 71 DAFI 71 FITC 71 TRITC rs Exported f Experimert Images Image Folders pmu pma pa I I Z2 DAFI ee FITC 22 TRITC Please select required image stack MitogreenE AR edT 1 Z3 DAPI Za FITC Z3 THITC Delete Exported Images ae ey 2 Click the reference to the image you wish to print In the case above TRITC Z1 has been selected This image is displayed in the viewer
60. your processed image or stack or click Cancel to close the dialog without saving any changes f Where do you want to sawe the mages 7 0 To an existing Image Set Processed Images C To anew Image Set res Refer to Chapter 7 Using a Processed Image Setfor further information WARNING When using the Apply to Selected function ensure you have selected the image set you wish to process in the gallery Leica FW4000 User Manual Maximum Projection X Note Maximum Projection is a Leica FW4000 product option that is only available if you have purchased the Enhancements module along with FW4000Z or FW4000TZ modules Maximum projection takes a Z stack and selects the highest intensity pixel in the Z plane for an X Y position The max projection you created is stored as a raw image If you perform another operation and save the image it will be a processed image 1 To set the maximum projection 3 1 Open the Image Viewer 2 Select the Image menu and click Max Projection to display the Image Viewer as shown Projection button in the Image Toolbar and display the gt Alternatively to set max projection click the Max Image Viewer as shown hia A lt gt File Edit View Image Database Window Settings ROI N oS senna Raw Images Image_T001_SO01_Z001_COMP tt 8444649824 Raw 241 2002 Leica FW4000 User Manual 189 190 Click Apply or Apply to Selected in the Image Vie
61. 0 User Manual 8 Click Start Leica Deblur and 3D Visualise executes the Inverse Filter deconvolution The status bar indicates the progression of the Inverse Filter 9 When the Inverse Filter method has finished a new XY Max Projection view appears This is the result of the Inverse Filter deconvolution Do not close the Inverse Filter result Untitled data set Compare it to the results of the Nearest Neighbours deconvolution from the next section Nearest Neighbours No Neighbours Use Nearest Neighbours No Neighbours with Widefield Fluorescence and Transmitted Light brightfield data sets only The Nearest Neighbours algorithm is the fastest algorithm available It works by deconvolving one image slice at a time As a trade off in order to achieve this speed it is less accurate than either the Blind Deconvolution or the Inverse Filter It should be used in cases where speed is most important Typical processing times are less than 1 second for a 256 x 256 single image slice and less than 1 minute for a 256 x 256 x 32 3D data set Pentium III 450 MHz Leica Deblur and 3D Visualise contains two methods for running the Nearest Neighbours deconvolution on a data set Processing Stack that runs the specified deconvolution on the entire image stack Processing Current Slice that is run while viewing one slice of an XY Slice Viewer of the image stack SS Note Do not close the Pollen deb data set Continue on to the sec
62. 01 W Timestamp attop at bottom J First frame is 0 mS J outline text Comment Movie 10 October 5 To timestamp each frame of your movie click Options click Timestamp and choose the position of the timestamp Its font and colour and define the first image to be time stamped Leica FW4000 User Manual 289 35 Note You may define the first image as being acquired at time point zero as opposed to the real time the image was acquired and for each successive image to be stamped relative to the zero time point S Note Movies made in the movie maker from single channel images will appear in colour 6 Click Write AVI to display the Choose a filename to save AVI to dialog Save the movie with an appropriate filename in a directory of your choice Choose a filename to save AYI to Save in E Desktop x El cH a Hetwork Neighborhood My Briefcase Save as tupe favi Files Cancel 7 Click Save to save the AVI or click Cancel to cancel the operation The Video Compression dialog is displayed Video Compression Compressor Full Frames Uncompressed Cancel i Compression Huality Configure al E AGU F key Frame Even E mames Date Rate a EB Se 290 Leica FW4000 User Manual 8 Choose the type of compression required for your movie system from the Compressor menu ideo Compression Compressor Lk Cinepak Codec by A adius Cancel Microsoft wideo 1
63. 071 2004 07 54 22 Time Positrons 3 We Limit images to those where Clear P Probe_ID 497 AND 2StackPos_IN BETWEEN 2 AND 30A stackPos_FL BETWEEN 2 90880060110829 AND 2 91119939589171 JAND Time_DT BETWEEN 14 01 2004 07 5419 AND 14 01 2004 O54 22 AND Timeseg IN 3 Time Sequence Number E f AND 1l 5 Insert c OF C AND C OR X Note If you use a combination of AND and OR in your selection you must insert opening and closing brackets to make the meaning unambiguous Otherwise the selection will not be what you intended X Note The function you select changes the range of information you must Supply in the right window 25 Click Apply to apply the image profile without closing the dialog If you are dissatisfied with the new profile click Clear and repeat steps 7 to 23 until you are satisfied 26 Click OK to accept the total image profiles or click Cancel to cancel the operation Cancel Leica FW4000 User Manual 119 Saving Selected Images When you have selected the particular images to review from the complete set you captured you may save them 1 To save the selected images you want to process 1 Click the Image Gallery File core menu and then click Save Select Images As to display the Select a folder for the Images dialog epee oe fee ee Bee Fete eet ie eee Se ae E ee ARREA 5 x estas ected Ima ages strc perier re T ptt Soe oe Eg Piagam Files Ej Leica
64. 2 91 Stack Lower 47 09 ke Limit mages to those where Clear P Probe_ID 491 AND 25tackPos_IN BETWEEN 2 AND 30A StackPos_FL BETWEEN 2 90580060110829 AND 2 91119939889171 Z stack Position BETWEEN 2 90880060110829 C AND m sop E nse and 2 91119939869171 C ANDI COR 14 Select Z stack Position in the left filter window 15 Click one of the AND or OR radio buttons 16 Click Insert 17 If necessary click a closing bracket to match the opening bracket in the previous AND or OR Leica FW4000 User Manual 117 118 18 Select Time in the left filter window 19 Click one of the AND or OR radio buttons 20 Click Insert Filter Counting 3 E arliest Time HCE 1 FA IMAGE 4701 2004 OF 54 19 HCE 1 Stop Latest Time 14707 2004 OF eb 2 Time Positions 3 mw Limit mages to those where Clear P Probe_ID 491 AND 45tackPos_IN BETWEEN 24ND 3 0F stackPos FL BETWEEN 2 905800601 10629 ANC 2 91119939859171 JAND Time_DT BETWEEN 14 01 2004 OF 54 179 SND 14 01 2004 OF 5424 AND Benes 5 meei and 14 01 2004 07 54 22 M AND ORI 21 Select Time Sequence Number in the left filter window 22 Type the required time sequence number in the right field 23 Click one of the AND or OR radio buttons 24 Click Insert Leica FW4000 User Manual Filter Counting 3 E arliest Time HCE 1 4701 2004 07 54 19 IMAGE 14 01 2004 07 5419 HCE 1 Shop Latest Time 1 4
65. 5 tif abe _O001 46 abe_OO01 47h abc 000146 tif abe 0001 49 ti abe 0007 500 tif abe_O00152 tif abc_O00153 tif abe 000154 tif abc 000755 tif abe_O0015 tif abe 000758 tif abc _000159 tif abe O00160 tif abc OO0162 tif abc 0001 63 tif 7 Cancel Reesor Le For each image imported click on the probe name and a drop down arrow will appear from which you may select the probe name you wish to use for the imported image Note The image size displayed in Leica FW4000 will be taken from the first image Imported Note Composite images generated in third party software may be reviewed here but can be rejected from the import by selecting IGNORE from the list of probes Note If the images imported into one experiment are of different dimensions you are not able to generate a composite image from them in Leica FW4000 Leica FW4000 User Manual 309 Deleting Experiments 1 To delete an experiment 3 41 Select one or more experiments which you want to delete in the archive view or one or more archived experiments to delete in the Retrieve view 2 Click the Delete button The Delete Experiments dialog is displayed X ea a BF BPAE cells 20x 89 23 09 2005 Default ser 3 Click OK to delete the experiment or click Cancel to cancel the operation gt Note In Retrieve mode the archive files are deleted immediately In Archive mode experiments are
66. Chapter One Introduction Chapter Overview This chapter contains the following topics Oe Oe Oo Oo DO Leica FW4000 User Manual How to Use the Leica FW4000 User Manual Style Reference System Hardware Configuration Software Installation Help Using Leica FW4000 Preparing Your Hardware and Samples Using Leica FW4000 in a Network Environment Configuring Leica FW4000 Stations ina Network Automatic Calibration How to Use the Leica FW4000 User Manual This manual is part of the Leica FW4000 User Manual and comprises two discrete areas containing information targeted at different user competencies You can either 4 Use the Quick Start Chapter 3 walkthrough of the major elements of the end to end process Leica FW4000 has an easy to use and intuitive interface therefore the walkthrough Is simple and matches the design features built into the application software Perform experiments by using Chapters 4 to 12 sequentially There is one chapter for each of the major steps required to perform an experiment followed by a specific chapter covering the image viewer When starting to perform an experiment you are recommended to start at Chapter 4 Then work through the User Manual until you complete the experiment or arrive at a natural point to pause When you restart dip into the appropriate chapter and continue working through the process You could run a complex and lengthy image capturing sequenc
67. Click Web Montage from within the Publish Web Montage Results dialog This displays the Specify a name for the web page dialog Look in E3 Documents E Files of type HTML Files Cancel Open as read only 2 Type the name of the web page into the File name field 3 Browse to an appropriate directory 4 Click Open Leica FW4000 User Manual 287 Making Movies 1 To make a movie that features your selected 3 images Tes 1 Click Movie Maker from within the Publish Movie Maker Results dialog This displays the AVI File Constructor dialog f ize AMF Ie Constructor wa cell 1 cab Rawlmagess image TOOT 5001 O02 CO3 ti i cell 1 cabAawlmages iImage TOOT 5001 2003 COT 2 Click Check file list to run through the files listed to check that the image files are of the same type and size You can watch the filenames as they are automatically scrolled until the end of the list 3 Atthe end of the check file run Check file list Write AVI and Options buttons become active You may interrupt the check file run by clicking Cancel 288 Leica FW4000 User Manual 4 Choose the number of frames per second by clicking Options and typing a number in the range 1 to 30 In the box TOOT 5001 001 abAawlmagessimage TOOT SOOT 001 COT ti abAawlmagessilmage TOOT SOUT O01 COZ tit ab Aawlmagesimage TOOT SOOT O01 COS aA awl manestlmane TANT SAAT Aone CAT hf Frames per second 1 3
68. Defaults upervisor E d P4900 Voll Gq DefE xp dire Volume Label Fw vol o Ea eee ime Documents eal Ca drewi SD Ea Drew hole v New archive database folder 6 Convert images to 8 bit format Compress Create New Folder Masimum File Size Wib Archive Archive a Subset of the Images 5 Check the Existing Folder radio button to archive the experiment to the existing folder You will need to click Yes to the dialog that asks you to confirm whether or not to overwrite the existing experiment 6 Check the New Folder dialog radio button to archive the experiment to a new folder The Archive Experiments dialog shown above Is displayed 7 Browse the filestore directory hierarchy to select the directory in which to archive your experiment 302 Leica FW4000 User Manual 8 Type the name of the new folder in the Create New Folder field that will contain your archived experiment and press Create New Folder 9 Click Archive 10 Optionally you can use the check boxes provided to specify additional options e Save images as 8 bit in the archive or in compressed format Save large experiments to multiple volumes of a removable storage device You may wish to archive only a subset of the images in which case a dialog Is displayed similar to the Select Required Images dialog in the gallery to enable you to specify which images are to be saved 11 Click OK to close the Archive Experiments
69. Factor and the Z Kernel Width are now saved for this specific data set Note Do not close the Pollen deb data set Continue on to the section Leica FW4000 User Manual Processing Stack 1 24 To process a stack 1 2 D Select Nearest Neighbours from the Launch menu and select Processing Stack The Nearest No Neighbors Parameters dialog appears The settings for the Haze Removal Factor 0 97 the Z Kernel Width 3 and the Nearest Neighbours algorithm were determined above in the Processing Current Slice section The Phase Content Expected should only be checked if the specimen is a Transmitted Light Brightfield data set and exhibits significant phase characteristics for example areas of the specimen appear brighter than the background For this data set leave the Phase Content Expected box unchecked Click Start The deconvolution will be performed on each slice of the entire stack A status bar will indicate the progression Compare the results of the Nearest or No Neighbours deconvolution with the original data and note how the deconvolution removes haze and sharpens features Examine the Maximum projections and the Slice Viewers of the original Pollen deb data set the Inverse Filter and the Nearest Neighbours results in both the XY and XZ views Close all data sets before continuing on to the next section Axial Blind Deconvolution S Note Use only with Confocal data Axial blind deconvolution also know
70. Fluorescent celli FITC 490 965 59 El BFAE Fluorescent cell Texas Red 596 3853 35 oo Leica FW4000 User Manual 35 36 To create a new experiment click the Start New Experiment button amp Refer to Leica FVW 4000 Quick Start Guide for further information on the procedure for creating a new experiment You can view the image gallery of the experiment currently selected in the Lab Book from the Create a New Experiment or Open an Existing Experiment dialogs To do this click the Image Gallery button OEE DEL DLYA When you have expanded the Lab Books for your experiments you can easily collapse all the hierarchies eT by clicking the Redisplay Data button Note When you create a new experiment in the Lab Book an image set called Processed Images is created automatically in the experiment This is the default image set used when the Saving Images dialog appears There are a number of places where this dialog is used after clicking Apply or Apply to selected when using the deconvolution or image enhancement tools Once you choose where the processed images should go the image set that is currently In session changes in the drop down list at the bottom left of the image viewer This provides a mechanism for you to switch image sets from the Image Viewer The new image sets are also available in the Image Gallery and in the tree view in the Publish Results dialog Refer to Chapter 7 Using a Processed Image Setfor
71. IMAGE Notify stop 15 19 26 gt Cancel 1 To build a time sequence 3 1 Design and write down the proposed script for your experiment taking into account all the components listed in the table above 2 Click Insert After Select the appropriate action and set the time period duration or count meen 3 Repeat step 2 until you have compiled the complete sequence that represents your experiment XS Note As you highlight the time sequence elements the appropriate element controls displayed on the left of the Time Sequence Builder window are activated Therefore when you wish to edit an element in the time sequence highlight the element and make the changes by using those controls or by typing into the text box X Note You must change non variable time sequence elements by insertion or deletion 92 Leica FW4000 User Manual 4 Click OK or Cancel to cancel the operation 5 Ifthe logic of the time sequencer Is flawed a message Is displayed before you can move to the next stage of your experiment you must correct any errors Alternatively you can check the logic of your time a gee sequence by clicking Check Again if the logic of the time sequencer is flawed a message is displayed however before you can move to the next stage of your experiment you must correct any errors m Click Delete to delete the highlighted time sequence Delete commands m Click the left pointing arrow
72. IPLab STK stk Bitmap bmp or PGM pgm formats The Dimensions will need to be entered by you the first time an 8 bit 12 bit 16 bit or 32 bit format data set is loaded Spacing Microns The X Y and Z Spacing is the size of 1 voxel in microns in the X Y and Z direction respectively The Z spacing Is often referred to as the step size X Spacing is the width of one pixel in micrometers Tip The X Spacing can be calculated by dividing the spatial width of the image frame in micrometers by its width in pixels X Spacing Is entered in micrometers and ordinarily includes a decimal point An accuracy of 3 or better is required Y Spacing is the height of one pixel in micrometers Tip The Y Spacing can be calculated by dividing the spatial height of the image frame in micrometers by its height in pixels Y Spacing is entered in micrometers and ordinarily includes a decimal point An accuracy of 3 or better is required 228 Leica FW4000 User Manual Z Spacing is the sampling distance in micrometers between adjacent optical sections Tip Typically the Z Spacing step size between optical sections is selected by the user collecting the data set The Z Spacing can be determined by dividing the axial depth of the data set in micrometers by the number of optical sections collected WARNING It is critical to have the correct Z Spacing step size within a 3 margin of error This should be
73. Image Adding an Annotation Changing the Annotation Toolbox Settings Displaying Annotations Clearing Annotations Saving Annotation Measuring an Angle rinting a Set of Results gt gt gt gt gt UHH E E E Leica FW4000 User Manual 153 Using the Measurements Dialog m 154 Click the Measurement button in the Image Toolbar to aa ATE open the Measurements dialog Measurements Full View E 7 D amp IV Apply Measure Frame New Save Print Copy Setup oe i M 4 Define Ftr election Layer 1 Profile Fitri Ftr2 Circle drag left mouse to determine size To change region name select row and enter new name 7 The measurements dialog allows you to perform measurements within the image and view the results on the screen You can also save and load results export them to Microsoft Excel and print them using an Excel template Excel must be installed for these options to be available The measurement selection allows you to apply the measurement criteria to the images selected in the gallery The identity of the image is shown in the measurement table in the Z N or T column This activity may produce many measurements that may be filtered by probe lf required you can collapse the Measurements dialog to make it easier to draw on the image Click the Shrink button to shrink the Measurements dialog 44 The dialog will shrink to contain a reduced set of
74. Image menu The Select Regions of Interest dialog will appear Refer to Chapter 8 Performing Measurements for information on the Select Regions of Interest dialog Annotation opens the Annotation Toolbox and allows you to annotate an image with various items including text scale bars shapes and straight line distances Annotation may be saved with the image or to a specified file This is useful in defining an annotation template which can be used with many images Refer to Chapter 8 Annotating your Image for information on annotation Leica FW4000 User Manual 133 a p Th T AN 134 Calibrate allows you to calibrate the system Using a stage as micrometer and a live image draw a line corresponding to a known distance on the micrometer Click this button to display the Calibration Refer to Chapter 4 Setting up an Environment for Experiments for more information on calibrating from the Live Image Setup dialog Refer to Chapter 8 Calibrating an Image for more information on calibration p Measure Distance opens the Measurements dialog and bia allows you to perform various measurements on the image Refer to Chapter 8 Performing Measurements for more information on measuring distance ib Remove Background removes the background from the 29 image The Remove Background dialog allows you to adjust the threshold and specify the display colour Click and hold down the toggle button to compar
75. MDW E3 LeicaFw 4000 EJ DefaultSupervisor Mi New Expernmernt Te H Sl Wages File Type Bitmap Create Folder Cancel 2 Select the file type from a choice of Bitmap JPEG or TIFF 3 Create a new folder or browse to an existing folder as appropriate 4 Click OK to save the images or click Cancel to cancel the operation 120 Leica FW4000 User Manual Defining the Image Gallery Settings 1 To define the look and feel parameters for the Image Gallery 3 1 Clickthe image gallery Gallery tie Beh menu and click Settings to display the Gallery Settings dialog which is superimposed on top of the Leica Image Gallery see Pea Thumbnails Set the thumbnail attributes Picture width pels E4 Picture Height pels E4 Label Background Color fo UF Cancel Apply 2 Redefine Picture width and Picture Height by using the up and down arrows Leica FW4000 User Manual 121 122 Redefine the Label Background Colour as required by clicking the Label Background Colour box choosing the required colour from the colour palette and clicking OK The Gallery Settings dialog now appears again with the new settings defined Click Apply The Image Gallery viewed behind the gallery settings dialog changes to reflect the newly defined attributes If you are dissatisfied with the effect repeat steps 2 to 4 until you are Satisfied with the Leica Image Gallery attributes
76. MM MRH HEE EA SAA aie RE EEEE REET Era HEERE EEEREN Log on as different User es S r Ses Show Sig ri On oe ee ra per ot ee i e EEE a EEP SEER ae ee a Agee N TE Sonane Sone nee r en F ce oe E a H ECE ech eo E EE E E E S E S iti ete ee siete perth a RERE ERN i ines S EEE Se 2 ei mport ope Experiments oe ferent pee roa pee User Mame Level ID Deleted Logged On Machine Default ser 1 False ee DefaultSupervisar ee IEE Reinstate eases Leica Delete Cancel When you are logged on in supervisor view you may add delete or reinstate users S Note When a user is deleted from the system only that user or a 20 Supervisor can access their experiments Therefore either you will have to reinstate that user before you can for example archive or delete that experiment or the supervisor will have to do It Leica FW4000 User Manual Changing Passwords 1 To change your password 3 1 Click Change Password on the Manage Users dialog 2 Type your password into the Password field 3 Confirm your password by typing the identical password into the Confirm Password field 4 Click OK to accept the new password or click Cancel to cancel the operation User Name E efauktSuperisor Password Confirm Password Cancel Changing Users Details The Manage Users dialog allows you to Change a user s level of ID
77. Notice that the Notify element is not indented If it were aligned with the IMAGE element above by pressing the R button twice then the Notify would be part of the Every 5 seconds step and there would be many occurrences of the message on the log Sometimes this is desirable see below but not in this case The Notify element displays the given message on the log but will replace an occurrence of the character sequence T with the current time so you can produce additional time stamps during an acquisition gt Build an example that takes twelve samples with five second waits in between them then pauses until you tell the sequence to continue Follow with the acquisition of twelve further IMAGE samples with fifteen second waits in between each image capture Start f How otar now Counting 12 Notify samples to go C Wait 5 seconds A uYy _ _ _ IMAGE Until stop S Fause Daye om 30 Counting 1 EEE ENIA Wai 15 seconds eji 1 Wait gt Cancel 15 46 49 X Note The first Counting 12 element is subtly different from taking twelve samples every fifteen seconds because the time to take each sample will extend the duration of the experiment X Note The character sequence C in a Notify during the acquisition this will be replaced by the current unexpired Count in any Counting block 6 Note In this example there is no metronome Con
78. Rendering Range values are Values Results 0 to 100 The Max Projection Rendering Range 0 to 100 The Min Projection Rendering Range 0 to 100 Sum Projection Rendering Range 20 to 100 The default Rendering Range of Voxel Gradient Best Focus and Alpha Blending The default Alpha Value is 0 5 Load Settings This feature allows you to load settings previously saved using the Save Current Settings As feature Load Settings include all fields found in the Standard and Expert Settings Save Current Settings As This feature allows you to save the Standard and Expert Settings as text files for later use with another data set The file is saved with an set extension This section of the tutorial assumes you have the FitcDapi_crop tif data set open and that the Standard and Expert parameters have been set up as described in the proceeding sections on Standard Settings and Expert Settings 1 To save the current settings 3 1 With your data set as the active view from the Settings menu select Save Current Settings As Leica FW4000 User Manual 241 242 In the File name field type the name you would like for these settings For example Test_parameters set Click Save You have now set and saved the Standard and Expert Settings for deconvolution Close all views before going on to the next tutorial Leica FW4000 User Manual Tutorials for Operations Menu Items Crop the Stack This function takes a selected reg
79. Verify that the Disable PSF Constraints is disabled This removes the limitations placed on PSF gt Verify that the PSF Waist Is 1 This is the size of the narrowest part of the PSF usually measured in Airy Disc diameters The default settings are 1 for Widefield and 3 for Confocal m Click OK to close the Expert Settings dialog Operation Settings The Operation Settings in the main File menu allow you to specify Information regarding the File directory and the Rendering Range for various projections of a data set Files You can choose the location of the temporary files or the startup directory by either typing in the new location or browsing to the new location The startup directory is the location in which the software application will look for image sets XS Note You are advised to place the temporary files directory on a drive with at least 1 gigabyte of free space Rendering The Rendering tab allows you to set the range of values a pixel may have depending on the projection chosen or the threshold values for a projection The projection rendering ranges can be set for Max 240 Leica FW4000 User Manual Min and Sum Projections The threshold values can be set for Voxel Gradient Best Focus and Alpha Blending projections XS Note You are advised to leave the Rendering settings at their default values You may want to change the Rendering settings in the future for noisy or photo bleached data sets The default
80. Window HardwYre Setup Help Sica MICROSYSTEMS Leica FW4000 Cancel Add K eneossssssssssssssssssssssssssssssssss x Capture gt gt Review gt gt Process gt gt Publish gt gt Archive gt User DefaultUser DefaultUser BPAE Fluorescent cell 2 cal Number of Images 63 Image Disk Space 34 836 KBytes Approximate Total Duration 00 00 00 Astart oe e Kisu Suppo GJFeica Pr i Leica FW 89 Paint Shop S 7 Fw4000 c LS ORO WH 16 08 Zl AAi gt Click Close to close Leica FW4000 gt Click Lab Book to open the Lab Book Leica FW4000 User Manual 17 General Administration m To manage various aspects of system hardware position the cursor in the centre of the Desktop and right click the mouse This action displays the User Management menu To display the Desktop properties click Display Properties Toggling the Show Title Bar field shows or hides the Desktop title bar To show or hide the status bar at the bottom of the Desktop click Show Status To show or hide the main menu at the top of the Desktop click Show Menu To show the optics data and filter tools click Show Microscope For a description on using these tools refer to the section Viewing Optics Data Leica FW4000 User Manual Administration Tools m nb To show or hide the initial sequence of logon screens click FILE at the main menu and toggle Show Sign On To
81. a FW4000 User Manual 89 experiment actually started at 13 50 but becomes much longer and unreasonable if the actual experiment start time is later than 14 00 Tip Use fixed times of a day when building a time sequence with circumspection A warning is given at run time when the experiment encounters a fixed time of day that has already passed the system can only assume that the experiment is intended to use the next occasion that this time occurs which could be the following day Special circumstances may require fixed times for experiments but generally times relative to the start of the experiment are preferable wherever possible When times are entered in the various editing boxes the system will report these in a friendly phrasing 5 minutes instead of 00 05 00 but on the experiment log the times will be in the 24 hour clock representation which is how they are held on the database Checking the Time Sequence Logic 90 Clicking the Check button allows you to see what will happen with a particular Time Sequence by simulating the capture process you Simulation results Sequences captured 1 Time elapsed 0 00 09 Note This successful result could be invalidated if you change the experiment setup later to have a very long exposure time with the 5 second cycle too short to allow each IMAGE operation to complete The metronome will in this case miss a beat Therefore you are advised to check the time se
82. acquiring your images check the Turn Lamp off after acquisition box Tip You should check the Turn Lamp off after acquisition box when there is a long time delay between capturing images or sequences of images Also in these cases you might want to specify lamp delay When you have defined a short time sequence leave this box unchecked n To allow sufficient time for the lamp to be at its optimum brightness set an appropriate Lamp Delay measured in seconds This is important after you switch on the lamp during a long time sequence when capturing brightfield images gt To cause a bleep to be sounded when a new dialog appears or other event completes check the Enable audio warning box Unchecking the box turns off the bleep sound n To specify that the microscope details are to be read from the microscope rather than entered manually by the user when creating a new experiment check the Read objective and data from microscope stand check box Refer to The E Relative Focus Correction description on page 49 of this manual for a description on correcting the relative focus 32 Leica FW4000 User Manual Changing the Database Location The Database tab allows you to set the paths for the location of the IM1000 database gt Click the Database tab in the Preferences dialog Program Database C Program Files LeicaF4000D ata ample DatabaseIM_ afl Default Root Folder for User Folders C Prograrn Files Lerc
83. agnification changer that is currently selected for the experiment Objective and magnification changers are set in the Live dialog in the Advanced tab If your experiment was performed with the wrong objective recorded you can change it afterwards in the Lab Book Leica FW4000 User Manual 13 Manually calibrating the system for a given objective A user may decide to manually calibrate the system for a given objective Information about the nature of the calibration is recorded in the user column of the Calibration tab Calculated means the calibration factor was calculated automatically by the system or from a user defined calibration e FW4000 means the calibration factor was manually defined by a user You calibrate the system manually the calibration for all other objectives will be recalculated using the measured calibration as the basis for all other calculations You calibrate more than one objective manually the FW4000 will use the first objective calibrated as the basis for all other derived calibration values n To delete all manual calibrations click Clear All calibrations 1 After you have defined a calibration factor manually you must reset the calibration values in the table to derived 1 Uncheck and check again the automatic calibration box 2 Click Apply 14 Leica FW4000 User Manual Chapter Two Administration Tools Chapter Overview This chapter contains the following topics 4
84. an angle in the image you will need to select the Alternative toolbox style Leica FW4000 User Manual 179 180 4 Specify a style for the micron marker Choose from Horizontal bar Vertical bar Double bar Box 5 Specify whether you want the micron marker to appear on the left or the right of the image 6 Specify the line width in pixels 7 Check Save Colours and Settings if you want to retain your settings after you exit Lerca FW4000 8 Click OK to apply the settings and close the dialog or Cancel to close the dialog without saving the settings Leica FW4000 User Manual Displaying Annotations n To display annotations click with the right mouse button in the main image window or a gallery image and select Display Annotation from the right click menu s T0 view annotations ensure that the Display Annotations box is checked gt Alternatively you can use the keyboard shortcut Ctrl D to display annotations Clearing Annotations 1 To remove an annotation 1 Open the View menu 2 Select Clear Annotation Saving Annotation 1 To save an annotation overlay to disk 1 Open the View menu 2 Click Save Annotation Leica FW4000 User Manual Save Annotation to Disk i 2 x Save in E Images e fe ef EJ ea JImages D _ Stack Example Bovine Pulmonary Artery Cells calibrated qan a Bovine Pulmonary Artery Cells qan Bovine Pulmonary Endothelial cells
85. ary 400040 ata Default Folder for Archive Cancel Change the program if required by clicking the Program Set button This displays the Set Database Program dialog where you can browse and select a new location You can also type the location of the database directly into the text box This allows you to point IM1000 to a database file in a different location and or to a different database This feature is useful if you are accessing the database over a network or if you have created your own database The new database must already exist in the named location Leica FW4000 User Manual 33 34 Click on the Database Set button to change the location of the IM1000 database This displays the Set Database Program dialog where you can browse and select a new location The current location is displayed in the text box alongside the button You can also change the location by typing the new location into the textbox Click on the Default Root Folder to change the location of the IM1000 default root folder This displays the Set Root Folder Location dialog where you can browse and select a new location The current location Is displayed in the text box alongside the button You can also change the location by typing the new location into the textbox Click on the Default folder for archive to change the location of the Leica FW4000 default archive folder This displays the Set Archive Location dialog where you can browse and select a new
86. ation such as Median Noise Reduction Leica FW4000 User Manual 1 To select a smoothing type 3 1 Select White to smooth lighter features in the image 2 Select Black to smooth darker features in the image n To select a smoothing amount move the slider to set the amount of smoothing The larger the value the more severe the effect n To choose between different smoothing operations choose an Operation from the drop down list Available operations are Median Noise Reduction Morphological Fillholes Matrix Averaging Weighted Averaging Spot Removal Leica FW4000 User Manual Removes noise that is one or two pixels in size without drastically affecting the sharpness of larger features Fills in small features up to the size specified by the Amount slider Details that are smaller than the size specified will be removed in the resulting image while the sharpness of the edges of larger features is kept intact Blurs the image The amount of blurring is set by the Amount slider and affects all features in the image This is an alternative form of the matrix averaging that gives a slightly softer smoothing preserving more of the image information for a given amount This option allows you to remove spots from an Image once it has been acquired The Amount setting controls the spot removal factor This defines how bright in relation to the surrounding pixels the spot must be before it is removed In general th
87. aved Image_TOO2_5S001_ 021_C01 tif Saved Capture Sequence Complete Time Sequence ended Defaultlser T empE pCB 43P6 31 5844D efaultL ser Free Disk Space C bs Disk Space Needed C f Try Again mage Viewer Live Image T Enable Audible Warning n To launch the Image Viewer directly from the Capture Image dialog click Image Viewer 35 Note You might want to view the captured images after each time sequence to ensure that you have captured the images you want Leica FW4000 User Manual 101 102 Note This way of working provides a hands off automated image capture sequence such as time sequence or Z stack image sequences method Alternatively set the image frame and exposure you require whilst the image is Live and click Stop Click the Image Viewer button to display the Image Viewer where you will see the captured image displayed Adjust the size and screen position of the Image Viewer so the image viewer and XY dialog are open alongside each other To exit from this stage in the Experimental Process click the Close button in the bottom left corner of the Capture Images dialog Leica FW4000 User Manual Using the Image Capturing Tools 1 To stop or pause capturing images in flight 3 1 Click Stop to display the Capture Interrupt stop dialog You have paused or interrupted a capture Press Continue to resume acquisition ane Press STOP to stop acquisition
88. avi aStacktwinkle avi stopwatch avi List files of type favi Files avi E c 0285 Network Leica FW4000 User Manual Folders c program tes leicaasmdw fo oc gt Program Files B Leicad SMD CG Archive Images Cg DefaultUser C Images Drives 293 294 19 20 21 Set the parameters in the Video Clip Player dialog tools palette to the values you require to obtain the best effect when viewing the video of images from your experiment Use the double arrow buttons below the movie window to run the movie forwards and backwards You can stop the movie at any time by clicking the Stop button this is the square button in the centre of these controls Also you can step through the movie one frame at a time by using the single arrow buttons Click Exit to close the Video Clip Player dialog 22 Click the button a cross in the top left corner of the AVIFile Constructor dialog Leica FW4000 User Manual Chapter Twelve Archiving your Experiments Chapter Overview This chapter contains the following topics 4 4 Leica FW4000 User Manual Introduction Archiving Experiments m Selecting Experiments in Archive Using Properties m Archiving Experiments Retrieving Experiments from Archive Importing Tiff Images into Leica FW4000 Deleting experiments 295 Introduction 296 During the course of setting up performing and manipulating images for you
89. by setting the upper and lower limits for the Z position XS Note The Z stack is a Leica FW4000 product option that is only available if you have purchased the FW4000Z or FW4000TZ modules To define the Z stack click Z in the Process flow or the Live Image Setup dialog The Z Stack dialog is displayed Hescale Set Upper 2 Spacing Number of planes Leica FW4000 User Manual 7 78 To set the upper limits for the Z position 1 With the mouse drag the green plane to the desired upper limit value 2 Setthe upper limit value by clicking Set Upper 3 Drag the green plane to the desired lower limit value 4 Setthe lower limit value by clicking Set Lower Tip The recommended way of working Is to have a live image visible in the XY dialog Use the focus control of the microscope to see when you have found the upper limit of travel you wish to define and then click the Set Upper button Repeat this process for the lower limit Type the Number of Z Planes required in the box provided The Z Spacing is calculated and displayed automatically 5 Click the Z Spacing lock or the Number of Z planes 1 lock button U Note The purpose of the locks is to fix the number of planes and calculate the Z planes or vice versa Both the Z Spacing lock and the Number of Z planes lock may both be off but only one may be on at any time 6 Click the up and down arrow buttons to move the focal plane one pla
90. caling values by Interaction with the sliding bars Directly typing in the max and min values required Interacting with the grey level histogram To rescale an image click on Rescale from the Live Image Setup dialog pre Live Image T4 Z1 DAPI 512 x512 No D Image Size Image Viewer Settings LabBook Calibration Adiust Contrast neh m 55 00 Offset e 63a F Micr LEE PLN 8 TR RERUN ERTSINS PRES NEE OR USOC NECN ET AUER RATION AE areae BlackLevel 000 0 Leica FW4000 User Manual X Note The Rescale option will only be available for monochrome images of greater than 8 bit depth Defining the Image Size The main image display represents the available acquisition area of the CCD camera Select a source region as required for images from the camera The current values for the image source position and size for the X and Y coordinates are selected when you select the required sub region X Note For cameras with slower image readout rates using a smaller region for focusing will improve the display update and allow easier focusing of the specimen 1 To set a predefined image size 3 1 Right click the mouse in the image area This displays the Image Sizing menu 2 Click any of the top four menu selections or click Custom and Z Fit To Window then draw your own image area Show Saturation When you select Custom the Frame Colour cursor changes Show Framed Image 3
91. ck To display the live image allowing you to monitor the image during the experiment check the Show live image during capture box Note Auto contrast is used only on high resolution cameras that have more than 256 possible grey levels To save the colours assigned to the image by the software when a monochrome camera is capturing the image check the Save Colour Leica FW4000 User Manual During Capture box n To autoload and launch the Image Gallery when you open an experiment check the Open Gallery During Capture box Mapping Keyboard Functions The Keyboard tab allows you to map the keyboard function keys to the six steps in the Experimental Process flow 1 To map the keyboard functions 3 1 Click the Keyboard tab in the Preferences dialog Process Control Hardware Control Setup Shutter Capture Filter H ext Review Filter Previous Process Focus Up Publish 3 Focus Down Archive stage Lett Stage Aight Stage Up Stage Down Keyboard Short Cuts Enabled eee ue gt Cancel 2 Click the function key menu for Setup 3 Highlight the function key you wish to use to represent Setup 4 Repeat steps 2 and 3 for each of the Process and Hardware Leica FW4000 User Manual 29 Control functions 5 Click OK or click Cancel to cancel the mapping WS Note You can also set the distance the stage moves during stage left right and so on movements in the Stage Step box WARNING Do not use NUMPAD keys
92. clicking the mouse to show a tools menu This menu allows you to copy and change the settings for the files in the Batch The Tool menu items are Copy Deconvolution Settings Paste Deconvolution Settings Change Settings Change PSF File Copy PSF File Settings and Paste PSF File Settings These features may also be accessed by moving your cursor over the files in the Batch and right clicking the mouse Copy Deconvolution Settings This feature allows you to copy the setup and deconvolution parameters from one of the files in the Batch Leica FW4000 User Manual 26 7 To copy deconvolution settings 1 Select the file of interest from the Batch by clicking on it 2 Select Copy Deconvolution Settings from the Tool menu This automatically copies the files parameters If you paste the copied settings file into a text editor for example Windows Notepad you will be able to view the actual settings for the deconvolution Paste Deconvolution Settings 268 This feature allows you to apply the setup and deconvolution parameters from one file into one or more files in the Batch This feature is generally used when similar files are collected using the Same microscope setup To paste deconvolution settings 1 Select the file s from the Batch that you would like to copy the deconvolution parameters to In this case the PollenPA deb file 2 Select Paste Deconvolution Settings from the Tool menu This automatically Pastes the Pollen
93. cross the depth of an image This only works on images with depth gt 1 The image intensity value often fluctuates erroneously because of random flicker from the camera shutter or the lamp instabilities Flicker occurs with nearly all widefield microscope systems It is due to several causes Most Cooled CCD cameras have a randomness in their shutters speed This shutter speed fluctuation causes variations of the order of several milliseconds typically from one exposure to the next The effect can be seen best in a side view projection of a data set Leica FW4000 User Manual 247 Tutorials for Movie Menu Items Leica Deblur and 3D Visualise allows you to create a movie sequence To create a movie a series of frames are generated and then displayed in a contiguous manner A movie sequence consists of a series of rotations through a specified range of images and about a specified axis You may also set contrast settings the type of the projection the number of steps the Start and End Angles and the Anchor axis axis of rotation The movie generated can be played later in Leica Deblur and 3D Visualise or exported as an AVI file Movie Maker 248 The Movie menu of the 3D Viewer provides the tools needed to generate a movie You have two options to generate a movie Use the Quick Movies option under the Movie menu which allows you to generate a movie quickly by spinning the data about the X or Y axis by predetermined angles
94. defines the width of a border surrounding each subvolume This border is the region where the subvolumes are processed to seam them together This guardband prevents artefacts at the seams The possible values are integers from 0 to N 2 where N is the width or height of the XY field in pixels whichever is smaller Generally the larger the guardband the fewer the artefacts and the better the image quality However deconvolution time increases with guardband size so a default value is set which minimises deconvolution time and eliminates artefacts in most cases The default value is 10 This number may be increased if seaming artefacts appear If so first increase the number to 15 then 20 and then 25 until the seaming artefact is gone Z Guardband Pixels 238 The Z Guardband specifies the number of slices that will be added at the top and bottom of the subvolume This Guardband prevents artefacts at the seams of these subvolumes The possible values are integers from 0 to N 2 where N is the depth of the XZ or YZ field The Z Guardband should never be larger than the subvolume overlap region A value of 6 is adequate for most image stacks Leica FW4000 User Manual Leave the default setting of Low for the Noise Smoothing setting Noise Smoothing Noise Smoothing allows you to indicate the amount of noise you see in the data The Noise Level control on the Standard Settings form allows you to adjust this for different data A set
95. displayed in the status bar at the bottom of the Image Viewer dialog 174 Leica FW4000 User Manual Calibrating an Image If you wish to calibrate an image you may use a stage micrometer and a live image and draw a line corresponding to a known distance on the micrometer When the mouse button is released the Calibration dialog is displayed X Note When the system hardware is set up correctly FW4000 calibrates itself automatically on start up 13 To define image calibration 1 Click the Calibrate button in the Image Toolbar 2 SONU EEEE 2 Using a stage micrometer and a live image draw a line by clicking and dragging with the left mouse button corresponding to a known distance on the micrometer The mouse cursor is shown on the right side 3 When the mouse button is released the Image Calibration dialog is displayed and a calibration scale appears on the image File Edit View Image Database Window Settings C2 ike GAd owe WOO m Calibrate image jas Draw a calibration line on the image then enter the actual distance below Pixel distance 373 0 pp Actual distance Moo fum New calibration factor 0 0027 re Peale Raw Images v Image_T001_S001_2001_COMP tit 844 x 649 x2 Leica FW4000 User Manual 175 176 4 Use the Pixel distance text box to type in the actual length of the calibration line you have just drawn 5 Also select the correct measuremen
96. e This function allows you to paste a file that was copied into the Batch queue Help This function launches the online Help files Adding Files to the Batch 1 To add files to a batch 3 1 Select Batch Process from the Deconvolution menu 2 Select the Batch Processing form from the FILE menu and navigate to the Tutorial Data directory 3 Open the Batch folder and select the required files 266 Leica FW4000 User Manual S 4 Note There are three methods of adding a file to a Batch 4 With the file selected right click the mouse and choose Copy Move your cursor to the Batch box and select Paste The file appears as the first file in the queue With the file selected click and drag the file over to the Batch box and drop It into the Batch queue Open Pollen deb in the Leica Deblur and 3D Visualise viewing window and select Start 3D Deconvolution from the Launch menu Press the Batch button located at the bottom of the Launch 3D Deconvolution dialog The file is automatically loaded into the Batch To see that the file has been loaded select Batch Process from the Batch menu The file will be under the Image File Name list While the cursor is over one of the selected files depress the left mouse button and drag the selected files into the Batch queue Note Do not close the Batch Processing dialog Continue on to the next section Accessing Tools Ro Note You can access a variety of tools by right
97. e that does not require your attendance overnight Leica FW4000 User Manual Style Reference The following conventions have been used to make this manual easier to read Menus FILE bold capitals Menu Commands Buttons Open OK bold Dialogs References Chapter 1 italics Notes Tips Warnings Appear as follows and contain References and Instructions additional information Note Additional information that should be noted Refer to References to other relevant material Tip Tips to help you use the program more productively WARNING Important information to which you should pay careful attention m One Step instructions Operations that can be performed in a single step 12 Step by step instructions Numbered instructional steps Information that only applies in certain situations is enclosed in boxes Leica FW4000 User Manual 3 System Hardware Configuration The hardware configuration is described in the Leica FW4000 Release Notes This document also contains important information concerning the Installation of the hardware A Refer to The Le ca FW4000 Release Notes for information on the required hardware configuration Leica FW4000 User Manual Software Installation Leica FW4000 is provided on a CD ROM and must be installed onto your hard disk before it can be run 1 To install Leica FW4000 from the CD ROM 3 14 Ensure that Windows is running 2 Insertthe CD into your CD ROM drive 3
98. e the brightness or type a value into the text box Note The Lamp level slider is not displayed for other probes m Specify a Gain value The current value is displayed in the box to the right of the slider The increments on the Gain control change depending on which camera is connected gt Specify an Offset value from 0 to 100 percent of the full range available for the current camera The current value is displayed in the box to the right of the slider Leica FW4000 User Manual 75 76 Change the White signal scaling The current value is displayed in the box to the right of the slider Change the Black signal scaling The current value is displayed in the box to the right of the slider Click Set to Standard Values to reset the camera gain and offset values for the current camera Click Averaging x frames to specify the number of images used to create the final image Multiple frames can be used to prevent noise To set objective magnification level use the next and previous Objective buttons eee 10 x Binning represents the binning mode of the image The Image Type represents the m IEE Image Type 5 BitMono bit depth of the image You select BEEE e REEE AT OEE o Bit Wong the appropriate value from the pel to Standa 16 Bit Mono drop down menu 8 Bit Colour Leica FW4000 User Manual Defining the Z stack The Z stack allows you to acquire images at different focus positions automatically
99. e Dabbe ker Sete S82 GA me Wee WOO _ fees Ee patina ape ain window gengi yrke GAAN Boo a F Tile Vertical arranges all open Leica FW4000 windows in a non overlapping horizontal pattern one above the other which allows you to view all the windows at once m Ee mM Mz RacAg 2 gag p imaga Min SON 20 EOMP N eka Fiewimager imag TOEN SE An COMP ee riswimese imaga TON SON An CP ts Full screen displays only the active Leica FW4000 window as a full screen Image Fit Image to Window adjusts the image size to fit into the Image Viewer window Leica FW4000 User Manual 141 Zoom gt To magnify an image by zooming click the Zoom button in the Image Toolbar As you left click in the image the image size is increased so that you can see it in more detail Conversely as you right click in the image the image size is decreased so you can see more of the Image in one screen If the image is too big for the image window use the scroll bars to move around in the image n Alternatively use the Pan facility to move the image around in the image window Click the Pan button in the 4 Image Toolbar You can then click part of the image and drag it within the image window To switch off Pan mode click the Pan button again Erasing 1 Toerasea defined area a 3 4 Click the Erase button in the Image Toolbar ee EO ee 2 Define an area to be erased by holding down the left mou
100. e following values Selection Setting Data to Deblur Region of Interest box already checked Colour selection All Channels Deconvolution Parameters Increase the Total Iterations number to 30 PSF Correction Factor Unchanged Super Res Factor Unchanged Image Info dialog settings Unchanged Noise Smoothing Low Expert tab Default ROI tab Default Frames tab Default Note The image size defined in the Image Info dialog settings are measured in pixels that is 256 x 256 Note The Noise Smoothing setting should remain as Low as this image Leica FW4000 User Manual does not contain a great amount of background noise 4 Click OK to begin the deconvolution process S Note To interrupt the deconvolution press the ESC key and wait for the current iteration to finish 5 When the 2D Blind Deconvolution is complete the 2D Deconvolution Results dialog appears with three tabs Results Channels and Save 2D Deconvolution Results In the Results tab you are able to view the result of the deblurring process at each Iteration by using the scroll bar in the View Iterations field You have the option to display the PSF during the viewing process by checking the check box next to Display PSF You also have the option to alternate between the current Iteration number result and the original image by clicking on the Original Current button Within the Adjust Deblur Parameters dialog you may choose to do further iterations by clickin
101. e in the new name Select the required region from the list Type a name into the Region name box Click OK to apply the changes and close the dialog or click Cancel to close the dialog without applying the changes gt eo oS gt Alternatively to change the name of a region highlight the name of the region and overtype the new name 164 Leica FW4000 User Manual Changing the Colour of a Region 1 2 3 To change the colour of a region 1 Click the Setup button in the Measurements dialog to display the Set Up Measurements dialog AEU Alternatively right click the colour and select a new colour from the displayed colour palette 2 Select on the Regions tab 3 Select the Region name Note The Regions List contains a list of all the regions in the order that they were created 4 Right click on the region name to display the Colour dialog 5 Select the required colour 6 Click OK to close the Colour dialog 7 Click OK to apply the changes and close the Set Up Measurements dialog or Cancel to close the dialog without applying the changes Alternatively to change the colour of a region right click in the region and select the new colour from the colour palette Leica FW4000 User Manual 165 Clearing Regions of Interest 1 To clear a region 31 Click View in the main menu and Show Coloured choose Measure Objects to display the Show Greys cale Measurement dialog Measure
102. e lower the amount the more spots will be removed A typical value would be 20 197 The following table gives some suggestions on which operation to use when Try Type Amount _ Reduction alternative Remove small Morphological White 1to5 features and Fillholes maintain hard edges for larger features Blur unwanted Matrix Averaging N A 1 to5 small detail Spot Removal To accept the enhancements you have selected 1 Click Preview to preview the changes without making any permanent alterations 2 Click and hold down Toggle to view the original image Using this button you can compare the two versions of the image before deciding whether or not to retain your changes 3 Click Apply or Apply to Selected in the Image Viewer to save your enhanced processed images The Saving Images dialog allows you to define the processed image folder or the defined image set as the location for saving your Images 4 Click OK to save your processed image or stack or click Cancel to close the dialog without saving any changes Leica FW4000 User Manual F Where do you want to sawe the images 7 To an existing Image Set Processed Images C Toanew Image Set Aw Refer to Chapter 7 Using a Processed Image Setfor further information 5 To sharpen only a defined part of the image open the Image Viewer Settings menu and click ROI Leica FW4000 User Manual 199 Enhancing the Contrast of an Image Before an image
103. e maximum projection of images check the Generate MIPs after each capture box This generates a maximum projection image from each Z stack and displays it in the gallery To display an image after it is captured and the first and last image refers to first in a time z stack sequence and so on check the Show Image Viewer after Capture box with the none first centre or last option selected Note If you have selected one of first centre or last from the Show Image Viewer after Capture menu then the image gallery display is overlaid with the image viewer Tip To automatically display only the image gallery set the Show Image Viewer after Capture menu to none To automatically show the image viewer after taking a single camera image check the Show Image After Capture in live image setup box Note This allows you to continue capturing images whist viewing the images that have already been captured alongside in the image viewer Check Show Grey levels as percentages to show the grey level value as a percentage rather than an integer in the range 0 to 255 To map the grey levels in an image from the camera to the 256 grey levels that are displayed select one of the options from the Auto Contrast Images during Capture dropdown list The options are Use the contrast values defined for each probe Auto contrast each image separately Auto contrast an entire stack using the same contrast values for each image in the sta
104. e number in the End Slice dialog When you click OK the Save Multi Frame Deblur dialog appears Use the default name for the resulting data set or type Leica FW4000 User Manual a name for your resulting data 5 Click on the Save button A 2D Deconvolution will run on your data set and upon completion you will be prompted to save the newly created file After you have clicked on the Save button another dialog appears asking you Do you wish to export as multiple files each of a single slice 6 If you select Yes Leica Deblur and 3D Visualise shows the naming extension it will append to the data set The new data set will be saved under a name indicating the Start and End slices along with the number of iterations in Its title Inverse Filter Use the Inverse Filter feature with Widefield Fluorescence and Transmitted Light brightfield data sets only The Inverse Filter is a one step non iterative deconvolution method based on inverse filtering theory It utilises optimal linear filtering Inverse Filter is one of the simple deconvolution methods offered by Leica Deblur and 3D Visualise The feature is useful for obtaining quick results but is not as accurate as Blind Deconvolution Inverse Filtering is typically more robust than the Nearest Neighbours or No Neighbours deconvolution methods The execution speed of the Inverse Filter is between that of Nearest Neighbours and Blind Deconvolution The Inverse Filter should be used in
105. e the Colour dialog 6 Check Thick Lines if you want the lines to appear thicker Leica FW4000 User Manual 159 160 7 Click OK to close the dialog and save the changes or Cancel to close the dialog without saving any changes To use thick line when drawing your measurement line check the Thick lines box Leica FW4000 User Manual Changing the Number Format 1 To change the number format 3 1 Click on the Setup button in the Measurements dialog to display the Set Up Measurements dialog sisi aE gt Alternatively click Settings in the Measurement dialog and choose Setup 2 Click on the General tab Number format OF Cancel 3 Click the Number format menu button to display the available formats 4 Choose a number format from the drop Ee down list 5 Click OK to close the dialog and save the changes or Cancel to close the dialog without saving any changes Leica FW4000 User Manual 161 Making Measurements Using the Feature Layer Using the Feature Layer to define a region of interest allows you to define complex and large regions for a variety of probes 1 To make a measurement using the Feature Layer 3 41 Click the Setup button in the Measurements dialog a to display the Set Up Measurements dialog a n Alternatively click Settings in the Measurement dialog and choose Setup 2 Click on the Features tab F mn amp Regions Features Parameters Limits Classify
106. e the image before and after removal of the background Refer to Chapter 9 Removing the Background for more information on removing the background Make a composite fluoro image Adjust Contrast allows you to adjust the gamma contrast and brightness of an image using the Image Contrast dialog Measure Intensity allows you to measure the intensity of the kak probes at different points in the image Refer to Chapter 8 Measuring Probe Intensity for more information on measuring intensity Leica FW4000 User Manual Sharpen allows you to sharpen the image Smooth allows you to perform noise reduction on the image Save the image you are processing currently This tool enables you to save an image through a number of processing stages Thus the sequence of processed images can be included in reports and published documents Deconvolution removes the out of focus information from the image using a 2D deconvolution algorithm the maximum intensity pixels from the entire stack Brightfield inverts the image making dark pixels light and pseudocolours the image to black and white i Max Projection is a single image from the Z stack that uses Pan allows you to move the image around in the image window Previous Stage Position moves to the previous stage position if any Next Stage Position moves to the next stage position if any Leica FW4000 User Manual 135 Z stack Playback Toolbar 136 T
107. elect one of your green channel images from the multi channel folder and click Open A dialog appears with the question A sequence of files associated with this selected file is detected Do you wish to load the entire sequence 4 Select Yes Select Green from Channel Selection and click Browse O1 6 Browse and select one of your blue channel images in the multi channel folder and click Open A dialog appears with the question A sequence of files associated with this selected file is detected Do you wish to load the entire sequence 7 Select Yes Select Blue from Channel Selection and click Browse oo 9 Browse and select Malaria _Blue 0 tif in the multi channel folder and click Open A dialog appears with the question A sequence of files associated with this selected file is detected Do you wish to load the entire sequence 10 Select Yes 11 Click OK in the Import Multi channel TIFF dialog A progress indicator will begin showing the importing of the red green and blue channels The output from the XY Max Projection of the three channels is displayed Note For a two channel data set select the Blue channel in the Disable Channel box and navigate to the Red and Green channel locations and load them into the Import Multi channel TIFF Images that corresponds to the File Locations fields Leica FW4000 User Manual Viewing the Channels and Slices of a Multi channel Data Set This function allows y
108. elected Users may also add their own customised fluorochromes Size and position of the image on the camera s chip Z stack and choose the spacing between the acquired images Discrete positions on the sample where you require images to be captured Time sequencing routines used to run the experiment Sd Refer to Chapter 4 Setting up an Environment for Experiments P Leica FW4000 User Manual 55 Capturing Images gp 56 Clicking Capture in the experimental setup causes the image capture sequence you set up earlier to run automatically You may stop the sequence at any time and view the images taken during the sequence run Clicking Try Again runs through the sequence again from the start 7 fimage_T004_5001_2001_CO2 tif Image T004 5001 7002 CO2 tif Start me H Run for 00 00 20 H Thursday 14 March 2002 12 01 03 User Name DefautSupervisor 81 Image _TOO4_SO01_2003_C02 ti Detaulupenvisor Every 00 00 05 Image_TO04_SO01_Z001_CO3 ti camer a i ese Tt s001 20 CY m 661 Image_TOO4_S001_2001_CO4 tit Elapsed Time e HTE 1 Image T004 S001 Z007 CO4 tif Image_T004_5001_2003_C04 tif Image_T004_5001_2001_C0S tif Image_T004_5001_2002_C0S tif Image_T004_5001_2003_CO0S tif Capture Sequence Complete Time Sequence ended DefaultSuperisor My latest Experiment Finished Free Disk Space C 378 003968 bytes Disk Space Needed E not known butes Close riage Viewer Live I
109. ends them to the top and bottom of an image It is not the same as adding blank slices to the top and bottom of a stack as mentioned below An extended data set will have false slices attached to its top and bottom These false slices serve the purpose of a buffer to keep the guardband region from overlying meaningful features thereby keeping the meaningful data from being obscured in the deconvolution process This tutorial continues with the Neuron deb data set from the Crop tutorial above To crop extended slices 1 Select Extend Slices from the Operations menu or click on the Extend Slices button on the Toolbar The Extend Image dialog appears Note You have the option of extending the data by using false slices or by using the Zero Pad option which when selected gives you the ability to add blank slices devoid of any information 2 Inthe Extend Depth box type the value 4 for the Top field value 3 In the Extend Depth box type the value 4 for the Bottom field value 4 The new Depth value will be displayed in the New Dimension frame 5 Leave the Zero Pad box unchecked Leica FW4000 User Manual 6 Click OK The extended data set will be displayed Click on the XZ view button and notice the difference between the real data slices and the false slices just generated 7 Close all views before going on to the next tutorial Optical Density Correction This feature corrects fluctuations in the image intensity values a
110. ent mouse kidney s Esperment Don Expenment Don BFAE cells 20x BPE celle 20x BRAE Fluoresc BRPAE Fluoresc BPAE Fluoresc checking the in Default Experim A The available archiving features include 4 4 4 4 4 Refer to Chapter 12 Archiving Experiments Archiving an experiment Retrieving an experiment Exporting an experiment to a networked PC or removable storage media Importing an experiment Deleting an experiment Leica FW4000 User Manual 61 62 Leica FW4000 User Manual Chapter Four Setting up an Environment for Experiments Chapter Overview This chapter contains the following topics Introduction Defining Live Image Acquisition gt gt Leica FW4000 User Manual Multi focus Acquire Adding or Replacing Images Rescale on Images After Acquire Defining the Image Size Setting up the Camera Defining the Z stack Selecting Fluorochromes Defining Stage Positions Defining Time Sequencing Routines Checking the Time Sequence Logic Time Sequence Controls Building a Time Sequence Building a Time Sequence Example Tutorial 63 Introduction nb 64 Setting up the environment for your experiment requires you to set up parameters in five areas These parameters allow you to set up Live images by defining the X Y image acquisition parameters The Z stack and choose the spacing between the acquired images Fl
111. er The new image sets are also available in the gallery and in the tree view in the Publish Results dialog ie Where do you want to save the images 7 f To an existing Image Set Processed Images C To anew Image Set es This dialog appears after clicking on Apply or Apply to all in the Image Viewer when you are using the deconvolution or image enhancement tools Leica FW4000 User Manual 139 Windowing tools The Window menu allows you to cascade arrange image windows arrange buttons fit images to the current window size or fit the window to the current image and close windows 1 To cascade and tile windows window Settings 3 1 Open the Leica Image Viewer Tile 2 Select the Window menu 3 Click the appropriate function Tile Vertical Tile Horizontal Cascade Full Screen Fit Image to window Cascade arranges all open Leica FW4000 windows in a cascading overlapping pattern The title bars of the windows remain visible feia J l File Edt View Image Database Window Settings Fille Edit View Image Database Window Settings Raw Images Image_TOO1_SO01_ZO01_COMP tt 6444649424 Raw 00 080 42 140 Leica FW4000 User Manual Tile Horizontal arranges all open Leica FW4000 windows in a non overlapping side by side pattern which allows you to view all the windows at once pen Fie Ed View rae Gite Window Settings 2 Ee Ame WHF BOO Pie ce Veew Imag
112. er WL 2 Stack Upper_FL Z stackLower FL stackSpace FL In some cases for example when you choose either ExpDate_DT or ExpTime_DT you can further delineate your data by supplying a date or time on the right boxes 300 Leica FW4000 User Manual 4 Click the Function menu arrow and BETWEEN click the required function BETWEEN 5 Click Apply to apply the data profile without closing the dialog If you are dissatisfied with the profile repeat steps 2 to 11 until you are satisfied You can apply a more complex filter using the AND or OR radio buttons However do not use both AND and OR because the results will not be as expected 6 Click OK to accept the total data profiles or click Cancel to cancel the operation Archiving Experiments 1 To archive an experiment 3 41 Highlight the experiment to be archived 2 Click the Archive button The Archive Experiments dialog is displayed i HHT x Do you wish to archive the following results ExpDate_DT PAP My Experiment OF 0801 Remove from Lab Book OK Cancel 3 If you wish to remove the data from the laboratory workbook check the Remove from Lab Book box Leica FW4000 User Manual 301 4 Click OK to display the Assign Archive Database dialog Archive ta Exishnig Folder New Folder Bigk xp ewl mpored Set up new archive volume on external storage Gad J Default Experiment Aver Sa Fwa vol Ld
113. erent time sequences by clicking Advanced When selecting values of Z or T for compositing the systems displays all available values from your experiment in the scroll menu ia m TE i ESEE Fie Edi View Nee ce y ROI Ei zE lat 7 Display at Time Position jo I Display at Position Moo Adjust Al Reset Simple i Create Create All i Create composite for each z plane Raw Images Image _TO01_ S001_2001_COMP tif O44 u bdd x 24 Haw ULLULULULU 2 To define where the new composite image should be placed in the Z stack click Display of Z position 192 Leica FW4000 User Manual Sharpening an Image 1 To sharpen an image 3 1 Open the Image Viewer 2 Select Image from the Image Viewer main menu and click Sharpen to display the controls for sharpening an image m Alternatively click Sharpen in the Image Toolbar to display the controls for Sharpening the selected image A File Edit View Image Database Window Settings QM 2k e GAm ewe WOO Preview G white Enhance Local Co C Black 1 To select an image to be sharpened 3 1 Select White to sharpen lighter features in the image 2 Select Black to sharpen darker features in the image gt To select a sharpening amount move the slider bar to adjust the sharpening amount The larger the value the more severe the effect Leica FW4000 User Manual 193 24 194 To cho
114. erm Channel is used for delineating data that was collected at different wavelengths for a data set This menu item will be used for data that is imported as multi channel data You may arbitrarily set any of the colour channels to any of the emissive wavelengths collected The channel description set up below are guidelines for you with respect to which channel will correspond to what wavelength Red Channel To access this feature click the FILE menu and select Import RGB Data The red channel function normally represents the longest emissive wavelength collected for the data set 1 To select the red channel 3 1 From the FILE menu select Open or from the Toolbar click on Open File 2 Fromthe Tutorial Data directory select Multi channel folder Select one of your red channel images and click Open 4 From the Channels menu select Red Channel An image set consisting of only the red channel will be generated wo Green Channel The Green Channel function represents the intermediate length wavelength collected for a 3 channel data set or the shortest wavelength collected for a two channel data set 224 Leica FW4000 User Manual To select the green channel open the Channels menu and select Green Channel An image set consisting of only the green channel is generated Blue Channel The blue channel function represents the shortest wavelength collected for a data set n To select the blue channel open the Channels me
115. ese menu selections are identical to the actions launched by the buttons described below Setup Point Small Square Medium Square Circle Ellipse Line Area Measure Objects Delete Selected Region Clear Regions Leica FW4000 User Manual m Select the Small square button and then click the required point in the image gt Select the Medium square button and then click the required point in the image 1 To draw a circle 3 i1 Select the Point button and then click the required point in the image HHHO olco Select the Circle button Ol E e be 2 Clickin the image where you want the top left most point of the circle to be Do not release the mouse button 3 Hold the left mouse button down and drag the circle to the required size 4 Release the mouse button to finish 1 To draw an ellipse 3 1 Select the Ellipse button o 2 Clickin the image where you want the top left most point of the ellipse to be Do not release the mouse button 3 Hold the left mouse button down and drag the ellipse to the required size 4 Release the mouse button to finish Leica FW4000 User Manual 171 To draw a line 1 Select the Line button C 2 Click in the image where you want the line to start by either Holding the left mouse button down and draw a freehand line Releasing the mouse button and click again to draw a Straight line You can do this many times to create a number of nodes joined by s
116. ew box Close all data sets before continuing on to the next section Leica FW4000 User Manual 215 3D Viewer The 3D Viewer accessed from the VISUALIZATIONS menu displays a graphical presentation of the data set This feature allows you to create oblique slices orthogonal slices movies apply different colour maps change projections adjust subvolume size rotate the data and to view data in the anaglyph stereo mode 216 To create graphical representations in the 3D Viewer 1 With a suitable data set loaded navigate to the SmallHip folder open the SmallHip avz data set Select the 3D Viewer from the View menu The 3D Viewer window opens with your data set in the Orthogonal Slices view of its XYMax projection In the upper left corner of the 3D Viewer window the red line represents the X axis the green line represents the Y axis and the blue line represents the Z axis of the data set Note The 3D Viewer has several menus items for example View Projection Axis Oblique Movie Colour Map Save and Options These menu items may also be accessed through the Control Panel found under Options From the Axis menu select Go to View gt XZ The data set will be rotated to the XZ Orthogonal Slice view 3 To rotate your data set Click anywhere in the 3D Viewer window Hold down the left mouse button and move your mouse The data set will rotate in the same direction your mouse moves Stop your mouse movement w
117. file This header file contains the necessary information to playback the movie By selecting this file the entire movie will be loaded 3 Press the Play button to play the movie The movie plays once through then automatically stops 4 To stop a movie that is currently playing press the mm Stop button 5 Press the Loop Mode button to put the movie playback T into Looping Play Press the Play button and the movie 4z will continuously play in a Looping motion until the Stop button is pressed 6 Press the Rock Mode button to put the movie playback into Rocking Play Press the Play button and the movie will continuously play in a Rocking motion until the Stop button is pressed Note Do not close the Colrpollenrs avm movie Continue on to the next section Leica FW4000 User Manual Concatenating Movies This tutorial continues with the movie from above in the Open Movie section 1 To concatenate movies 3 1 Select Concatenate Movies from the Movies menu 2 The Concatenate Movies dialog appears Click the Add a Movie button and navigate to the directory where the movie files are stored 3 Select the first movie to be added in the concatenation and click the Open button The movie will be placed first in the Movie List 4 Click the Add a Movie button again and select the another avm to be opened Once the Open button is clicked Leica Deblur and 3D Visualise will add the avm to the Movie List in the second place
118. further information To delete an experiment 1 Select Users or a particular user in the tree view 2 Select an experiment in the right hand view 3 Click the Delete icon Note Ordinary users can only delete their own experiments To delete an image from the Lab Book 1 Click the Raw Image or Processed Image radio button which Leica FW4000 User Manual 4 p 6 displays the Tree View node 2 Highlight the images to be deleted 3 Click the Delete icon 4 Click OK in the Confirm Deletion Dialog box To undo a delete action click the Undo icon ie Note You can undo only the most recent Delete Experiments action that is the ones deleted the last time you pressed the Delete icon To print the details of the experiment selected in the tree view click the Print icon Click the Properties icon to change which details of a pm users experiments are shown in the right hand panel a i when a user is selected in the tree view Also you can filter the experiments that are shown Refer to Chapter 12 Archiving Experiments for further information Note You can search for all experiment performed on a certain day or search for experiments that have z stacks To access some extra functions right click on the experiment name to bring up the menu options Unlock experiment Use this to unlock an experiment when the database has locked it as a result of a problem You rarely need to use this option Unl
119. g on the Further Processing button or you may return to Setup to rerun the 2D Deconvolution with different parameters Within the Use Current Iteration Settings to Deblur dialog you may choose to process the Full image or Multiple Frames of the Full Image or ROI Region of Interest You may click the Use ROI dialog to enable it and then click the Full Image button to process that region of interest throughout the entire data set Leica FW4000 User Manual 257 A E AEAEE n T 20 Deconsalubon esws Soo TEAT E han linur BY LEralizne 30 20 al a C Dip FSF Ciia Cwe hirl Joba Fara nsei 10 ue eran r urhe eed shun ln heip Jc Curent Ioan zhat Dic blr Follnaeqe D UFC M Den PEF M Aliz fial otrrt 2 E d Te JaA Arel Time Lapse Images 258 You also have the option of selecting a region of interest and applying the region of interest to only part of a time lapse data set To apply an ROI to only part of a time lapse data set 1 Select 2D Deconvolution from the Launch menu The 2D Deconvolution dialog appears with the Standard tab displayed Select the Multiple Frames option from the Data to Deblur frame To set the range of images you would like to process select the Frames tab and in the Multiple Frames frame select Frame range Type the desired starting Frame number image number in the Start Slice dialog and type the desired ending Frame number imag
120. ge 1 To reorder the image display sequence a a Click the left mouse n h select A one of five experimental parameters Heete an arrow is superimposed upon the parameter you want to move 2 Move the parameter buttons to the sequence you want by dragging and dropping the selected buttons to left or right individually 3 When you have moved the buttons to the order you want click Reorder to confirm the selected order Reorder 4 The image display order changes to match the reordering of the parameters 5 Repeat steps 1 to 3 to compare different images until you are Satisfied that you have a suitable set gt To display only the images in the selected treeview node click the images in the left Lab book View and select combinations of probe as you require gt Note If the captured images are unsuitable then start again by reviewing the environmental parameters and possibly the time sequencing X Note The parameter Reorder button appears only when you change the parameter order 110 Leica FW4000 User Manual XS Note Once you have defined an image set to view and process you can change the display order of the images in the gallery To reorder the images resort the images by changing the sequence position of the button Leica FW4000 User Manual 111 Previewing Composite Images The composite images viewed here have been produced by mixing output from a selection of the probes The composite images allow
121. gion 1 FITC Grey Level 150 Region 1 TRITC 2 wW Hg ra S 3 t3 H R 43 g tg it it 2 oO ao Pa E0 oo as v5 ao E zw 2 i H Gal o a m D j Q 4 e DD ree oT oc o Gal o m 2 D 0 Line drawing right click to complete Note The Profile button toggles the grey scale level measuring tool on and off 2 Note You are able to Load and Save the profile by using the File menu in the Measurements dialog De 173 Leica FW4000 User Manual Measuring Probe Intensity You can view the intensity levels of all the probes in the image stack at any point in the image To view the intensity level of a probe 1 Select the required image in the Image Gallery 2 Select View in the Image Viewer main menu and click Measure intensity to display the Probe Meter dialog n Alternatively click the Measure Intensity button in the Image Toolbar to display the Probe Meter dialog i Z amp Ii DAPI FITC Texas Red 3 Click any point in the selected image to view the intensity for that point or hold down the left mouse button and move the mouse around in the image to view the intensity levels at any point in the image instantaneously The probes shown in this diagram will depend on the settings for the current image that is the filters that have been set up for this image SS Note The current X Y position is
122. he Z stack playback toolbar allows you to play reverse and step through the images in the Z stack To run up through the images in the Z stack click the Run gy Upwards button To step upwards through the Z stack one image at a time click the Step Upwards button Stop button To step downwards through the Z stack one image at a F To stop the playback of the Z stack display click on the ul time click the Step Downwards button see To run down through the images in the Z stack click the Run Downwards button hl Leica FW4000 User Manual Time Sequence Playback Toolbar The Time sequence playback toolbar allows you to play reverse and step through the images In the time sequence To run up through the images in the Time Sequence click dd the Run Backwards button m lo step backwards one image at a time click the Step E Back button To stop the playback of the Time Sequence click the Stop button T m lo step forwards one image at a time click the Step j Forward button m To run down through the images in the Time Sequence pp click the Run Forwards button Customising Your Image Toolbar You may add and remove toolbar buttons and therefore functionality from the image toolbar 1 To customise your image toolbar cre 3 1 Click Settings inthe mainmenuand click Customise Toolbar This displays the Customise Tools dialog Leica FW4000 User Manual 137 F 1 x A
123. he image and enter it isss into the Lab Book The image is displayed in the Live i32 Image Setup dialog Leica FW4000 User Manual 65 Alternatively set the image frame and exposure you require whilst the image Is Live and click the Camera button Aw Refer to Chapter 2 Setting General Preferences for a description on how to cause the Image Viewer to open automatically from the Preferences dialog X Note This way of working is aimed at users with basic image capture requirements perhaps only capturing one image When you set up an experiment where Images exist already the Leica Capture dialog appears where you can choose the action to take Expernment Jims Experiment Dont Touch 05220 Images already exist for this experiment what do you want to do Delete existing images and continue Continue and Add Images to Experiment Continue in view Only with changes disabled Create anew esperiment 05 Cancel To exit from this stage in the Experimental Setup process click in the top right corner of the Live Image Setup dialog 66 Leica FW4000 User Manual Adding or Replacing Images You are able to add or replace images in an experiment at different sessions The implication is that you can capture images on a tissue Sample over a period of time without running that experiment continuously within the Leica FW4000 application The time interval between sessions may be defined as week
124. hlight the user to be deleted 2 Click Delete 3 Click OK to confirm the deletion or click Cancel to cancel the operation 4 Click OK to accept all the changes you have made in the Edit User dialog or click Cancel to cancel the operations X gt Note The names of deleted users are listed in the Edit Users dialog but do not appear in the list of users available from the initial logon screen 1 To reinstate a user 3 1 Highlight the user to be deleted 2 Click Reinstate 3 Click OK to confirm the reinstatement or click Cancel to cancel the operation 4 Click OK to accept all the changes you have made in the Edit User dialog or click Cancel to cancel the operations Leica FW4000 User Manual 23 User Password Management Users may change their own password as and when required gt To change your password click FILE at the main menu and Manage Users This sequence displays the Change Password dialog File Viet Setu sal e pture Revie a a ee A a E abt TARR PEE titititi a ss co iit sass tet EEE ERREK Hiti E SEREK i Hinh ieee FE Hi E ray ets FEEF fee Log on as different fee S na oa mae nana aoa SE sata eS sh i L EEF eo Re eee M es Oy gN ee ee te ae rei PERE PE EEFE EE EEFE feet Becerra aces nn aaa Rinne HEESEN en Set te te eee ie Or F ere se EEEE EEEE FEER EE TET ete Sieh fl ee n Har it ate Ha T E poe ee ee ee ae a A AA EEE AA BEREA EEEE dr Sense ot
125. ialog Leica FW4000 User Manual Importing Experiments from OF luoro Single experiments Z Stacks or time lapse experiments acquired in Leica OFluoro may be imported into Leica FW4000 Once imported the experiment may be treated as though It was acquired in Leica FW 4000 and a user may utilise all of the Leica FW4000 features with the imported imagery n To import an experiment from QFluoro click the Import button The Import Experiments from OFluoro System dialog is displayed Single Image Experiments All experiments in folder Z Stacks Sequence Experiments Merge to single experiment qc E3 Program Files New Experiment Hame Leica FW4000 User Manual 307 To import an experiment from Leica OFluoro browse for the required Leica OFluoro image or experiment and select either Single Image Experiment or Z stacks Sequence Experiments from the Import Experiments from QFluoro System dialog To import all images in a folder check All Experiments in Folder Each image file name will be imported as a separate experiment To rename your experiment do so at this stage and proceed to import the experiment s by clicking Import in the Import Experiments from OFluoro System dialog Importing Tiff Images into Leica FW4000 308 You may import monochrome and colour tiff images to Leica FW4000 from another source This allows you to import images not captured by Leica FW4000 and to make an experiment of those i
126. ick Apply to view the new Alpha projection 5 Place the Alpha Blending Projection next to the Voxel Gradient Projection and compare the differences in their appearance Best Focus 222 This function takes parallel rays perpendicular to the viewing Surface and cast them through the 3D image The voxel encountered along each ray with the greatest local contrast is selected for display in the resulting 2D image This produces an image constructed from the most in focus features from the volume A Best Focus projection creates an image out of the regions within a volume that best stands out against their neighbours This is useful for observing more subtle features in an image which may be not as pronounced in other projections Leica FW4000 User Manual 1 To obtain the best projection 3 1 Make the Max Projection of your data set the active view 2 Select Best Focus from the VISUALIZATIONS menu Leica Deblur and 3D Visualise will display the Best Focus projection of the data set 3 Adjust the Min and the Max intensities by clicking Options on the File menu 4 Close all views before going on to the next tutorial Leica FW4000 User Manual 223 Tutorials for Channels Menu Items For this tutorial you may use your own data type or you may follow along with the recommended data to use All multi channel data regardless of what microscope It came from or its data type will be separable into its own channels Xo Note The t
127. if quantitation and dynamic range are not Important The TIFF format allows the deconvolution results to be easily imported into other software The TIFF format uses no compression and is an 8 bit format The Leica Deblur and 3D Visualise File deb output will require four times the disk space of an 8 bit or TIFF output Deconvolution Performance The Deconvolution Performance field allows you to select from two Deconvolution Performance settings These settings affect the performance with regard to the stability resolution and the speed of the deconvolution You can choose how quickly the results are returned versus the resolution desired The Unchecked setting which refer to stability resolution and speed respectively allows the deconvolution to have the highest stability and resolution It is generally used when working with relatively thick samples or noisy data sets The Checked setting provides higher speed with a small trade off in resolution Minimum Image Intensity Removal Minimum Image Intensity Removal automatically calculates and removes the erroneous background intensity level in the data The 234 Leica FW4000 User Manual most common cause of this erroneous background intensity is the electronic dark current background electrical signal level of the photodetector or CCD camera Other causes are the bias voltage of amplifiers in the camera back scattered light that penetrates the emission filter and non specific dye
128. iginal view or by rotating your data with your mouse to a position that you want the movie to start from Select Start Point from the Movie menu Use your mouse to rotate the view to a position that you want as the end point for the movie Select End Point from the menu Select a Step Angle from the available options under the Step Angle option from the Movie menu Click on Play Movie The movie will play in the direction in which you selected your Start Point and then the End Point Again you may play the movie in Loop mode Rock mode and in the Opposite direction Close all views before continuing on to the next section Note You may also select a Mid Point when generating a movie To do so follow the same steps as explained above for setting the Start Point and End Point Leica FW4000 User Manual 249 The last option under the Movie menu Is Create a Movie This feature opens an untitled movie file which may be saved It provides all the options to Play the movie Stop the movie Rock mode Play and Loop mode Play Before you are able to access the created movie file you must close the 3D Viewer dialog Open a Movie 4 250 3 To open or play a movie 1 Select avm from the Movie menu Set the Files of type to avm 2 Select Colrpollenrs avm from the Movies directory and click Open The first frame of the movie is displayed Note The avm file extension indicates a Leica Deblur and 3D Visualise movie header
129. in a stage position list 3 41 Locate the area of interest 2 When you have located the area of interest and adjusted the focus return to the Visit Sites dialog 3 Select the position you want to modify by clicking anywhere in this row 4 Click Replace 5 If you want to add a comment to help you identify this position later on type a comment into the Comment box of the dialog before clicking Replace n To remove a position in a stage position list select the position you want to remove by clicking anywhere in this row Click Remove To remove all positions from the stage positions list you will need to select each one in turn and click Remove gt To replace a position in a stage position list select the position you want to replace by clicking anywhere in this row Click Replace To replace all positions from the stage positions list you will need to select each one in turn and click Replace gt To move the stage from one position to the currently selected position click Move To m When you have completed all the required activities click OK or Cancel to cancel the operations 86 Leica FW4000 User Manual As you define new positions that position is shown as a small red cross on the stage plan You can move round the stage plan by using the Zoom In and Zoom out buttons When the cursor is moved over a red cross a comment on that stage is displayed as a pop up message n To add or change a stage comment highlight the
130. ing with a manual microscope without automated Z control check the Manual focus adjustment check box The system calculates the total travel Check the Threshold check box if you wish to manually threshold the image during the acquisition Repeat the above steps for each of the probes you wish to capture in the multi focus acquisition mode Click the Acquire icon to start capturing for the current probe only saving in the lab book if required or the Capture icon to start the capture for all probes The resulting image will be one single image consisting of a maximum projection of all planes acquired Capture Note Use of the Multi focus Acquire and Z Stacking features of Leica FW4000 require that the Z travel limits have been set up in the software These may be accessed from the Hardware Setup menu Initialise focus 72 Leica FW4000 User Manual Setting up the Probes This section describes how to set up the camera for use with each probe 1 To set the camera exposure time for 3 each selected probe 1 Selecta probe by clicking the appropriate probe button 2 Click Live While an image is being captured the Live button is highlighted in red and the Shutter 2 button changes T 3 Move the slide to the required exposure time 4 Stop the acquiring by clicking Acquire again 216 ee XS Note You have a choice here You can click the Live image again to stop being live because the fu
131. ingle frame or a complete multiprobe stack to take place every five seconds S35 Note This is a large experiment There is no restriction on how long the experiment should run so a maximum elapsed time of 24 hours is assumed n Add a Run element The example restricts the run to 10 minutes Start now Run for 10 seconds 5 seconds 15 36 21 gt Cancel Leica FW4000 User Manual 95 96 Note How the elements are indented This means that the Every five seconds instruction keeps control doing an IMAGE every five seconds and will not release control until the 10 minute time period is achieved Add a Notify element to produce a message on the log and on screen during the image acquisition process Notify starting the time elapse Every 5 seconds MAGE Ne a i corr Note There will be only one occurrence of the Notify message because of the indenting protocols See the previous Note Add two Run blocks one after the other with an hour s wait in between Start after 5 minutes Run for 10 seconds Notify starting the time elapse Every 5 seconds IMAGE Notify First sequence done Wait 1 hour Run for 30 minutes Wait 30 minutes Delete Check Ss i A ea AaS i T The first Run block takes images at a higher rate than the second The Notify element displays a message First sequence done after the first Run block has finished Leica FW4000 User Manual X Note
132. ings main menu the first of the component images Is selected automatically You can select the required component image by clicking it in the Image Gallery Note You must remove the background from an image before you can define a region of interest If you have not done this already you will be prompted when you click OK Refer to Chapter 9 Removing the Background tor information on removing the background from an image Leica FW4000 User Manual The Select ROI dialog allows you to select a sub region of the image This means that you can limit processing to specific parts of the image by using an ROI processing mask The ROI mask tells Leica FW4000 which pixels are to be affected during processing of the image You can define any number of shapes in the ROI mask using simple mouse editing The resulting mask image can be thought of as a transparency which can be placed over the image You then specify which parts of the image you are interested in by drawing on the transparency 1 To define a region of interest 3 41 Select the required component image 2 From the Image main menu choose Setup ROI The Select ROI dialog appears eS i Action Select Regions Select All C Reject Regions Clear Apply Threshold Edit Mode Clip Mode Display Show mask outlines Show full mask i i Cancel Undo ROI is drawn region Leica FW4000 User Manual 147 gt gt a 148
133. ion of an image see Select Region and creates a new document based on the pixels that fall inside that selected region 1 To crop the stack 3 1 Start in upper left corner of the view to select a region Re Rend r 2 Press the left mouse button down and simultaneously drag the mouse to form a square similar to the one demonstrated in the Leica FW4000 User Manual 243 Re figure above Once the square is formed release the left mouse button The size of the above cropped area is approximately 100 by 100 pixels Note The cropped region s dimensions are concurrently displayed in the lower left corner on the main window status bar 3 D Select Crop from the Operations menu and click on the Crop the Stack option or from the Toolbar click on the Crop button The image will be cropped and displayed in the viewing window Select Save As from the File menu type a file name to save the cropped image Click Save Slice by Slice Cropping 1 24 244 To crop using the slice by slice method 1 Open a suitable data set in a slice viewer and from the Operations menu choose Select Region and select the Rectangle as the cropping tool Click within the Slice Viewer projection and place your cursor in the upper left corner of the area of interest Click and hold down the left mouse button while dragging your cursor to the right and downwards A dotted line rectangle will be created select Crop from the Operations menu
134. ith the left button still depressed to stop the rotation If your mouse Is moving while you release the left mouse button the data set will continue rotating Clickin the 3D Viewer window to stop the auto rotation Select Cube Surface from the View menu From the Oblique menu select Display Slice The Oblique Slice appears on the XY plane To move the Slice hold down the Shift key left click on Leica FW4000 User Manual the slice and move your mouse gt To crop the volume or move an Orthogonal Oblique Slice hold down the Ctrl key left click on the plane slice and drag m lo rotate the Oblique Slice hold down the Shift key left click on the Oblique Slice and drag n To zoom the displayed image right click on the image and drag from bottom right to top left or vice versa Enhancement Depending on the display of the machine which is running the software images may appear too dark or in other cases too bright To compensate for these differences you can perform a Gamma correction on a particular view 1 To perform a gamma enhancement 3 1 With a suitable data set loaded select Image Enhancement from the View menu he Image Enhancement dialog appears 2 Type a value of 1 2 in the Gamma field You will notice the change in the image within the Image Enhancement dialog SX Note A Gamma value of 1 0 restores the image to its original state Values greater than 1 brighten the image whereas values below 1 darken the i
135. itis a odes aaa set a as ac TEREE J Pe S i BERE BEN Import ample Experiments Pate Aa A F FE P E E EEEE EEEE EFEFEF A a a eae sna popes a e ks n REEE S F Carte gene fe ste Hr TEF ih EEEE ee S ete xl Lee rene etait de FEFE FE eee eet FEFEFE EE Bh ttt FEFEFE enin FEE User Mame E efaulkllser Password ee Contirm Password Cancel 35 Note Only the password details are active Only a supervisor has the ability to add new users as the tabs in the dialog are not available when a level 1 user is logged on 24 Leica FW4000 User Manual 1 To change your password 3 1 Type your password into the Password field 2 Confirm your password by typing the identical password into the Confirm Password field 3 Click OK to accept the new password or click Cancel to cancel the operation Leica FW4000 User Manual 25 Setting Preferences The Preferences menu allows you to tailor the system and hardware preferences to suit your experimental environment gt To set system preferences position E H Setup ea mee eve innanananane Tai abies tetit aniis IEE anihin the cursor in the centre of the SEES SESS SERS desktop and right click the mouse or gt cael coors Logon as different o click FILE at the main menu and amenn E Show Sign o o E Preferences This sequence displays cee ee r T ccs i the Preferences dialog A Users o o ee ee ae E ee ee n ht EE iat
136. layed Sum Projection This function takes all the voxel values along each parallel ray perpendicular to the viewing surface and sums their intensity values This creates a projection of all the summed values Sum Projections provide volumetric representations of the data in which more information from the data is considered than with the Maximum projection Background noise will also be included in this type of projection To generate a Sum Projection for your data set either click on Sum Projection or select Sum Projection from the Projections menu Voxel Gradient This function finds the first voxel that is above the intensity threshold for each parallel ray perpendicular to the viewing surface then computes the dot product of the gradient at this point These dot product values make up the pixel intensity values in the 2D projection This function is ideal for examining the surfaces of objects 1 To show the Voxel Gradient 3 1 Right Click on the XZ Max projection view of your data set and select 3D Viewer from the menu 2 From the Projection menu select Voxel Gradient XS Note The Voxel projection creates a shaded iso surface for a volume at a specific threshold value X Note The default threshold for the minimum percentage box of the Voxel Gradient shading is 20 This threshold can be set on the Control Panel of the 3D Viewer under the Options Menu or in the Operation Setting from the main File menu in Leica Deblur
137. ld Selection Numerical Aperture field 1 4 Refractive Index Oil 1 515 Modality dialog Fluorescence 6 In the Emissive Wavelength frame check that the wavelength for each channel is displayed 7 Check that the channel first channel is set with the correct Probe type and wavelength 8 Check that the channel 2 second channel is set with the correct Probe type and wavelength 9 Check that the Automatic Deconvolution Settings is disabled 10 Type a value of 100 into the Total Iterations field 11 Type a value of 50 into the Save Interval field enter This performs 100 iterations of deblurring saving the 50 and 100 iteration Leica FW4000 User Manual 253 14 15 16 17 18 19 In the Output File Format check that the setting is on TIFF 16 bit This is the same format as the Input File Tick Performance checkbox for faster processing Set the Noise Level Check that the Minimum Image Intensity Removal is set to No Check that the Special Specimen Type is blank Check that the Use Recommended Expert Settings is enabled Click OK The 3D Blind Deconvolution starts The results are displayed in the main viewing window Close all views before continuing on to the next section 2D Deconvolution 254 Leicas Deblur and 3D Visualise deconvolution allows you to apply the Blind Adaptive Deconvolution to a single two dimensional image The 2D Deconvolution algorithm is also capable of improving the resolution of
138. lected a sensible set of images 1 To prepare images for publishing 3 1 Open the Leica Image Gallery 2 Select the individual images you want to publish 3 Click the Image Gallery Gallery menu and click Export Selected Images to Publishing 4 Close the Leica Image Gallery beet poe E E PN or t t ttt AELA BEA BA IN BA IN BA BE BA B BA BE OA B E i oF art al FA FTE me eet cents es ee EEEH i kar A eel ee eee E ee ee f Taf z i yo E E BEGGES E E EE E ERRERA ea Pubs E ae a H ERAGE oa EHI E eid e ARBECA AQE o e Acti ese E ES Se posite e T a EEE FEE z Hae t eee LES ork To select or clear images for processing and publishing right mouse click a branch of the Lab Book view to show the context sensitive menu 124 Leica FW4000 User Manual Select all images in node Clear all images in node Display Image Details gt To add a new image set Right click on the Experiment node at the top of the tree view 2 Click Add new Processed Image Set and enter the name of the image set in the Define Image Set dialog 3 Click OK Select all images in node Clear all images in node Display Image Detalls Add new Processed Image Set To change the name of an image set 1 Right click on the image set name 2 Click Rename Image Set and enter a new name for the image set in the Define Image Set dialog 3 Click OK Leica FW4000 User Manual 125 Existing Image Se
139. location The current location is displayed in the text box alongside the button You can also change the location by typing the new location into the text box Leica FW4000 User Manual Opening the Lab Book The Lab Book is the central controlling element for choosing the experiment to work in Also it is the interface to the Leica FW4000 database The structure of the Lab Book for each experiment is identical and is set up for you by default It contains your data captured images and results for each of your experiments The Open Existing Experiment dialog shows the structure of a Lab Book You can reach the Lab Book from the Leica FW4000 Desktop without having to restart Leica FW4000 gt To open the laboratory book click Lab Book The Open Existing Experiment dialog is displayed deve Raw Images Processed Images 0 Date 25 05 2002 Time 14 39 31 Raw Image Folder Raw Images Frocessed Images Measurement Result Conclusione and Mot Study fasi El Jims Experiment Dont Ti m El Jims Experiment Dont Ti m dims F neriment oe BPAE Fluorescent cell 2 cab 65 F aT 1 L ix C Program FilessLeicaPyy 4000D atasD etant sern BPAE Fluorescent cell Processed Image Folder C Program Files LeicaF w 4000D ata DetaultUsersBPAE Fluorescent cell Documents Movies L Wavelength nm Exposure ms Point Spread Functie he Structure SESE DAFI 359 231 13 a BPAE
140. m a smoothing operation first for a stronger effect Automatically evaluates the intensity distribution of the image and adjusts the contrast and brightness to give a more evenly weighted distribution Automatically adjusts the contrast curve to a logarithmic distribution improving the visibility of dim features Automatically adjusts the contrast curve to an exponential distribution reducing the visibility of dim features Automatically adjusts the contrast curve to a distribution based on the square root of the intensity increasing the visibility of dim features Inverts the image Leica FW4000 User Manual Tip In most cases the Adjust Contrast Brightness option will give the best result Try increasing the contrast level to increase the range of intensities then decrease the brightness level to help suppress the lower background signals gt To sharpen only a defined part of the image open the Image Viewer Settings menu and click ROI 1 To accept the contrast changes you have made 3 4 Click Apply or Apply to Selected in the Image Viewer to save your enhanced processed images 2 Click OK to save your processed image or stack or click Cancel to close the dialog without saving any changes Where do you want to sawe the images 7 f To an existing Image Set Processed Images C Toanew Image Set Cancel Aw Refer to Chapter 7 Using a Processed Image Setfor further information Image Equalisation You
141. mage Try Again W Enable Monitoring Show Stage Plan Note This way of working provides a hands off automated image capture sequence such as time sequence or Z stack image sequences method Refer to Chapter 5 Capturing Images Alternatively set the image frame and exposure you require whilst the image is Live and click the Camera button Click the Image Viewer button to display the Image Viewer where you will see the captured image displayed Adjust the size and screen position of the Image Viewer so the image viewer and XY dialog are open alongside each other Note This way of working is recommended during optimisation of the image acquisition parameters Leica FW4000 User Manual Reviewing Images The Image Gallery displays thumbnails of the component and composite images which you can select and magnify The options Supplied with the gallery allow you to The gallery is an optional component 4 4 gt gt AN Combine images Change the order in which the images are displayed by probe fluorochrome time point acquired and Z position Select and deselect images for inclusion in processing or publishing activities Redefine elements of the experiment Recall experimental details Use filtering options Launch an image viewer E RN feia Fie Gallery ode ye G J BPAE cells 20x magnification Z stacl SN Refer to Chapter 6 Reviewing mages Leica FW4000 User Manual 57
142. mage Gamma values may be between 0 1 and 10 0 3 You may also adjust the image Black Level and Brightness by using the scroll bar next to these adjusters Xo Note The Brightness and the Black Level values are used in a histogram stretching intensity filter For 8 bit data this filter sets all the pixel values that are greater than the Brightness parameter to 255 and all the pixel values less than the Darkness parameter to 0 Pixel values which fall in between the brightest and darkest parameter values are linearly scaled between 0 and 255 Image Enhancement may be done on a single colour channel Red Green or Blue or on All Channels by activating the appropriate choice under Leica FW4000 User Manual 217 4 218 Channel 4 To return to the original data click Cancel on the Image Enhancement dialog 5 Click OK to close the Image Enhancement dialog Note When applying Enhancement to a Slice Viewer projection it is possible to change only the slice being displayed Note When viewing in a Slice Viewer the maximum and minimum values for Brightness and Black Level are the true maximum and minimum values of the entire data set For other projections the Brightness and Black Level values represent the maximum and minimum intensity values of the view Leica FW4000 User Manual Tutorials for Projections Menu Items Maximum Projection S This function takes parallel rays perpendicular to the viewing surface a
143. mages that may be loaded You can process and measure the images Imported into the experiment using Leica FW4000 tools Composite images generated in a third party piece of software may be viewed during the import step but if imported they may not be used for image processing Note If you attempt to import tiff images from Leica OFluoro that have lost their accompanying nfo file they must be treated as third party tiffs that is each image must be assigned as a probe To import tiff files 1 Browse for the location of the images you wish to import in the Import Experiments from QFluoro System dialog The tiff images will appear in the window on the right 2 Select the images in the list that you wish to import checking all experiments in the folder if you wish to automatically select all Use the shift key to select all images between selected items or the control key to select freely from the list 3 Click Import and a preview of the first image is shown Leica FW4000 User Manual Importing abe 0001 46 tik 4 f Single Image Experiments 2 Stacks Sequence Experiments a La od Program Files EJ LeicaF w 4000 C DefaultSupervisor 4 DefaultUser Logs _ Sample Database New Experiment Name image no Imaget Probe probe name All experiments in folder Merge to single experiment abe_O001 39 tif abc_O001 41 abe 0001 42 tif abe_O001 43 hr abe ODOT 44 tif abe 00074
144. ment and familiarise yourself with the Leica FW4000 software Leica FW4000 User Manual Lab Book The Lab Book is the central controlling element for choosing the experiment to work in It is also the interface to the Leica FW4000 database The Lab Book structure is set up by default for your data captured images and results The electronic files are structured very much like a laboratory workbook and contain the following areas or topics for your data E sperimenta E etails ME Users aap H A DefaultSupervisar Hame EPAE cells 20s magnification B A DefaultUser Raw Images BEF EFAE cells 20x magnification stack Processed Images 0 o he Conditions Date 25 05 2002 Time 14 09 02 2 a fe nee Raw Image Folder C Program FilessLeicaP 4U fi Processed Image Folder C Program Files LeicaF w 40 i Measurement Results Study fe Documents nane 3 Wavelengt _ P omposite OP a FITC 490 1268 16 i og Texas Aed 2002 05 oe Point Spread Functions Structure E BPAE cells 20x magnification Z stack1 mE BPAE Fluorescent cell 0000000 gaaon Lee aL PAVE Fluorescent eella vA Refer to Chapter 2 Opening the Lab Book for more information on using the full set of facilities available with the Lab Book Leica FW4000 User Manual 53 Experimental Process This chapter gives you an overview of the six major elements in the Experimental Setup that you will use when performing an experiment Setup gt
145. ment images or add them to the experiment In addition to the original images Leica FW4000 User Manual 127 128 Leica FW4000 User Manual Chapter Seven Processing Images Chapter Overview This chapter contains the following topics Fundamental Principles of Image Processing Starting the Process Environment Image Toolbar E m Z stack Playback Toolbar m Time Sequence Playback Toolbar Customising Your Image Toolbar Using the Processed Image Set Windowing tools Erasing Send Images to Leica QWin No Neighbours and Nearest Neighbours Deconvolution Defining a Region of Interest 8 Applying an Overlay Leica FW4000 User Manual 129 Fundamental Principles of Image Processing 130 When you acquire images with Leica FW4000 the brightness and contrast of the image are affected by various factors These factors Include Camera integration time Video levels Camera gain and offset The sharpness and resolution are dictated largely by the quality of the optical microscope and camera that you use to acquire the image Leica FW4000 provides many facilities to help you acquire high quality images Certain image processing techniques are available to allow you to enhance images after they have been captured These include the ability to Highlight features of interest by eliminating background fluorescence Adjust image brightness and contrast to improve visualisation of detail
146. ments dialog Setup Alternatively click Settings in the Measurement dialog and choose Setup 2 Select the Excel tab f i G Regions Features Parameters Limits Classify General Excel Excel Template C Program Files LeicaF 40005 7 ernplates B ook 1 nlt ia if Send ColummHeadings I Send Reference Data Print Grid Lines Sheet First Colurmr fi First Aow Paper size for printing measurements OF Cancel Type the name of the required Excel Template into the Excel Template box Alternatively click the button to browse to locate the required template Leica FW4000 User Manual 5 Check Send Column Headings if required 6 For templates that use more than one Sheet select the sheet from the drop down list 7 Specify the First Column and First Row numbers if required 8 Click OK to apply the changes and close the dialog or Cancel to close the dialog without applying the changes XS Note You are able to Save the Excel files by using the File menu in the Measurements dialog Leica FW4000 User Manual 169 Making a Measurement Manually Use the toolbar along the bottom of the Measurements dialog to select the kind of shape you want to draw on the image m 170 I Apply Measure Frame J m eo ae oe Alternatively click Settings in the Measurement dialog and choose Setup Click on the appropriate menu item to perform the action you desire The actions from th
147. mes dialog allows you to Select the fluorochromes used by each image Add fluorochromes Create fluorochromes Modify fluorochromes Specify the colour used to represent each fluorochrome Select the sequence in which the fluorochromes are displayed on dialogs and captured during acquisition sequences Define the wavelength of light used to capture an image of a particular fluorochrome gt gt gt Oo OM To select Fluorochromes click The Setup Fluorochromes dialog is displayed ml Hemove The Select Fluorochromes dialog see the screen below allows you to organise the fluorochrome database by adding and deleting fluorochromes and entering detailed information Available fluorochromes are listed in the centre area of the dialog where detailed information for the selected fluorochrome is also displayed Leica FW4000 User Manual m lo change the sequence of image display in the Image Setup use the Move Up and Move Down blue arrows to physically move a fluorochrome in the list n To exit from this stage in the Environmental Setup process click the button in the top right corner of the Setup Fluorochromes dialog 1 To add a fluorochrome to the active list 3 4 Click Add Empty For Brightheld BGR EH FLU 2 Double click Name for the fluorochrome you wish to add to the Setup Fluorochromes dialog or highlight the fluorochrome required and click Add 3 Click OK or Cancel to cancel the opera
148. mount of RAM required by the deconvolution application It should be turned Off only if the deconvolution application is producing rigid box like artefacts in your data Dynamic Subvolumes The Dynamic Subvolumes selection allows the deconvolution application to subdivide the data set into the largest size the processing computers 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 deblur a data set You should verify that the following fields are set to their default values The Subvolume overlap Pixels is 10 The Guardband Pixels is 10 The Z Guardband Pixels is 6 Leica FW4000 User Manual 237 Subvolume overlap The Subvolume overlap setting Pixels determines the number of pixels that the montaged subvolumes will overlap The possible values are integers from 0 to N 2 where N is the width or height of the XY field in pixels whichever is smaller An overlap of 10 or 25 pixels usually works best If the result of the deconvolution contains artefacts having rigid lines edges or an obvious grid structure start with a value of 10 If doing so reduces the problem but does not eliminate it then increase this number again Overlapping regions are deconvolved twice so making this number too large for example 100 will increase the deconvolution time Guardband Pixels The Guardband size
149. must ensure that each experiment is opened and Sync d for each set of images used for batch processing Remember that deconvolution may have made some intermediate results as well Sync will collect all of them for the current experiment Leica FW4000 User Manual 213 Tutorials for the Deblur View Menu Items Single View 214 This Deblur feature allows you to choose the view in which the data set is presented The data can be viewed from three orthogonal perspectives The choices available are XY ZY and XZ where X represents the image width Y the image height and Z Is the depth or may be thought of as the optical axis in microscopy applications When a choice is made the single view that is generated will be for the currently active projection For example if a Sum Projection is the active view selecting ZY will generate a ZY Sum Projection of the view XY isthe view ofthe face of the data set or the front view of each slice XZisan edge like view of the data set or of one of its slices It can be thought of as a horizontal slice through the XY view ZYisa side view of the data set or each slice It can be thought of as a vertical slice through the XY view To view a vertical slice through the XY view 1 With a suitable data set loaded for example the TLBview tif data set select XZ from the Single View option under the View menu Note Transmitted Light Brightfield data will open in the Min Projec
150. n as 1D deconvolution is a form of blind deconvolution especially customised for speed It is designed to be the fastest method for deblurring a confocal data set and is used for applications where improving the resolving power is desired only along the optical axis This is a typical scenario for confocal data collection where empty lateral magnification is often avoided the X and Y dimensions show no distortion or haze Typical processing Leica FW4000 User Manual 263 1 24 264 times are in a range from two to five minutes with a 256 x 256 x 32 data set Pentium III 450 MHz To deblur a confocal data set 1 Select Axial Blind Deconvolution from the Launch menu Leica Deblur and 3D _ Visualise executes a one dimensional deconvolution of the data set A status dialog appears indicating the progression The Untitled deconvolution results appear on the screen upon completion Generate an XZ View by clicking on the XZ view button on the Toolbar Untitled deb XZ Max projection appears on the screen Generate a Slice Viewer of the Untitled1 deb XZ Max projection by clicking on the Slice Viewer button on the Toolbar The median slice number approximately 34 will be displayed Compare the axial deblurred result to the raw data set Leica FW4000 User Manual Tutorial for Batch Menu Item This function can be launched directly from the Deconvolution menu and selecting Batch Process The Batch process is an alternative ch
151. nction changes when you are live that is the arrows change to a red square or you can click on the Camera button to acquire the image 5 Click the next probe button and adjust the slider to the required value 6 You can set up the correct exposure for another probe by repeating steps 1 to 3 but selecting a different probe 7 Repeat these steps until you have set the exposure time for each probe you wish to use n Alternatively you can select a different probe while a current sequence is running by clicking another probe button Leica FW4000 User Manual 73 Setting up the Camera and Microscope The Advanced Setup dialog image is dependent on the camera in use To access this dialog select Advanced from the Setup Live Image dialog This dialog has two tabs one for camera setup and one for microscope setup a 19 00 Camera Offset __ f 5 85 me Lamera Gain Image Type 8 Bit Mono Binning fi F Set to Standard Values Averaging 1 frames Colour Setup Auto Exposure Auto Expose Standard Lamp fO 09 Black Level Vi rite Level 74 Leica FW4000 User Manual Mag Changer 1 x Aperture Diaphragri Field Diaphragri Lamp E in r009 z Black Level Vi rite Level 1 To define the Brightfield lamp brightness 3 1 Select the Brightfield probe 2 Use the Lamp level control to m specify the brightness of the lamp Use the slider to chang
152. nd cast them through the image The maximum voxel value encountered along each ray is taken for the projection pixel value and the resulting image is made up of each maximum voxel value This projection highlights edges and prominent bright features To use maximum projection 1 With a suitable data set is loaded in the XY Maximum projection view select XZ from the Single View option under the VIEW menu Choosing different views will display the image from different perspectives 2 Set XY Max Projection as the active view by left clicking in the view Note The XY Max Projection view is the standard default view except for transmitted light brightfield data which opens in the XY Minimum projection view Note The status bar on the lower left corner of the main window will display the active view and projection Minimum Projection This function takes parallel rays perpendicular to the viewing Surface and cast them through the image The minimum voxel value encountered along each ray is taken for the projection pixel value and the resulting image is made up of each minimum voxel value The Minimum projection provides volumetric representations in which foreground intensities tend to be suppressed This projection highlights edges and prominent dim features Leica FW4000 User Manual 219 To use minimum projection select Minimum Projection from the Projections menu The Minimum Projection will be automatically calculated and disp
153. nd smoothness suppresses snowy like noise due to low light levels 3D Blind Deconvolution is a method of deconvolution that adapts itself to the real PSF of the microscope system which can be significantly different from the theoretical PSF and from the previously measured PSFs due to specimen and Instrument variations The Leica Deblur and 3D Visualise Blind Deconvolution system adapts to PSF changes within a specimen itself Thus the deconvolved results are superior to those methods which utilise theoretical or previously measured PSFs which do not require the point spread function PSF of the system to be explicitly known prior to the deconvolution 1 Using 3D blind deconvolution 3 1 With an appropriate data set loaded select Launch 3D Deconvolution from the Launch menu The Launch 3D Deconvolution dialog appears Tip Before starting a deconvolution it is good practice to verify the Deconvolution Settings 252 Leica FW4000 User Manual 2 Onthe Image Dimensions frame verify the following Dimensions Pixels Microns Width 90 6 3 Height 120 8 4 Depth 50 1 5 3 Verify the Spacings values are Dimensions Microns X 0 07 Y 0 07 Z 0 15 4 in the PSF Dimensions frame ensure that the PSF Dimensions fields are disabled To enable these fields select the Non blind method from the Deconvolution Methods section X Note For this tutorial we have selected the Adaptive Blind method 5 On the Microscope frame Fie
154. ne per click until the value you require is displayed 7 Click Set Upper or Set Lower to confirm the focal plane value To display the image at either end of the Z Plane range gt click the relevant Set Upper or Set Lower arrow The focal plane colour changes as you click either of the two arrows Focal plane Colour Upper Red Lower Blue Current Green Leica FW4000 User Manual When you set the upper or lower values such that they are not visible on the scale within the cube click Rescale This action repositions the family of Z planes back in view and scales them to give a good graphical representation within the cube Aescale as the centre of the stack click Set Centre X Note Once you have decided on the mid position typically the most in focus image or the zero position set on a DM microscope and clicked Set To define the current position of the Z stack Set Centre Centre the number of planes required will be divided above and below this centre point X Note To use any feature in Leica FW4000 requiring automated movement of the microscope you must ensure that the upper and lower focus limits are set This can be done by selecting Initialise Focus from the Hardware Setup menu Leica FW4000 User Manual 19 Selecting Fluorochromes 80 This stage in setting up the parameters for your experiment allows you to set up the fluorochromes that are relevant to your experiment The Setup Fluorochro
155. nt by multiple users on a network Access of the same experiment by one user on different stations In the network Leica FW4000 installation normally saves images in the local file system and information about the images and all experimental data ina local SQL server database Leica FW4000 connects to the database by using information stored under a named system data set You can change this information so that the data source Is another SQL server somewhere else on the network Images and other data are stored in the file system at locations you determine when an experiment is created If you place this data on a network drive that is mapped to a drive letter then other users can access this data and the database will record information about these files using the mapped drive letter so that Leica FW4000 will behave normally 10 Leica FW4000 User Manual Configuring Leica FW4000 Stations in a Network Each workstation in the network must map each network drive to be accessed If one workstation is assigned to act as the server this workstation must also map the network drive on which the database resides as the same letter drive as all other workstations in the network 1 To set up network working 3 1 Runthe Network Assistant Application This application needs to be run on each workstation in the network and must be run on the server workstation first 2 Launch the Network Assistant application by browsing to C Program Files
156. nu and select Blue Channel An image set consisting of only the blue channel will be generated S Note Close all views before going on to the next tutorial All Channels This function allows you to view the Maximum projection of a Multi channel colour image Multiple probes can be mixed from the File menu by clicking Import Multichannel Tiff A Multi channel image can contain either two or three channels You can assign any one of the three colours red green or blue R G B to any one of the channels when loading a data set Each pixel in the Multi channel image will consist of a triple intensity value R G B This triple value corresponds to the way pixel values are stored For a two channel data set you may disable the channel not desired You may assign any Channel to a sequential data set The emissive wavelengths and their associated channel colours are suggested for simplicity 1 To view the maximum projection of all channels 3 1 Under the FILE menu select Import Multi Channel Tiff which launches the form that is used to mix multichannel data interface 2 The Disable Channel selection is None by default This is the setting used for a 3 channel data set The Channel selection will Leica FW4000 User Manual 225 226 be set on Red Note Your Tool s Option in Window s Explorer for hide file extension for known file types must be unchecked in order for you to view the tif extension 3 Browse and s
157. ock all experiments Use this to unlock all experiments when the database has locked them as a result of a problem Allow deletion Leica FW4000 User Manual 37 This option is checked by default Uncheck it if you want to prevent your experiment being deleted Reset gallery options Use this to reset any image selection options that have been applied in the gallery by the Select required images option Allow changes This option is checked by default Uncheck it if you want to prevent experiments being changed in any way Display experiment details This option is only available when you click Conditions and displays information about the status of the experiment ZS Note When Allow changes and Allow deletion are unchecked the icons on the experiment change to a no entry or a red key symbol 38 Leica FW4000 User Manual Point Spread Function Management Leica FW4000 applications have facilities for creating and managing point spread functions for use in deconvolving captured images with Leica Deblur and 3D Visualise You can use a captured experiment such as an image of a bead as a Point Spread Function PSF You can attach a PSF to an experiment for use later to improve the Captured images 1 To implement Point Spread Function management n 3 4 Click the Point Spread Function Management button a This action displays the Point Spread Function Management dialog Leica FW4000 User Manual 39 fil D ix
158. of images for processing ensure that you have selected a sensible set of images Refer to Chapter 6 Preparing Selected Images for Processing for further information Leica FW4000 User Manual Removing the Background 2 To remove the background from the image 1 Open the Image Viewer 2 Select the Image menu and click Background Removal 3 Alternatively click Remove Background in the Image Toolbar feia E Fie Edt View Image Database Window Settings 2 FE cA Ng WOO i E amp a amp ill Preview Toggle 16 47 Raw Images Image_T001_5001_2001_COMP tif 1024 x 1024 x24 Raw 2 4 2 7 0 0 2 4 To adjust the threshold level move the slider from left to right You will see the image change as you make this adjustment Alternatively you can type the threshold value you require into the box to the left of the threshold slider Leica FW4000 User Manual 187 188 A 5 Click Preview to preview the changes without making any permanent alterations 6 Click and hold down Toggle to view the original image Using this button you can compare the two versions of the image before deciding whether or not to retain your changes 7 Click Apply or Apply to Selected in the Image Viewer to save your enhanced processed images The Saving Images dialog allows you to define the processed image folder or the defined image set as the location for saving your images 8 Click OK to save
159. oice to starting the deconvolution process When chosen the Batch function docks the data set and their settings in a queue for processing at a later time Batch Process m Click on Batch Process in the File menu Start S This function starts the deconvolution process for the batch files in the queue Once the Batch process has started you may cancel the processing of one or more of the files by pressing the Cancel button on the progress box This cancellation will only apply to the file currently being processed and will not effect the previously processed files or the files yet to be processed Note This Menu item will not become available unless there are image stacks in the queue Batch Processing Form This function allows you to navigate to a folder and select the files you would like to add to the Batch Pressing the Explore button launches Windows Explorer Once the file of interest is located you have the option of clicking on the file and dragging it into the Batch queue or copying the file and pasting it into the Batch queue Cancel This function closes the Batch feature without starting the processing on the files Leica FW4000 User Manual 265 Clear List This function allows you to remove all the files in the Batch queue Delete This function allows you to remove the selected file from the Batch queue To use this feature first select the file to be deleted then press the Delete button Past
160. omposite ae Choose the Image to Align and the Reference image from the drop down lists Leica FW4000 User Manual 205 206 Select an area for alignment by dragging the mouse over the area you want to align on either the Image to Align or the Reference image The selected area will be displayed in the Set Alignment area on the right side of the dialog Use the slider bars to move the Image to Align in relation to the Reference image The effect is displayed in the Set Alignment area of the dialog Click OK when you are satisfied with the changes To close the dialog without saving any changes click Cancel To reset the values to zero click Reset amp Before you can see the effect of the alignment you must recombine the images To do this click the Show Composite button Leica FW4000 User Manual Converting an Image to Brightfield S33 Note This option is only available for the component images 1 To convert an image to brightfield 3 1 Open the Image Viewer 2 Select Brightfield from the Image Toolbar y Apply Apply to Selected Reset Raw Images gt Image_T001_5001_Z001_CO1 tif 844 649x16 Raw 960 Scaled 100 0 1 To accept the brightfield changes to your images 3 14 Click Preview to preview the changes without making any permanent alterations 2 Click and hold down Toggle to view the original image Using this button you can compare the two versions of the image
161. only seen if you have purchased a licence to run Leica Deblur and 3D Visualise If you have not purchased this licence the Image Viewer is automatically launched Leica FW4000 User Manual 131 Leica Image Viewer Image toolbar Z stack playback toolbar Leica File Edit View Imad Database Window Settings r Raw Images v Image _T001_5001_Z001_COMP tif 8441649124 Raw 15 7 9 8 0 8 AA There are two toolbars Time sequence playback toolbar he Image toolbar The Z playback toolbar The Time sequence playback toolbar 132 Leica FW4000 User Manual Image Toolbar The Image toolbar allows you to perform a variety of operations to enhance the image R Select allows you to draw a selection box on the image by holding down the left mouse button and dragging This box can subsequently be copied to the clipboard or moved to the Print dialog Zoom allows you to zoom in and out of the image Left clicking zooms in and right clicking zooms out Erase erases the defined area Define an area by holding down the left mouse button and drawing around the required area To remove all parts of the image except the defined area draw around the area with the right mouse button held down Define Regions allows you to define a region of interest ROI for the fluorochrome currently displayed using the current ROI settings The defined region can be used for image enhancement by selecting Use ROI from the
162. oolbar T0 open the Annotation Box click the Annotation button Tatsi in the image toolbar to display the Annotation toolbox fh Leica FW4000 User Manual 177 178 To switch off displaying the Annotation Toolbox click Annotation in the Image Toolbar Changing the Annotation Toolbox Settings You can change the settings on your Annotation Toolbox by using the Annotation Settings dialog gt To change annotation toolbox settings click the Annotation Setup button in the Annotation Toolbox Leica FW4000 User Manual S 3 Annotation Settings Options i Transparent shapes Tool style f Standard CO Albtermative Micron marker style Horzbar Yertbar Double bar Box Use fied size Fixed size i OO Default micron marker position f Left C Right Allow adjustment Settings W Save colours and settings Line width 3 Cancel Annotation module Version 1 7 23 40 Dec 2002 Copyright c 1995 2002 Leica Microsystems Imaging Solutions To customise the annotation toolbox 1 Choose from two options Check Use Line for Calibration if you want to use the Draw Line button for calibrating the system This is not 4 usually necessary Check Transparent Shapes if you want to display only the outlines of shapes If this box is unchecked shapes will appear filled in 2 Select a style for the toolbox Choose from either Standard or Alternative Note If you want to measure
163. open the Lab Book click FILE at the main menu and then click Open Experiment To import a sample experiment click File at the main menu and then click Import Sample Experiment This opens a dialog from which you can choose to open one of 12 sample experiments pean N A n m n uu n n nnn n onnon n nn u n nn n n n n n n nn on n a nan nn n n er nn n n nn nn nn n n nn a n on a nn nn nan nn n a oa 3 le View ture Revie ef ae ae stasenenenenes ttit ARRI titititi EER aE E EEE at ie fee Log on as A user RE a En eee ee ren show Sign On i ee E ESEE ACA ee ies ca EMER A EEEE EEEE EEEE EAEE oM lanage 2 Users PETTITT EEE EEEE feces itt ate annie er HHAH a sort 5 Sample Ex eriments ie ee os cbs bs seh xpi RE FER opi st a n Mei i a Se abet peer Exit oe ee oe eS ae Be Note There are 12 sample experiments available that you can use for demonstration purposes Note On initial start up an Import Sample Experiment dialog appears until you close the dialog permanently If you wish to use a sample experiment you can access the dialog through the Import Sample Experiment option Leica FW4000 User Manual 19 Supervisor s Tools for Managing Users gt To manage users passwords and ID levels click FILE at the main menu and Manage Users This sequence displays the Manage User dialog aia He a nn nn n n n mn ma min ma min tes File View au Torrt HA E ELF atn a Liana ae F Er E
164. ormance you may Start with a value of 40 for the number Total Iterations Consider the following criteria when selecting a number of Total Iterations Values Results 1 40 Minimum use if speed is the most important consideration 41 70 Recommended use for optimal trade off between speed and robustness 71 100 Maximum recommended under normal conditions use If resolution Is the most important consideration Save Interval The Save Interval field allows you to select the number of Iterations that will occur between storage of deconvolution results on the disk The possible values are integer from 1 to the number of Total Iterations For example if the number of Total Iterations is 20 and the Save Interval is 5 the deconvolution will be saved at 5 10 15 and 20 iterations The deconvolution application automatically checks for available disk space before beginning deconvolution Leica FW4000 User Manual 233 Output File Format The Output Settings File Format field allows you to select the format In which deconvolution application will save the deconvolution results The formats available for deconvolution are TIFF 16 bit TIFF sequence 16 bit SEQ SIK Use the Leica Deblur and 3D Visualise File deb to retain the most accuracy and dynamic range especially if quantitative accuracy Is Important For visual output typical monitors only support 256 greyscale levels so the 8 bit selection may be acceptable
165. ose between different sharpening operations select an Operation from the drop down list Available operations are Enhance local Improves the contrast of small details in the image contrast while generally retaining the overall contrast of the image Use the Amount slider to set the size of detail you are targeting for enhancement Extract small Pulls small features out of the image up to the size details specified by the Amount slider Only those details that are smaller than the specified size will be present in the resulting image Using small amounts 3 to 10 for example is an effective way of picking out sharp details in a larger area of fluorescence while larger amounts 11 to 20 are useful for removing areas of uneven background fluorescence Tip The resulting image may not have as much contrast as the original image To correct this try an Auto Stretch Intensity operation accessible in the Contrast tab The following table gives some suggestions on which operation to use To Sye Type Amount Enhance banding Enhance local Black 2to6 patterns contrast signals background shading To accept the selected sharpening enhancements 1 Click Preview to preview the changes without making any permanent alterations 2 Click and hold down Toggle to view the original image Using this button you can compare the two versions of the image before deciding whether or not to retain your changes 3 Click Apply or Apply
166. ou to view the channels and slices of a multi channel data set X Note Keep the data set open from the previous section 1 To view the channels and slices of a multi channel data set 3 1 Select Slice Viewer from the VISUALIZATION menu or click the Slice Viewer button You may navigate through the slices composing the red channel 2 Close this Slice Viewer projection before going on to step 3 SS Note When a colour is selected from the Channels menu the view projection formed depends upon the currently active view projection 3 From the View Channels menu select Green Channel or click the Green Channel button to view the green channel 4 Select Slice Viewer from the Projections menu or click the Slice Viewer button You may navigate through the slices composing the green channel 5 Select All Channels from the Channels menu or click the All Channels button to view the Slice Viewer projection of the multi channel data 6 Select Close All from the Windows menu A dialog appears with the question 7 Untitled is not saved Do you wish to save it 8 Select No This will close all open views Leica FW4000 User Manual 221 Tutorials for Settings Menu Items Image Dimensions The Image Dimensions are the Width pixels Height pixels and Depth slices The Width Height and Depth will automatically be set for a data stored as TIFF tif Leica Deblur and 3D Visualise avz OcuView ocu BioRad Pic pic
167. ouse pointer The controls you can use to build your time sequence include Time Sequence Builder components IMAGE command Instructs the software to perform the defined experiment fun Runs for a defined period of time C Until stop Until stop Until aspecified time Fora period of time Leica FW4000 User Manual Waits for a period of time before executing the next Sets the number of times the last action or nested sequence Is repeated The time frequency executing the last action or nested sequence Free format message to be displayed at this point in the overall sequence These change the position of a highlighted element in the time Move Down sequence Pause command The sequence pauses until it is restarted Move right command Moves a selected number of instructions to the right This action will change the overall sequence as you will create a nested sequence Move left command Moves a selected number of instructions to the left This action will change the overall sequence as you will remove a nested sequence Delete command Deletes a selected sequence component SS Note You should refer to the list of Time Sequence builder actions available when you build the time sequences later in this chapter You can set up a time sequence that has fixed times of day in some of its steps A sequence that says Run Until 14 00 00 is reasonable if the Leic
168. ow Run for 00 00 10 TE 1 Image TOOT S001 4003 _CO1 tif Saved Image _TOO1 S001_ 4004 _CO1 tf Saved User Mame Image_TOO1_ 5001_4005 CO1 tf Saved Every 00 00 05 Image _TO01_5001_ 006_CO1 tf Saved Experiment HEB Image TOOT SO01_4007_CO1 tif Saved Image_TO01_5001_4008_CO1 tf Saved Image_TOO1_5001_4009 CO1 tf Saved Elapsed Time SECS Image TOOT S001_4010_CO1 tif Saved Image TOO S001_2011_CO1 tt Saved Image TOOT S001 4012 CO1 tf Saved Image _T001_5001_2013_C01 tif Saved Image _T001_5001_2014_C01 tif Saved Image _T001_5001_2015_C01 tif Saved Image_T001_5001_2016_C01 tif Saved Image _T001_5001_2017_C01 tif Saved Image _T001_5001_2018_C01 tif Saved Free Disk Space IC bytes bytes Disk Space Needed C Try Again Live Image When the time sequence program is complete you will see an appropriate message at the end of the run details and the Stop button will become a Close button See the end of time sequence run dialog Image_T002_5001_2011_C01 tif Saved Image _TOO2 SO01_ 012_C01 t Saved User Name Image TOO S001 7013 CO1 ti Saved isinsin Every 00 00 05 Image TOO2 S001 7014 CO1 ti Saved epee HEB 1 Image TOO S001 7015 CO1 ti Saved P IMAGE Image _TO02_5001_7016_CO1 tif Saved Ss HEE 1 Image TOO2 S001 2017_C01 tif Saved Bree nls Notify Image TOO S001 7018 CO1 tif Saved HTE 1 Image TOO 5001 2019 CO1 tif Saved Image _TOO2_5S001_ 020_C01 tif S
169. p Z3Comp Z4 Comp Z5Comp Z6 Comp v ZF Comp w ZE Comp w Z9Comp Z10Com w Z1 Com v Z12Com MEES Leica FW4000 User Manual 113 Building a Profile for the Images to be Selected 114 The Select Required Images dialog allows you to select particular images to review from the complete set you captured by defining the precise criteria that Leica FW4000 uses to find those images The dialog allows you to build a profile of the setup criteria for the images to be selected and match the captured images from the image gallery with that profile As you select different parameters to build the profile for the image you want to select the information displayed in the Filter area of the Select Required Images dialog changes After setting up each of the filter criteria you build up the total filter profile by clicking Insert The profile appears in the field in the centre of the filter area To build the image profile for images you want to process 1 Click the Image Gallery File menu and then click Select oe FE Required Images to display the Select Required Images dialog Note When you have an active image selected from the Select Required Images dialog you are returned to display all images Then you can toggle between a view of all images and the previous selection Note In this mode a context sensitive button appears at the bottom of the dialog to enable more effective toggling between the two Image views
170. p down menu available in the Cube field 6 Type a Comment if required 7 Select the Method required for this fluorochrome for example Brightfield or Fluorescence 8 Click OK to add the new fluorochrome to the list of fluorochromes or click Cancel to cancel the operation The Select Filter Cube dialog allows you to specify or modify the fluorochrome filter used in each filter position The filter associated with each filter rotator position is established by automatic examination of the microscope Leica FW4000 User Manual 83 1 To set up a filter cube 3 41 For each filter cube double click the required fluorochrome from the list of appropriate fluorochromes in the Select Fluorochromes dialog This displays the Select Filter Cube dialog x ae seri 2 Define all the filter cubes as appropriate 3 Select the filter cube you would like to use by clicking on the Cube Name or Cube Position field 4 Click OK or Cancel to cancel the operation The selected filter cube is added to the fluorochrome list gt To change the colour representation of a fluorochrome filter double click the colour for the fluorochrome filter you wish to change Select the appropriate colours from the extensive colour database displayed or design your own colour Click OK to apply the new colour gt To change the defined filter for a fluorochrome in the default list double click on the filter name for example RGB in the Select fluo
171. quence after you have set up the imaging characteristics Leica FW4000 User Manual It is possible for you to build sequences that do not have sensible meanings The Check button Scans a time sequence to identify any blocks that do not actually capture any images This situation usually results when steps are deleted in a sequence without taking care of the left over indentation Corrects some of the easier indentations to make meaningful sequences Identifies expressions that are incomplete for example Run where no time limit or stopping time has been set Time Sequence Controls Consider that the system has one referee a Aun for 10 minutes element and one metronome an Ervery 5 Seconds element The metronome is responsible for causing things to happen the referee blows the whistle to signal at the time to Stop There is only one referee and only one metronome in the system so a later Run or a later Every element will override the effects of an earlier one Two controls are required as the metronome has to be set to beat at a regular interval while the Wait timers can be set individually to any time period desired This gives more control to the experimenter but requires a little more thought in setting up the sequence Leica FW4000 User Manual 91 Building a Time Sequence To set up the time sequencing click T The Time Sequence Builder dialog is displayed Run tor 10 seconds Every 5 seconds
172. r experiment you may accumulate large amounts of data When colleagues also perform experiments they will accumulate large amounts of data The combined effect of many people performing experiments means that huge amounts of data are accumulated and reside in your laboratory s filestore To ensure that old experiments do not unnecessarily occupy and fill filestore you are strongly recommended to archive these experiments to an appropriate medium Archiving manages your experiments by using two tools available through the Archive Experiments dialog Archive Retrieve Archive stores an experiment on a storage medium but keeps a record in the database as to where you stored it You use Retrieve to quickly get the experiment back as the archiving system automatically requests the return of an experiment from the location where it is stored The other options available through the Archive Experiments dialog allow you to sort and select experiments so you can easily find the experiment you wish to archive or retrieve Leica FW4000 User Manual Archiving Experiments lo archive your experiments click Archive in the Experimental Setup process flow The Archive experiments dialog is displayed and the archiving tools are ready for you to use Check the Archive radio button Seica 5 TERE IN ame YC by Owner WE fal Results C Retrieve E Owner ES i Default SET El DefaultUser BPAE cells 20s El
173. raw gan a Lung Resistant Protein qgan Pine deconvolved gan Plant section gan File name Plant section qarn Save as type Annotation qar Cancel A 181 E n ood XS Note Annotations are automatically saved when you save an image However it is useful to be able to save and load specific annotation files You might for example use an annotation file as a template This might contain the name and address of your laboratory or other information that is required to appear frequently You can then include this information on an image without having to type it again Measuring an Angle lf you want to measure an angle you will need to change the style of the Annotation Toolbox 1 To change the style of an Annotation box Label ya 3 41 Click the Annotation button in the Image toolbar to ff display the Annotation Toolbox Annotation 2 In the Annotation Toolbox click the Annotation Setup button to open the Annotation Settings dialog wl 182 Leica FW4000 User Manual Annotation Settings Options E Transparent shapes Tool style Standard Alternative Micron marker style Horizbar Yertbar Double bar Bos Use fixed size Fixed size 100 Default micron marker position Lett C Rigt Allow adjustment Settings W Save colours and settings Line width 3 Cancel Annotation module Yersion 1 1 23 40 Dec 2002 Copyright c 1995 2002 Leica Microsystems Imaging
174. rm deconvolution on an image 1 Open the Image Viewer dialog 2 Select the Image menu click Deconvolution to display the Deconvolution Settings dialog Leica FW4000 User Manual 143 Alternatively click Deconvolution in the Image toolbar to display the Deconvolution Settings dialog Nw corns File Edit View Image Database Window Settings 4 C2 eS GAmuwWse HOO Deconvolution Settings Haze Removal 4 t f A Smoothing 4 t f Spot Removal a fio Preview Toggle 3 Specify the amounts of Haze Removal and Smoothing by using the sliders or by typing directly into the boxes 4 Click Preview to display the results of the operation in the main image window without changing the image permanently 5 Click and hold down Toggle to view the original image Use this button to compare the image before and after the enhancement 6 Click No Neighbours or Nearest Neighbours to display the Saving Images dialog 144 Leica FW4000 User Manual F l mm Where do you want to save the images f To an existing Image Set Processed Images To anew Image Set Cancel 7 Specify an existing image set or create a new image set to which you want to add your images 8 Click OK to close the dialog and apply the changes or click Cancel to close the dialog without saving any changes X Note When you create a new experiment in the Lab Book an image set called Processed Images is created automatically
175. rochromes list From then on any new experiment will automatically be set with the correct filter position for the defined microscope and filter positions will only require changing if filters are added or removed from the microscope 84 Leica FW4000 User Manual Defining Stage Positions The Visit Sites dialog allows you to pre set the position of the stage Once you have set up the stage positions you can use this dialog to move between these pre set positions You can also save a list of positions for subsequent re use n To define stage positions click N The Visit Sites dialog is displayed a Ban z Move To Remove Replace Hide Flan The Current Position area atthe top of the dialog displays the current position of the stage Leica FW4000 User Manual 85 Tip You are recommended to display the Live Image Setup and Visit Sites dialogs together Then you can use the live image as a view the co ordinates of which are recorded in the Visit Sites dialog Taking this approach will help you to design an effective stage plan 1 To add a position to the stage position list 3 41 Locate the area of interest 2 When you have located the area of interest and adjusted the focus return to the Visit Sites dialog 3 Select the Comment box and type the name for the new stage position to identify it at a later time 4 Click Add 5 Click OK or Cancel to cancel the operation 1 To replace a position
176. s days or hours To add or replace a specific image click either the Add or Replace radio buttons in the Live Image Setup To continue adding images to an experiment 1 Click Setup from the main menu 2 Select Continue and Add Images to Experiment from the Setup dialog 3 Click OK to display the Live Image dialog The Add radio button is selected automatically and the Replace radio button Is greyed out 4 Select the image capture sequence required from the drop down in the Live Image dialog a a a ee ee Fi ed a pete ES ee sy se EES setup Capture Review Process Publish Archive swindon rardwa Experiment Jims Experiment Dont Touch 05219 Images already exist for this experiment What do vou want to do 7 Cancel fe add gt Replace One Probe One Probe All Probes 2 Stack One Probe 2 Stack All Probes Note The animated Cancel Add status icon appears on the Desktop Leica FW4000 User Manual 67 To change the probe in use click the change probe Icon Note The change probe icon is a short cut to the Setup Fluorochromes dialog Refer to Chapter five Selecting Huorochromes for a description of changing and selecting probes Rescale on Images After Acquire 68 You can set the maximum and minimum grey level values for the scaling of the image after capture Subsequent images acquired will be scaled according to your settings You may set the max and min s
177. s Shift Correction dialog Use Focus cormection during automatic acquisition sequence Focus Correction 1 0 0 8 0 5 0 3 0 0 0 3 0 5 Relative Z Shitt 0 6 1 0 DAFI FITC Sek Positions Leica FW4000 User Manual 1 0 0 6 0 5 0 3 0 0 U 3 Measured 7 0 5 0 8 1 0 TRIT conc 49 50 ed Click Set Z Positions displays the X Y dialog Adjust the microscope until the image is in focus Repeat this process for each probe These values are then stored and can be applied if desired by accessing the Preference section on the Hardware tab Use Relative Focus Correction either by Selecting the option from here Switching it on by checking the box under the Settings menu on the Live image screen Leica FW4000 User Manual Chapter Three Quick Start into Experimental Setup Chapter Overview This chapter contains the following topics e Introduction Lab Book Experimental Process Leica FW4000 User Manual Setting up an Experiment Capturing Images Reviewing Images Processing Images Publishing Results Archiving Experiments 51 Introduction 52 Leica FW4000 has an easy to use and intuitive interface The environment provided allows you to perform experiments and tasks quickly and easily Tip When you have read through this chapter and Lerca FW4000 Quick Start Guide create a test experi
178. se button and drawing around the required area 3 Release the mouse button to delete the image area defined Send Images to Leica QWin 142 Leica QWin exports the current image to Leica s QWin image processing application for further detailed measurement and analysis Leica QWin is available for purchase separately Note You should have Leica QWin installed and running before you use this option Leica FW4000 User Manual No Neighbours and Nearest Neighbours Deconvolution Ne 24 Note The 3D deconvolution by nearest neighbour is a Leica FW4000 product option that is only available if you have purchased the FW4000Z or FW4000TZ modules There are two types of deconvolution No Neighbours and Nearest Neighbours which are accessed from the Image viewer and discussed in this chapter Deconvolution offered in Leica Deblur and 3D Visualise which pops up when you click on the process arrow Refer to Chapter 10 Advanced Deconvolution ftor more detailed information on using deconvolution techniques Refer to Chapter 2 Point Spread Functions Management for more information on the PSF dialog The Deconvolution button allows you to apply both No Neighbours and Nearest Neighbours deconvolution The difference in operation between the two types of deconvolution is Noneighbours deconvolution applies to one image Nearest neighbours deconvolution applies the selected parameters to the selected stack To perfo
179. sider that you are using an egg timer that is started after each IMAGE operation and which holds up the experiment for the specified time Leica FW4000 User Manual 97 98 Leica FW4000 User Manual Chapter Five Capturing Images Chapter Overview This chapter contains the following topics Capturing Images Overview Using the Image Capturing Tools Leica FW4000 User Manual 99 Capturing Images Overview A The image capturing sequence performs the set of programmed time sequences you designed when setting up the time sequencing program Refer to Chapter 4 Defining Time Sequencing Routines The key features of the image capture process are Run the image capture program automatically on entering this element of the end to end process Enable monitoring Stop or pause capturing images to modify the sequence or view the images captured so far into the sequence Rerun the time sequencing program if the set of results is deficient in some way To start capturing images click the Capture button in the Environmental Setup process flow 100 Review gt gt gt Process 2 gt gt Publish gt gt gt Archive _ The Image Capturing dialog appears When the dialog first appears there is a short time interval before the time sequencing runs When the time sequencing runs you can see progress of the run in the right window Leica FW4000 User Manual Start n
180. sing 4 Close the Leica Image Gallery Settings Display All Images Export Selected Images to Image Processing Export Selected Images to Publishing Select All Images Ctrl 4 Select Deselect Current Image Ctri I Clear Image Selection Ctrl C Delete selected Images Ctrl D Leica FW4000 User Manual 211 Click the process arrow in the Environmental Process flow to display the Process Images dialog PES DAES AS OATS OSS Blind Deconvolution send selection for processing cend to Bach M processed data 1 To select the action to be taken on a set of images when exporting 3 them to Leica Deblur 1 Click to Batch in the Process Images dialog 2 Open the drop down menu adjacent to the Batch check box to Batch fw Blind Deconvolution hake oe ae Non blind Deconvolution 30 Timelapse Maker Inverse Filter 3 Select the required processing feature from the menu 4 Press Send to send the selection for processing You may repeat steps 1 to 4 for each experiment from which you wish 212 Leica FW4000 User Manual to select images by opening that experiment The Batch queue can store requests from many exeriments gt Alternatively you can press View to open the Image Viewer to use the process tools available To return processed sets of images to the experiment folder in which it was created use the Sync button SS Note After batch processing has completed you
181. ssing Adaptive PSF Deconvolution Fired PSF Deconvolution PSF Processing Aral Stretch Factor 1 PSF waist Ain Disc s 1 Disable PSF Constraints crcl Help Object Section This section of Expert Settings affects the handling of the object data The default values are ZMontage is deselected disabled XY Montage is selected enabled Dynamic Subvolumes is selected enabled Leave these fields at their default values Montage The Z Montage setting allows the deconvolution application to break the data set into sections along the optical axis and to deconvolve these subsections separately The valid settings are e On checked e Off unchecked 236 Leica FW4000 User Manual The default for this option is Off It should only be turned On for image stacks with a large Depth setting for example gt 100 slices This option reduces the amount of RAM required by the deconvolution process It may also be useful in rare cases where the sample thickness is so large that the PSF changes dramatically along Z In such cases Z Montage allows the blind deconvolution to find different PSF solutions for different depths XY Montage The XY Montage option allows the deconvolution application to break the data into sub volumes along the XY dimensions The valid settings are e On checked e Off unchecked The default for this setting is On This option reduces the a
182. t Unit You can choose from Microns typical Millimetres Centimetres Nanometres and Inches 6 Click OK to apply the new settings or Cancel to abandon the changes 7 You are then prompted to update the system calibration as shown on the right side Click Yes to update the calibration or No to retain the previous calibration settings Note The Calibration button in the Image toolbar changes the calibration for the current image only Note Only people with supervisor level access may calibrate the system Users may calibrate the current image only Leica FW4000 User Manual Annotating your Image The Annotation Toolbox provides facilities for you to annotate an image You access the Annotation toolbox by clicking the Annotation button on the image toolbar The buttons in the annotation toolbox are Select Delete object Change colours Draw line Straightline Fy Arrow ine mye Change font fpg Mutt tne tex _ nce Slilinse aria ine airs p eairation scale Adding an Annotation The Annotation Toolbox allows you to add pointers text and calibration markers to the image Use annotation to draw attention to and provide further information on interesting features within the image The annotation is saved as a separate layer of information and does not alter the image itself but can be saved with the image Separate annotations are maintained for each image window S Refer to Chapter 7for more information on the image t
183. the Properties dialog Details Fiter What fields do you wish to see Available Properties Selected Properties Cancel 2 Build up a set of selected properties by highlighting a property in the Available Properties list and clicking the single right arrow button The highlighted result moves into the Selected Properties list Repeat this action until you have selected and moved the required properties into the Selected Properties list 3 If there are any results in the Selected Properties list in error you can return those results to the Available Properties list by clicking the single left arrow button 4 Ifyou wish to move all properties between the two lists click the double right or left arrow button 5 When you are satisfied with your choice of selected properties click the Filter tab Leica FW4000 User Manual 299 Filter Tab The Filter tab allows you to restrict the number of experiments displayed in the archive dialog using search criteria 1 To build up the filter profile for an experiment to be archived 3 41 Select the Filter tab from the Properties dialog Details Filter i Limit records to those where Owner YO DefaultUser op Owner DefaultUser OK Cancel Apply i 2 Check the Limit records to those where box 3 Click the Filter menu arrow and click E sperment_IL the required filter Experiment IC Name WL Espate_ OT Espfime_OT Own
184. the region of 5 to 15 mm To apply the new margin settings click OK Click Cancel to close the dialog without saving any changes Right clicking on the image to be printed in the Print Page dialog displays the Print Options dialog This allows you to toggle the Show Annotation and Show Reference Data facilities by clicking Leica FW4000 User Manual 211 the appropriate radio button Print Options X Show Reference Data Cancel I lt Show Annotation Apply to all images Margins Lett sar Right oe Top mm Bottom 25 mm 9 Click Setup to display the Printer Setup dialog Print Setup Ei Ed WILMSLOW ASHP LaserJet 4050 Series ting Properties Status Ready Type HF Lagenlet 4050 Seres PS Where print Comment Onentation Portrait Paper Size Ad ig C Landscape SOUICE Automatically Select Network Cancel 10 Select the printer type used by Leica FW4000 Select a printer and click OK to apply your choice AS Refer to your Microsoft Windows documentation for information on using 278 Leica FW4000 User Manual the standard Printer Setup dialog and for using the Microsoft Windows Font dialog 11 Click Fonts to change the font used to print the reference data The standard Microsoft Windows Font dialog will be displayed 12 Click Print or click Close to close the Print page dialog together with the selected images in the frames Leica FW4000 User Manual 219
185. this setting that can each be used in succession with a difficult image to extract the most information from it The PSF images will be stored in a new experiment To define the location of this experiment select the browse button marked and browse to the desired location Leica FW4000 User Manual Changing the Microscope s Current Configuration You can change the current microscope configuration details It will already be set up to match your hardware and you will not normally be required to change these settings The dialog may be used to verify that the microscope configuration is correct test the connection between the computer and the microscope or alter the software configuration if the microscope options have been changed n To change the microscope configuration select Hardware Setup from the main menu and click Setup Microscope This displays the Setup Microscope dialog Leica FW4000 User Manual me Data in nitialise stage eee ene EEFE o ee ae Setup Microscop a i oy gangs an na ae HHN a as es Device ee AAA eny ER i Panra aaraa a aaa ar aE Aa aa aa aa PaE a AE a aa aa a aa A k sae EEE p 43 Leica Peripherals ad Stand Microscope a seen sH Peripherals er Stage Demo Focus Settings Lamps Filter Settings Shutter Configure Optics icrascope F oeer pe i EO eis
186. ting of Low indicates that noise can barely be seen in the data A setting of Medium indicates that the noise can easily be seen in the data and a setting of High indicates there Is a significant amount of noise Widefield data generally contains low levels of noise therefore the default setting is Low Confocal data generally contains medium levels of noise therefore the default setting is Medium Use Other to manually input the Noise Smoothing Factor to indicate that the amount of noise falls in between the settings of Low Medium or High PSF section In the PSF section there are two tabs Adaptive Blind Non Blind Non Blind tab The Non Blind tab allows you to select the PSF input from two choices Synthetic PSF or Measured PSF Adaptive Blind tab In the First Guess frame verify that Stretched Theoretical is enabled Axial Stretch Factor is set to 1 The First Guess frame has two options Flat Sheet that selects the initial estimate of the object used to initiate a blind deconvolution process This option uses for first Leica FW4000 User Manual 239 guess a volume filled with a constant value equal to the average of the entire image stack For very noisy data this is an appropriate starting point for the deconvolution Stretched Theoretical that is a theoretical calculated PSF lengthened along the Z axis to more accurately initiate the first guess for a non widefield data set gt
187. tion gt To display a predefined list of default fluorochromes from which you can select check the Show all Fluorochromes box m To delete selected fluorochromes click Remove Leica FW4000 User Manual 81 1 To change the sequence of image capture 3 4 Click the Advanced button in the Setup Fluorochromes dialog to display the Setup Fluorochromes Advanced dialog Sequence f Lambda then C 2 then Lambda 2 Toggle between the Lambda then Z and Z then Lambda radio buttons This radio button changes the order in which the images are captured For example Lambda then Z will go through the stack capturing all the DAPI images followed by all the FITC images Z then Lambda captures DAPI FITC DAPI and FITC 3 Click the Standard button to return to the Set Fluorochromes dialog aca Note The wavelength boundary for the fluorochrome selected in the list of fluorochromes ts highlighted in the Advanced section of the dialog 82 Leica FW4000 User Manual 1 To create a fluorochrome 3 1 Click Create in the Select Fluorochromes dialog This displays the Add Fluorochromes dialog Colour Flucrachrome Mame Excitation Emission Cube Method Comment Proteins 2 Click in the Colour field Choose a colour from the Colour Palette and click OK to return to the Add Fluorochromes dialog 3 Type a Name for your fluorochrome 4 Editthe values contained in the Excitation and Emission boxes 5 Choose a Cube from the dro
188. tion Leica FW4000 User Manual 261 Processing Current Slice 1 24 262 To process the current slice 1 2 Select Slice Viewer from the Projections menu The slice currently displayed will be the slice processed Select Nearest Neighbors from the Launch menu and click on Processing Current Slice The Nearest No Neighbor Slice Operation dialog appears The Haze Removal Factor the Z Kernel Width and the No Neighbours selections will be set at their default values Click the Apply button The Nearest Neighbours deconvolution is applied to the current slice Click the Restore button to return to the original view of the slice To use the No Neighbours algorithm instead of the Nearest Neighbours algorithm check the No Neighbors box on the Nearest No Neighbors Slice Operation dialog Adjust the results by trying different values for the Haze Removal Factor and the Z Kernel Width also try the No Neighbours deconvolution by clicking in the flag box to activate and deactivate it Set the Haze Removal Factor to 0 97 the Z Kernel Width to 3 and leave the No Neighbors box unchecked These values produce satisfactory results for the Nearest Neighbours or No Neighbours algorithm Click the Restore button to restore the optical section to its original view Click the Close button to exit from the Nearest No Neighbours Slice Operation dialog The algorithm chosen Nearest Neighbours and the values selected for the Haze Removal
189. tion view 2 Setthe XY Min Projection as the active view by selecting TLBview tif XY Min Projection MULTI CHANNEL from the Windows menu item or by clicking within the XY Min Projection view Note The status bar at the bottom of the main window will display the active view and projection Leica FW4000 User Manual Triple View Selecting the Deblur Triple View feature allows you to create three orthogonal views of your data set Each view is the Minimum Projection of the data On the top left of the Triple View display is the XY view on the top right is the ZY view and on the bottom left is the XZ view of the data set The TLBview tif XY Minimum Projection from the previous section should be active oD 1 To activate the TLBview tif X Y Min Projection 3 o Select Triple View from the View menu The XY ZY and XZ views of the TLBview tif data set are displayed in the Triple View box Make active the TLBview tif XY Min Projection data set From the VISUALIZATIONS menu select Slice Viewer The data set will be displayed in a Slice Viewer From the VIEW menu select Triple View The XY ZY and XZ Optical Slice Viewers are displayed in the Triple View box The red lines indicate where each slice Is in relative position to the others in the data set To remove the red lines click Hide Crosshair To view the red lines click Show Crosshair To close the Triple View click on the X in the upper right corner of the Triple Vi
190. tire feature connected to the ROI that is if part of a QO feature is inside the ROI the whole of that feature will be included in the ROI mask 7 You can now draw with the selected tool on the image to define a region You can define several regions if necessary 8 Click OK to close the Select ROI dialog XS Note The information area at the bottom of the dialog will display information about the selected modes 9 Use the Display area of the dialog to specify how the ROI mask should be displayed on the image Show mask outlines Displays only the outlines of included features Show full mask Displays the entire features 10 Click Overlay Colour to change the colour in which the ROI mask appears This will display the Colour dialog Click a colour and click OK to close the Colour dialog to apply the new colour 11 Click OK to close the dialog and apply the ROI mask Click Cancel to close the dialog without saving the changes Click Undo to cancel the most recent operation Leica FW4000 User Manual 149 Below you can see an example of features selected by an ROI mask The lines around the features indicated the defined regions Applying an Overlay sp Click on the Image in the main menu and Ee hetebase Window select the type of overlay display you require for your experiment from one of the options Contrast Background Removal Brighthield Sharpen Smooth Deconvolution Composite Mas Proyection Setup All
191. to Selected in the Image Viewer to save Leica FW4000 User Manual your enhanced processed images The Saving Images dialog allows you to define the processed image folder or the defined image set as the location for saving your images 4 Click OK to save your processed image or stack or click Cancel to close the dialog without saving any changes Where do you want to save the images 7 To an existing Image Set Processed Images To anew Image Set s A Refer to Chapter 7 Using a Processed Image Setfor further information 5 To sharpen only a defined part of the image open the Image Viewer Settings menu and click Use ROI Leica FW4000 User Manual 195 Smoothing an Image 1 24 196 To smooth an image 1 Open the Image Viewer 2 Select the Image menu and click Smooth to display the controls for smoothing an image Alternatively click Smooth in the Image Toolbar to display the controls for smoothing an image Lica an File Edit View Image Database Window Settings RO S OSS ame Efe Reset Preview Toggle 00 fe White Median Noise Redi C Blaci By removing noise from an image through smoothing you can sometimes improve the effect that other subsequent image processing steps will have For example the Auto Contrast enhancement in the Image Contrast dialog often gives a better result for very noisy images if performed after a smoothing oper
192. to move a command to the left m Click the right pointing arrow to move a command to l the right X Note Notice that toggling between the L and R buttons allows you to adjust the indenting and nesting of sub sequences in your time builder Leica FW4000 User Manual 93 Building a Time Sequence Example Tutorial 94 This tutorial will guide you through building a complex time sequence We will start with a simple example and add time sequence elements and build a complex sequence Build the simplest possible time sequence that contains the elements Start now IMAGE and Stop Start now stop 15 29 03 aR Cancel This sequence starts as soon as the Capture button is pressed runs for 10 seconds and takes images at five second intervals What IMAGE does will depend on other settings for the experiment It may be a single frame from the camera or a complete Z stack of many planes in several fluorochrome probes In this example the Start element is selected so you can set a Start Time of day by selecting the At button or you can set an initial delay by using the After button Leica FW4000 User Manual Add the Every element into the previous example Burn C Until stop lt Start now Every 5 seconds C After Delete Check 15 32 29 Cancel The Every element causes an IMAGE operation which could be a s
193. torials for Channels Menu Items Red Channel Green Channel Blue Channel All Channels Viewing the Channels and Slices of a Multi channel Data Set Tutorials for Settings Menu Items Image Dimensions oo E E EHEHEHE EE E Leica FW4000 User Manual 209 Spacing Microns Data Background Deconvolution Settings Standard Settings Expert Settings Operation Settings Load Settings Save Current Settings As Tutorials for Operations Menu Items Crop Tutorials for Movie Menu Items Movie Maker Opena Movie Concatenating Movies Tutorials for Start Deconvolution Start 3D Blind Deconvolution m 2D Deconvolution Inverse Filter Ei E Nearest Neighbours No Neighbours Axial Blind Deconvolution Tutorial for Batch Menu Item Batch Process Leica FW4000 User Manual Exporting Images from Leica F W4000 to Leica Deblur and 3D Visualise XS Note This Chapter is applicable only to users of the Leica Deblur and 3D Visualise software This chapter describes the steps involved in passing image sets from the Leica FW4000 to the Leica Deblur and 3D Visualise Before you can process your images in Leica Deblur and 3D Visualise you must specifically select them and export them to the Image Viewer 1 To prepare images for processing 3 1 Open the Leica Image Gallery 2 Select the individual images you want to process 3 Click the Image Gallery Gallery menu and click Export Selected Images to Image Proces
194. traight lines You can also combine these two methods if required 3 Release the mouse button to finish To draw an area 1 Select the Area button D 2 Click inthe image where you want the area to start by either Holding down the left mouse button and draw a freehand area Releasing the mouse button and click again to draw a Straight area You can do this many times to create a number of nodes joined by straight lines You can also combine these two methods if required 3 Click the right mouse button to finish drawing the area The end of the line will automatically join with the start of the line As you finish drawing the shape the measurements will appear in the Measurements dialog To measure an object click the Measure button in the Measurements dialog Leica FW4000 User Manual gs iss Settin the In click Settings Alternatively gt Setup and choose dialog Measurement Foint Measure Objects Small Square Medium Square Circle Ellipse Line Area Measure Objects Delete Selected Region Clear Regions click the Profile button in the To measure the grey scale levels Measurements dialog nb Note The grey scale measurement tool is best used in conjunction with the line drawing tool 2 ile Edit Settin zeungs F Save Frame I Apply Measure tee m Copy Setup amp Print 0 New Region 1 DAPI Re
195. ts for this experiment Raw Images Processed Images Nem name for Image Set A oo oaeo Description TA Note This action allows you to change subsets of processed images and you can enter into a defined image set within the Lab Book The Lab Book offers you several ways of entering images raw processed and subsets 126 Leica FW4000 User Manual Replacing Images When you review images in the gallery and need to replace one recapture that image using the image capture tools To replace an image or images in the image viewer 1 Either Select the image to be replaced in the Image Gallery Ifyou are replacing more than one image select the node in the tree view containing the images to be replaced 2 Click the Replace Images icon to open the Live Image fe dialog T Note The status icon on the desktop shows the animated Cancel Replace icon Cancel Replace 3 Select the image to be replaced in the Live Image dialog 4 Capture the image To capture images at several different stage positions use the Capture icon on the main form Refer to Chapter 4 Defining the Live Acquisition for a detailed description of capturing a single image 5 Ifyou decide not to replace the images after all click the Cancel Replace status icon on the desktop 6 To replace the images press the Review icon and then select the Accept Replacement Images icon in the gallery Alternatively you can reject the replace
196. ue of 1 is the default tt does not change the image n Use the Contrast slider to adjust the relative contrast difference between the light and dark features in the image A value of 1 0 indicates that no adjustment is made Values less than 1 decrease the contrast while values greater than 1 increase the contrast Leica FW4000 User Manual 201 202 Use the Brightness slider to adjust the overall brightness of the image A brightness setting of 0 0 indicates that no adjustment Is made Values less than 0 indicate a darkening of the image while values greater than 0 indicate a lightening of the image Alternatively choose a contrast operation by clicking Operation from the drop down list Available operations are Adjust Contrast Brightness Auto stretch Intensities Histogram Equalisation Logarithmic Fit Exponential Fit Square Root Fit Invert Allows you to adjust the image contrast and brightness manually Use the Contrast slider to adjust the relative contrast difference between the light and dark features in the image Use the Brightness slider to adjust the overall brightness of the image A brightness setting of 0 0 indicates that no adjustment is made Automatically determines the minimum and maximum intensities in the image and adjusts the contrast and brightness as needed to ensure the image occupies the full sensitivity scale This Operation can be sensitive to noise so it Is sometimes useful to perfor
197. uorochromes selected from the fluorochrome database and define the settings to be used for each fluorochrome selected Discrete positions on the sample where you require images to be captured Time sequencing routines used to run the experiment This chapter advises you on how to set up each of the parameters and where applicable how the parameters interact with each other Start the setup process by clicking Setup on the Environmental Setup process flow AED gt Se gt a gt gt The Live Image Setup opens automatically when you click on Setup and displays a further set of process buttons You must check the check box where applicable before you can set up that aspect of the environment While the Live Image Setup is open you can Open the other dialogs and arrange them on the Desktop Arrange the sequence of the process icons on the screen by right clicking an icon and drag dropping It to the position required Leica FW4000 User Manual Defining Live Image Acquisition You define the image acquisition parameters by setting the X Y and Z coordinates together with setting up the camera and selecting a region for the field of view that will be captured gt To set up the X and Y coordinates click X Y The Live Image Setup dialog is displayed Image Size Image Viewer Settings LabBook Calibration Z E Rescale i Advanced m Click the Acquire button to acquire t
198. vailable Parameters Selected Parameters Select 4 Erase w Annotate ca Measure Measure Intensity E HOI F ve Calibrate a i Remove Background A Make Composite Contrast p al Save mage EL Deconvolution 2 Move toolbar parameters between the two lists until the set of tools you require for your experiment is listed in the Selected Parameters list Use the single ee and double arrows in the centre of the dialog to move the parameters gt Note Single arrows move the highlight parameter in the direction of the arrow Double arrows move the entire contents of the selected list in the direction of the arrow 3 Move the parameters to the desired order in the Selected Parameters list by using the up and down Aha arrows 4 Click OK v ea 138 Leica FW4000 User Manual Using the Processed Image Set When you create a new experiment in the Lab Book an image set called Processed Images is created automatically in the experiment This is the default image set used when the Saving Images dialog appears There are a number of places where this dialog is used after clicking Apply or Apply to all when using the deconvolution or image enhancement tools Once you choose where the processed images should go the image set that is currently In session changes in the drop down list at the bottom left of the image viewer This provides a mechanism for you to switch image sets from the Image View
199. velength for each channel 6 To change the wavelength select the channel of interest radio button and either type the wavelength nm or select the probe type 7 For the first channel Channel1 set Probe to FITC or type 456 for the wavelength 8 For the second channel Channel 2 set the Probe to DAPI or type 520 for the wavelength 9 Channel 3 will remain blank when working with a 2 channel data set Leica FW4000 User Manual 231 Sie ee ee ee Deconvolution Settings The Deconvolution Settings section has seven fields Oe Oo Oo o Automatic deconvolution settings Total iterations Save interval Output file format Deconvolution performance Minimum image intensity removal special specimen type Use recommended expert settings To setup the standard settings by using deconvolution methods 1 2 3 4 8 9 10 11 Check that the Automatic Deconvolution Settings are disabled In the Total Iterations field type a value of 100 In the Save Interval field type a value of 50 This will perform 100 iterations of deblurring saving the 50 and 100 iteration In the Output File Format verify that the setting is on TIFF 16 bit This is the same format as for the Input File Click the Best Medium Fast check box The Deconvolution Performance settings refer to the Stability Resolution and Speed of the deconvolution Verify that Noise Level is set to No Verify that the Special Specimen Type
200. wer to save your enhanced processed images Click OK to save your processed image or stack or click Cancel to close the dialog without saving any changes Leica FW4000 User Manual Channel Mixing nb Channel mixing allows you to adjust the colour intensity for each probe To adjust the colour intensity of each probe 1 Open the Image Viewer 2 Click the Make Composite button from the Image Toolbar The Channel Mixing dialog is displayed Alternatively click Image in the Image Viewer main menu and choose An to display the Channel Mixing dialog File Edt View Image Pe au Settings w Nw Zz 5 nw Q T Adjust All Preview err rer rere i Create composite for each z and time position i To anes the intensity of a probe hold the left mouse button down on the coloured bar and drag it up and down or click at the new level required To adjust all probe intensities simultaneously check Adjust All Leica FW4000 User Manual 191 n Click Reset to reset the intensities to their previous values n To apply changes click Create or Create all in the Image Viewer to close the Channel Mixing dialog and apply the changes You can close the Channel Mixing dialog without saving any changes by clicking the cross in the top right corner of the dialog gt If your experiment comprises Z stacks and or Time Sequences you can make composite images from different Z positions in the stack and from diff
201. when entering numerical information for controlling hardware for example when adjusting the focus position as this causes your stage to move unexpectedly Setting Hardware Preferences The Hardware tab allows you to set up the hardware for manual and automatic microscopes gt Click the Hardware tab in the Preferences dialog 30 Leica FW4000 User Manual es 2 ee ee l eh fein E aS Manual Filter Changer Initialise Focus Manual Shutter Close Shutter during Capture After each Stack We Turn Lamp Off After Acquisition Lamp Delay seconds Os seconds Use Relative Focus Correction Read Objective and data fram microscope stand Enable Audio Warning i Show Event Reports UE Cancel To change a filter with a manual microscope check the Manual Filter Changer box XS Note When the Manual Filter Change box is checked you will be prompted to ensure the correct filter is in position n To open the shutter and ensure that light is reaching the sample check the Manual Shutter box gt To Initialise the focus when your microscope has an automated X Y Stage click the Initialise Focus button or external control device gt Closing the shutter gives you options on when you want the camera shutter to close The options available are Leica FW4000 User Manual 31 Only at the end of capture After each stack After each sequence After each image gt gt n To turn the lamp off after
202. y dialog and display the Create Web Page dialog By Web Page Report Template Limit length of text to i characters Create web page Close 5 Select the appropriate report template for the number of probes you have used in your experiment by clicking one of the format buttons in the centre of the Create Report dialog gt Alternatively to select the report format click Browse to display the Select Template dialog Select the appropriate template and click Open Browse Leica FW4000 User Manual 285 Select Template Look in TemplatesHTML a do jE E saicFW4000 dot ite BF LeicaFw4000 2 dot My Recent Bf LeicaFw4000 3 dot Documents 8 L eicaFWw 40004 dot F W LeicaF w 40006 dot 1 BF LeicaFw4000 3 dot Desktop BF MosaicFw4000 dot a k as i k a F i biy Documents k File name Leica WA 000 4 dot Wy Metwork Files of type Templates Cancel Places Open as read only 6 Click Create web page to create the web page or click Close to close the Create Web Page dialog without creating the web page You will be asked to specify a file name and location to which the web page Is to be saved 286 Leica FW4000 User Manual Producing a Web Montage The web montaging feature allows you to select single composite and component images from different image sets and publish them as a web page montage 1 To montage your selected images for web publishing 1
203. you to identify more clearly activities occurring in your experiment Samples 1 To develop composite Images SiG 3 4 Move the Probe button so that tic EEE positioned at the right end of the four buttons the exact number depends on whether stage positioning zstack and time sequence are being used and click Reorder 2 Click on the Display Probes Composites radio button 25 Leica Image Gallery File Gallery xX 2 Ba C Display All Selected Images i Display Probes Composites Preview Composite l MitogreenERRedT1 v ZIDAPI v ZIFITC v ZITRIT v Z2DAPI v Z2FITC v Z2 TRIT v Z3DAPI v Z3FITC v Z3TRIT Z4DAPI v Z4FITC Z4 TRIT ct 2 E A subset of the available images has been selected Yew All Images 112 Leica FW4000 User Manual 3 Click the required Probe s to use for producing composite images 4 Click Preview Composite Preview Composite Preview Composite 5 Repeat steps 3 and 4 as you view a variety of composite images w Note This allows you to quickly preview composite images of different combinations of probes but only produces thumbnail images When you are happy with the combination of probes use the Image Viewer to define the precise composition of the composite images and to create the full size images 25 Leica Image Gallery Fie Gallery eL C Display All Selected Images f Display Probes Composites z MitogreenERRedT 1 Z1 Comp Z2Com

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