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1. You can set up your software s user interface in such a way that you can view the live image 1 and the images that have up till then been acquired 2 next to one another 00181 25072011 4 3 Acquiring HDR images Preparations HDR stands for High Dynamic Range Dynamic range relates to the capacity of cameras or software to display both bright and dark image segments Before acquiring an HDR image the necessary exposure range needs to be determined for the current sample The exposure range is made up of a minimum and maximum exposure time as well as several exposure times between them A recently determined exposure range will continue to be used for all HDR images until you let your software determine the exposure range anew It is irrelevant whether the exposure range had been determined automatically or manually If you are acquiring several images of the same or similar parts of a sample you don t need to determine the exposure range each time If you change the sample or adjust settings on the microscope it is recommended to determine the exposure range anew either automatically or manually Acquiring an HDR image with a manually determined exposure range With this procedure you set the minimum and maximum exposure time in the Camera Control tool window yourself Your software guides you through the process with relevant message boxes How much the exposure time is adjusted by is determined by your software w
2. 7 Check whether you are satisfied with the changed image resolution If not change the image resolution anew e You can first reduce the image resolution then save the report and then increase the image resolution again This is possible because each time that you open the Change Image Resolution command the image is transferred from your software to MS Word 139 Working with reports 8 Once you are satisfied with the changes to the image resolution save the report Take a look at the new file size in the Windows Explorer Updating placeholders The Update Placeholders command makes it easy to have any changes made to the images after the report has been created also shown in the report Please take note that all changes made in your software have to be saved if they are to be displayed when the Update Placeholders command is used In MS Word you open a report that you created some time ago In the meantime you had changed a lot of images in your image analysis software e g added measurements Now the report is to be updated so that it shows the newest version of all of the images 1 If you only want to update one placeholder select just that one 2 In MS Word use the Olympus gt Update Placeholders menu command e The Update Placeholders dialog box opens 3 Inthe Update Placeholders dialog box specify whether or not all placeholders should be updated 4 Select the Update fields linked with placeho
3. On the right you can see the preview image after the threshold values were set The image background is now black The colors in the preview image have changed because the image in the preview window is always displayed with the most possible contrast Viewing the results 13 In the Output group select the Image as new layer and Intensity Profile check boxes lf you want to output the intensity profile as a sheet select the Export to workbook check box 14 Select the ROIs that you defined on the cells The ROIs you have selected are highlighted in the dialog box 15 Close the Ratio Analysisdialog box with OK e If it wasn t already displayed the ntensity Profile tool window is displayed automatically now The tool window contains two intensity profiles one for each ROI you defined 108 Browsing the time stack Viewing the ratio image and the source image separately Life Science Applications PUTT CTE PTT ee eee eee eee eee ee ee er ere ee 13403 A Hwe 610 me CUR OO The intensity profiles show how the ratio value in both ROIs 1 and 2 changes over time The colors of the intensity profiles correspond to the colors of ROIs they describe e The source image is now a multi layer image and displays the concentration of calcium ions in addition to the image information 16 Use the File gt Save As command to save the resulting image Save the resulting image in the TIF or VSI file format Setting
4. User Manual cellSens LIFE SCIENCE IMAGING SOFTWARE Any copyrights relating to this manual shall belong to OLYMPUS CORPORATION We at OLYMPUS CORPORATION have tried to make the information contained in this manual as accurate and reliable as possible Nevertheless OLYMPUS CORPORATION disclaims any warranty of any kind whether expressed or implied as to any matter whatsoever relating to this manual including without limitation the merchantability or fitness for any particular purpose OLYMPUS CORPORATION will from time to time revise the software described in this manual and reserves the right to make such changes without obligation to notify the purchaser In no event shall OLYMPUS CORPORATION be liable for any indirect special incidental or consequential damages arising out of purchase or use of this manual or the information contained herein No part of this document may be reproduced or transmitted in any form or by any means electronic or mechanical for any purpose without the prior written permission of OLYMPUS CORPORATION Adobe and Acrobat are trademarks of Adobe Systems Incorporated and be registered in various countries OLYMPUS CORPORATION All rights reserved Version 510 UMA _cellSens19 Krishna en_00_01August2013 10 11 Contents About the documentation for your SOPWALEC ccccceeeeeeeseeeeeeeeeeeneeeeneeeseseneees 5 User INEA asa A EA a E EA EAE Nt 6 Maries ONEI OW oirn er E A E E oonete SoctaGee
5. open the Acquisition Settings dialog box Select the Acquisition gt HDR option in the tree view e You can find more information on this topic in the online help If you don t want to change any settings use the File gt Save As command to save the image Use the recommended TIF or VSI file format e These are the only formats which also save all the image information including the HDR entries together with the image In this way you can see e g whether or not an image was acquired using HDR Open the Properties tool window and look at the data in the Camera group 21 Acquiring single images Acquiring more HDR images without setting the exposure range anew If you have just acquired HDR images of the same or a similar sample as a rule it is not necessary to determine the dynamic range anew In this case you have already completed the preparations for acquisition Such as carrying out a white balance and set the HDR image acquisition settings correctly such as choosing the optimal algorithm used for output rendering anyway In such circumstances acquiring an HDR image is especially easy Do the following 1 Inthe Camera Control tool window select the Activate HDR check box 2 Click the HDR button in the Camera Control tool window to start the image acquisition e The image acquisition will begin After the acquisition has been completed the HDR image will be shown in the document group 3 Check the image
6. Only of the active image check box 118 Viewing the statistical parameter 10 11 12 13 Measuring images Close the dialog box with OK e Now the results for both images will be shown simultaneously in the tool window In the Measurement and ROI tool window click the Measurement and ROI Options button Select the Measurement and RO gt Results entry in the tree view e Inthe Statistic group you ll find various statistical parameters Select the Mean check box Close the dialog box with OK e Now in the Measurement and RO tool window under the measurement results the chosen statistical parameter 1 will by shown You can see there the mean value of the layer thickness for all of the measured images THK TI proita Adsa O9VOOPKEGA Fa 257 78 jam Fa a 264 16 pm ral 317 72 H a 228 08 um J 225 886 pm 317 72 um 266 92 um 00154 119 Measuring images 12 2 Performing an automatic image analysis Task Preconditions Setting options Setting threshold values You can use an automatic image analysis to perform numerous measurement tasks Several typical tasks and their process flow are described here Counting objects You have an image with objects that interest you You want to know how many of these objects there are in the image The objects that you want to count must not be connected but must be clearly separated from one another The for
7. The time stack will then be turned into a standard true color image This image shows the frame that is at that moment displayed on the monitor Image processing operations e g a sharpen filter affect either the whole image or only a selection of individual images You will find most of the image processing operations in the Process menu You can find more information on working with image processing functions in the online help The dialog box that is opened when you use an image processing operation is made up in the same way for every operation In this dialog box select the Apply on gt Selected frames and channels option to determine that the function only affects the selected frames Select the Apply on gt All frames and channels option to process all of the individual images Select the individual images that you want to process in the tile view You can find more information on this image window view in the online help Look through the thumbnails and select the images you want to process In the tile view the standard MS Windows conventions for multiple selection are valid An image processing operation does not change the source image s dimensions The resulting image is therefore comprised of the same number of separate images as the source image 00011 Time Lapse Movie Both the Time Lapse and the Movie acquisition processes document the way a sample changes with time What is the difference between the t
8. channel time stack for instance incorporates several color channels Every color channel incorporated in the image is reproduced with its own time stack Ch The multi dimensional images have their own navigation bar directly in the image window Use this navigation bar to define how a multi dimensional image is to be displayed in the image window 00009 5 1 Overview Acquisition processes Basic acquisition processes Complex acquisition processes Your software offers a wide range of different acquisition processes Note Your software is available in a variety of versions This online help contains the description of all acquisition processes For this reason it can happen that your software version doesn t have some of the acquisition processes described here 23 Acquisition process Snapshot Dx a Acquisition process Movie Acquisition process Time Lapse Acquisition process Acquisition process XY Positions MIA Acquiring multi dimensional images Basic acquisition processes Use the Camera Control tool window to acquire images and movies You can use your software to acquire high resolution images in a very short period of time You can use your software to record a movie When you do this your camera will acquire aS many images as it can within an arbitrary period of time The movie will be saved as a file in the AVI format You can use your software to play
9. e The button looks like this if the lamp is switched on e Inthe Selected components list you can also see that the lamp for this observation method will be switched on 12 Click the OK button to save the new observation method e The Microscope Control tool window will then contain a new button with this observation method s name e You can now use the observation method in the Process Manager tool window for the acquisition of a multi channel fluorescence image Defining observation methods for fluorescence channels Define an observation method for the acquisition of a fluorescence image It also makes sense to do this when you don t work with a motorized microscope since then the acquired image will be automatically colored with the correct fluorescence color The following hardware components belong to an observation method for fluorescence channels How you integrate these hardware components with the observation method is described in detail in these step by step instructions Hardware component Settings E Fluorochromes Assign the fluorescence colors Ka Mirror turret Choose the mirror cube to be used g Open the fluorescence shutter for the image Fluorescence shutter acquisition T Transmitted light lamp Switch off the transmitted light lamp 4 Use the Acquire gt Devices gt Device Customization command Activate the Observation Methods tab Click the New Observation Method ae button Give the n
10. gt Count and Measure Results command 12 In the Count and Measure Results tool window switch to the Class Histogram results view by clicking the Class Histogram tab e The results will be shown in the results sheet for every frame in the time stack You will then be able to immediately recognize whether and how the number of objects has changed over the time period 13 In the Count and Measure Results tool window switch to the Class Measurements results view by clicking the Class Measurements tab 14 There select the Object Count entry in the Measurement picklist and the Time entry in the Grouped by picklist e The histogram shows the measurement parameter s chronological distribution In the histogram you can always recognize which measurement value belongs to which frame 15 Move your mouse pointer onto the histogram When you move along the black line with your mouse the mouse pointer will alter its appearance and changes into a double arrow Keep the left mouse button pressed Now you can move the line and by doing so select different points in time The image belonging to the respective point in time will always be shown in the image window In the chart the changes in the number of objects over the time period will be shown Along the X axis the frames are mapped along the Y axis the number of objects Exporting a chart You can also export the chart and save it as a file This enables you to archive your data o
11. layout 1 Toolbars 2 Tool windows 3 Status bar 4 Menu bar The layout saves the element s size and position regardless of whether they have been shown or hidden When for example you have brought the Windows toolbar into a layout it will only be available for this one layout 00013 User interface 2 3 Document group The document group contains all loaded documents These can be of all supported document types As a rule the loaded documents are images Appearance of the document group 1 Document group in the user interface 2 Document bar in the document group 3 Buttons in the document bar 4 Navigation bar in the image window 1 Document group in the user interface You will find the document group in the middle of the user interface In it you will find all of the documents that have been loaded and of course also all of the images that have been acquired Also the live image and the images resulting from e g any image processing function will be displayed there 2 Document bar in the document group The document bar is the document group s header For every loaded document an individual tab showing the document name will be set up in the document group Click the name of a document in the document bar to have this document displayed in the document group The name of the active document will be shown in color Each type of document is identified by its own icon At the top right of ea
12. Close command in the context menu For the selection of documents the standard MS Windows conventions for multiple selection are valid 12 Closing all documents Closing a document immediately Generating a test image User interface To close all loaded documents use the Close All command You will find this command in the File menu and in both the Documents and the Gallery tool window s context menu To close a document immediately without a query close it with the Shift key depressed Data you have not saved will be lost Opening documents There are a number of ways in which you can open or load documents 1 Use the File gt Open command 2 Use the File Explorer tool window To load a single image double click on the image file in the File Explorer tool window To load several images simultaneously select the images and with the left mouse button depressed drag them into the document group For the selection of images the standard MS Windows conventions for multiple selection are valid 3 Drag the document you want directly out of the MS Windows Explorer onto your software s document group 4 To load documents from a database into the document group use the Database gt Load Documents command You can find more information in the online help If you want to get used to your software then sometimes any image suffices to try out a function Press Ctrl Shift Alt T to generate a color test
13. Experiment Manager to acquire several multi channel fluorescence images of a certain position on the sample at certain intervals Just like the Experiment Manager you can use the Process Manager tool window to handle complex acquisition processes The Experiment Manager can be used as an alternative to the Process Manager It s more intuitive for more complex processes and offers you more options e Inthe Experiment Manager tool window you can trigger a particular device at a particular time using the RTC to add a chemical to your sample or to heat or illuminate your sample for example e Only the Experiment Manager allows you to define loops within loops This makes it possible to repeat a fast time stack several times at particular intervals e The Experiment Manager allows you to use streaming to attain shorter intervals between two separate image acquisitions in a time stack than are possible with the Process Manager Prerequisites for using the Experiment Manager Prerequisite The Experiment Manager tool window is only available when you ve purchased the Experiment Manager solution in addition to your software This solution is only available with the highest software package Make sure that your software is correctly configured When you want to acquire multi channel fluorescence images it makes sense to define observation methods for your color channels before you define the experiment If you want to assign the fluorescence c
14. In this dialog box select the Apply on gt Selected frames and channels option to determine that the function only affects the selected frames Select the individual images that you want to process in the tile view Click e g fa this button m in the image window s navigation bar to switch to the tile view Look through the thumbnails and select the images you want to process In the tile view the standard MS Windows conventions for multiple selection are valid Select the Apply on gt All frames and channels option to process all of the individual images An image processing operation does not change the source image s dimensions The resulting image is therefore comprised of the same number of separate images as the source image 00012 Acquiring a Z stack A Z stack contains frames acquired at different focus positions That is to say the microscope stage was located in a different Z position for the acquisition of each frame Note You can only use the Z Stack acquisition process when your stage is equipped with a motorized Z drive Example You want to acquire a Z stack The sample is approximately 50 um thick The Z distance between two frames is to be 2 um Fa E r AS 1 Switch to the Acquisition layout To do this use e g the View gt Layout gt Acquisition command 2 On the Microscope Control toolbar click the button with the objective that you want to use for the image acquisition 3 Switch to
15. Manager tool window e The acquired fluorescence image is displayed in the image window by default after the experiment is finished In the image window all three color channels are superimposed on each other so that you see all three fluorescence images at the same time 6 Inthe image window take a look at the multi channel fluorescence image that has been acquired If necessary change the settings for individual commands in the experiment plan 73 Running experiments Acquiring a multi channel fluorescence image together with a transmitted light image Expand your experiment plan and acquire a transmitted light image as well with the brightfield observation method for example 1 Select the multi channel group in the experiment plan Use the mouse to enlarge the frame so that there s enough room inside the group for another image acquisition command 2 Click the small arrow next to the mage Acquisition button to open a menu Choose an observation method for the acquisition of a transmitted light image e g phase contrast differential interference contrast DIC or brighttield 3 Click once in the multi channel group to insert the element for the acquisition of the transmitted light image into the experiment plan It may be necessary to first enlarge the multi channel group s frame Next position the element to the right of the last fluorescence image acquisition 4 Connect the last fluorescence image acquisition
16. Ratio Image One color channel is divided by the other on a pixel by pixel basis The result is the ratio image in which the intensity is proportional to the ion concentration When the Fura 2 fluorescence dye is used the image acquired at the excitation wavelength of 340 nm is divided by the image acquired at the excitation wavelength of 380 nm Color channel 340 nm Color channel 380 nm 4 Viewing a ratio image The result is a multi layer image One image layer is the source image and the other image layer is the ratio image The ratio image displays the concentration of ions using pseudo colors The pseudo color image is superimposed on the source image so that you can see the structures in your sample and the concentration of ions at the same time You can change the display of the resulting image in the image window in order to view only the ratio image for example 105 Life Science Applications The process flow of a ratio analysis on a multi channel time stack 1 Acquiring a multi channel time stack 2 Carrying out background correction vy 3 Calculating an intensity profile When the ratio analysis is carried out on a multi channel time stack you can compute the intensity profile in addition to the ratio image The intensity profile displays the change in concentration of ions in a particular image segment You determine the image segment by defining a ROI You can define the ROI right here in the Ratio Anal
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18. The Manual Threshold dialog box will be closed 127 15 Open the Count and Measure Results tool window by using the View gt Tool Windows gt Count and Measure Results command e The results will be shown in the results sheet for every ROI The Area Fraction ROI column shows what percentage of the ROI s area is taken up by the phase 00350 Working with reports 13 Working with reports 13 1 Overview The illustration shows a report example that is made up of three pages and contains six images Process flow when you create a report The report functionality enables you to document the results of your work and to make them available to third parties You can publish reports as a DOC file or DOCX file for MS Word 2007 2010 or as a printout When a corresponding printer driver is available on your system you can also print reports to a PDF and send them to other users When working with reports you have to switch to the Report layout In this layout the Report Composer tool window that is required when working with reports is by default displayed The first step when working with reports is always opening or creating a new report instruction in your software In the report instruction you specify which images and which page layout are to be taken for the report The display and any further editing of the created report are done in the MS Word application program Therefore this program has to be installed on
19. The actual image data will not be changed Save the spectrally unmixed image if you need it 00361 96 Life Science Applications 11 3 Colocalization Examples for colocalization What is colocalization In the fluorescence microscopy it can occur that the fluorescence signals emitted by two parts of a sample e g molecules that have been stained with different fluorochromes interfere with each other In these cases the different parts of the sample lie very close to one another or one over the other The effect of the interference of fluorescence signals is termed colocalization In the digital image analysis the colocalization of fluorescence signals can be measured This is done by detecting pixels that have the same intensity in both color channels These measurements are carried out on multi channel images and are always valid for one channel pair 1 Superimposed signals in the green and blue color channels The colocalized pixels are displayed in white 2 Superimposed signals in the blue and red color channels The colocalized pixels are displayed in white 00705 26072011 97 Life Science Applications Measuring the colocalization Use the Colocalization button to start a measurement of colocalization You will find this button on the Life Science Applications toolbar This button isn t available in all software versions Measuring the colocalization on the whole frame 1 10 Load the mul
20. You can find more information on the navigation bar for multi channel images in the online help Creating multi channel images Your software offers you several ways of acquiring a multi channel image Use the Multi Channel automatic acquisition process to acquire a multi channel image Use the Image gt Combine Channels command to have several separate images combined into a multi channel image 35 Using the navigation bar Hiding the navigation bar Using the Dimension Selector tool window Breaking down a multi channel image into its color channels Reducing the color channels in a multi channel image Acquiring fluorescence images Displaying multi channel images A multi channel image contains much more data than can be displayed on your monitor 1 23 When you load a multi channel image into your software you ll see a navigation bar in the image window that provides you with access to all of the fluorescence channels e You can have the individual fluorescence images displayed separately or also as a Superimposition of all of the fluorescence images 1 e Should you have acquired a brightfield of the sample together with the fluorescence images you can make this brightfield appear or disappear 2 e The individual fluorescence images are monochrome For this reason you can change the color mapping however you like You can display the fluorescence channels in the fluorescence colors use a pseud
21. a Life Science Application toolbar displayed You can find the functions for j defining ROIs and for measuring the intensity profile on this toolbar 3 If necessary use the View gt Tool Windows gt Measurement and ROI command to have the Measurement and ROI tool window displayed The ROls that are defined in the current image are listed in this tool window 4 Use the Tools gt Options command Select the Measurement and ROI gt Dynamic ROI entry in the tree view Select the nterpolate linearly continue with current option You have now defined how a dynamic ROI behaves when you define its position on the frames Viewing the movement 5 Use the navigation bar at the top of the image window of the paramecia _ M4 re CO 6 Move the slide control slowly to the right to view the movement of the paramecium that is at the top left border of the image in the first frame e The paramecium first moves down and to the left Then it changes direction and moves up before finally disappearing at the left border of the image Defining ROIs Region 7 Display the first frame in the image window To do this use the navigation Of Interest bar at the top of the image window 8 Click the New ROI Rectangle L button on the Life Science Applications toolbar e The ROI is displayed in the sheet of the Measurement and RO tool window In the Type column the keyword ROI is added to the type name 9 With two mouse c
22. all of the microscope s motorized components will be brought into exactly the position that has been defined for these components in the observation method 4 Use the View gt Tool Windows gt Camera Control command to make the Camera Control tool window appear 5 Switch to the live mode To do so click the Live button in the Camera Control tool window e For motorized microscopes The reflected light shutter will be automatically opened This behavior will be specified when the observation method is defined For the shutter the Use for acquisition status should have been selected 6 Inthe Camera Control tool window select the Exposure gt Manual option Some cameras offer the SFL mode for fluorescence acquisitions e g the DP72 Switch this mode on 8 Optimize the exposure time Should the exposure time become longer than 500 ms reduce it by increasing gain To do this change the value in the Exposure gt Sensitivity field or use the Gain slide control 9 Bring the image into focus 45 Changing the fluorescence image s display Saving the fluorescence image Acquiring fluorescence images 2 F If your microscope stage is equipped with a motorized Z drive a focus regulator will be at your disposal in the a Microscope Control tool window 10 Finish the live mode To do so click the Live button in the Camera Control tool window e For motorized microscopes The reflected light shutter will be clos
23. and the exposure time will all be shown for each color channel Acquiring a multi channel fluorescence image together with a transmitted light image Simultaneously with the multi channel fluorescence image acquire a transmitted light image e g a phase contrast image 1 Define an acquisition process for a multi channel fluorescence image To do this follow the step by step instructions described above Click the Add Channel button Choose an observation method for the acquisition of a transmitted light image e g phase contrast differential interference contrast DIC or brightfield Click on the transmitted light channel in the Process Manager tool window e The channel has now been activated Your microscope will be set in the transmitted light mode Click the small plus sign next to the transmitted light channel e You ll now see a table with additional information about the transmitted light channel Make sure that the Transmission overlay check box has been selected Only then is the transmitted light image assigned its own layer that lies over the fluorescence channels Switch to the live mode 55 Starting the acquisition process Showing and hiding the transmitted light image in the image window Moving the transmission image with respect to the multi channel image 10 11 12 13 14 15 16 17 18 19 20 Acquiring fluorescence images Select manual exposure time i
24. be shown below in the Count and Measure tool window in the Object Count group Should you not be able to see this number click the small black arrow to make it visible If you have selected the Object Count measurement parameter the number of objects will also be displayed in the Count and Measure Results tool window s results sheet 121 Measuring images Counting objects that belong to different phases Task You have an image on which you define two phases You want to know how many objects there are per phase in the image In the image two phases are to be defined The first phase is to map the black objects within the blue round object The second phase is to map the blue round objects Preconditions The objects that you want to count must not be connected but must be clearly separated from one another The objects in both of the phases must have different intensity values by which one can differentiate between them Setting options l l l 5 1 In the Count and Measure tool window click this Options dialog box button to open the 2 Select the Detection entry in the tree view 3 In the Options group enter the value 5 in the Minimum object size field to specify the minimum object size By doing that you will rule out the possibility that individual pixels that may well belong to the phase but not to an object are counted as objects which would then falsify the results 4 Select the Measurements entry
25. column click on the column s header then select the Hide command in the context menu 4 Exit edit mode by clicking on any point in the MS Word report outside the workbook Changing image resolution By default all images of a report are transferred to MS Word reports with a resolution of 192 dpi In certain cases it can make sense to change the resolution of individual or all images in a report For example if you want to print the report you can raise the resolution Alternatively if you want to publish the report on the Internet you can reduce the resolution 1 Open the report in MS Word Decide whether you want to change the resolution of all images or just certain images 2 If you only want to change the resolution of one individual image select that image If you want to change the resolution of all images you don t have to select any 3 Select the Olympus gt Change Image Resolution command e The Change Image Resolution dialog box opens 4 Select the option you want in the Apply to group You can choose between Selected images and All images in report e The Selected images option is inactive if no images were selected while opening the command 5 Specify in the mage Resolution group how you want to change the image resolution If you choose the User defined option you can enter any resolution of your choice between 96 and 600 dpi into the DPI field 6 Click the OK button to change the image resolution
26. command with the transmitted light image acquisition command To do this click on a control point and while holding the left mouse button pressed move the mouse pointer to the other control point 5 Select the command for the transmitted light acquisition in the experiment plan e Inthe Experiment Manager tool window the Image Acquisition Camera Settings and Display groups are now shown e The Transmission overlay check box in the Image Acquisition group is available when the command selected in the experiment plan is linked to a transmitted light observation method 6 Select the Transmission overlay check box in the Experiment Manager tool window Now the transmitted light image is assigned its own image layer on top of the fluorescence images e This icon appears in the experiment plan on the element for the acquisition of the transmitted light image 7 Make the rest of the settings for the acquisition of the transmitted light image 8 Click the Start button located in the Experiment Manager tool window to run the experiment e Then together with your fluorescence images a transmitted light image will also be acquired and saved together with the multi channel fluorescence image The result of this acquisition process is a multi layer image that you can view with the Layers tool window The illustration shows an experiment plan for a multi channel acquisition with a transmitted light image 1 The transmitted light ima
27. commands for image acquisition with the FITC and TRITC observation methods as well and arrange the commands in a row e The three commands have to be connected to each other with a line This line has an arrow which unambiguously defines the order of the commands Depending on where you placed the commands in relation to each other they may already be connected 6 You can define the connector between two commands in the experiment plan manually To do so create a connector by dragging one of the control points on the edge of a command to a control point on the following command When doing this you always have to connect an output control point on the right of the element with an input control point on the left of the element 70 Running experiments e Your software automatically draws the connector in the experiment plan When you move elements in the experiment plan the connector between these elements is automatically adjusted Both experiment plans show the same experiment First an image is acquired with the FITC observation method and then an image with the TRITC observation method The green FITC element s output control point 1 is correctly connected to the red TRITC element s control point 2 The connector has been selected in both experiment plans and is therefore displayed in color Configuring the Define the exposure time and other acquisition settings for the image acquisition experiment plan agmmands 7
28. deconvolution filters essentially differ in how the point spread and noise functions are determined which are needed for the deconvolution of the image and the noise depression The more image data is used to calculate the point spread function the more precise the result will be and the longer the calculation will take 103 2D deconvolution Nearest Neighbor Filter Wiener Filter Constrained Iterative Filter Life Science Applications The 2D deconvolution filter uses a theoretical point spread function in which only the acquisition parameters are used but no image data You can apply the 2D deconvolution filter on all supported image types However the 2D deconvolution filter always affects only an individual frame As the amount of data processed is quite small the 2D deconvolution filter is very quick It makes the image appear much sharper but does not then permit any quantitative analysis of the image data The filter is especially well suited for TIRF images where the image information comes from a very narrow Z range of the sample The nearest neighbor filter employs a theoretical point spread function for the deconvolution in the calculation of which the data of the image under examination and of the two neighboring images of a Z stack are taken into consideration The point spread function is applied to the neighboring images The scaled sum of the neighboring images that have been processed in this way is then ded
29. documents cannot be included in a report LImage Fields D0811 0023 The illustration shows an example of a report instruction In the report two different page templates are to be used The first page template contains a single 131 Exchanging the document template Working with reports placeholder for an image the second page template contains two placeholders for an image After the page template the images that are to be inserted in the report page are displayed 6 Check the report instruction now You may still edit it and e g delete or shift documents or select another page template Creating a report 1 Ww Click the Create button You find this button in the Report Composer tool window e The report will be created Creating a report can take some time when large reports with many images and documents are involved Pay attention to the progress bar that is shown The MS Word application program will open automatically and display the new report In the example shown below the report has three pages The fact that the first page template only contains one image placeholder and two images have been selected in the report instruction automatically leads to the creation of two report pages If you want to you can still make additional changes in the MS Word application program To do so use the Olympus add in or the Olympus toolbar If you want to save the report inst
30. from different positions on the sample Add a delay 1 to your experiment plan before the acquisition of the second multi channel Z stack image Select the Wait for keystroke option for the delay for example Drag a time lapse loop around the whole experiment In this case add another command Move XY 1 before the acquisition of the first multi channel Z stack image Select the Absolute position option for this command This makes sure that the images in a multi channel Z stack image have a stage position In the example shown the acquisition of both multi channel Z stack images is repeated 4 times First the system acquires the multi channel Z stack image at stage position 1 and then it moves the stage The system acquires the multi channel Z stack image at the second stage position and then it waits for the time specified in the time lapse loop s interval field After the delay the acquisition resumes at stage position 1 The experiment will produce two multi channel Z stack in time lapse images 7 Switch to live mode and move to the position on the sample where you want to start the experiment 8 Click the Start gt button located in the Experiment Manager tool window to run the experiment Running experiments Adapting existing experiments You can view and edit the settings for individual commands at any time 1 Should the Experiment Manager tool window be hidden use the View gt Tool Windows gt Expe
31. image to select this image layer The name of the image layer in the tool window corresponds to the name of the image 2 Click once on the eye icon next to the ratio image 109 Optimize the display of the ratio image Life Science Applications e The ratio image is now not displayed in the image window You see only the source image 3 Inthe Layers tool window click once on the ratio image to select this image layer e When you select an image layer in the Layers tool window this image layer is automatically displayed 4 Click once on the eye icon next to the source image e The source image is now not displayed in the image window Now you see only the ratio image The illustration shows the Layers tool window with the image resulting from a ratio analysis On the left the ratio image 1 is not displayed On the right the source image 2 is not displayed 1 Display only the ratio image in the image window Use the Adjust Display tool window to optimize the display of the ratio image 3 Select the Auto Contrast option This makes sure that the ratio image is displayed in the image window with the most possible contrast With this setting all the colors in the pseudo color table you are using are applied to the ratio image The Histogram of all frames check box decides whether only the frame in the time stack that is currently on display will have its contrast optimized or whether the contrast will b
32. in the Resolution group Reduce the image resolution in the live mode When you use a higher binning in the live mode the frame rate will be reduced which enables you to focus better For this purpose select an entry e g with the supplement Binning 2x2 from the Live movie list located in the Resolution group Should you work with a color camera Switch on your camera s grayscale mode The Toggle RGB Grayscale mode button should now look like this ia You can find this button on the Camera Control tool window s toolbar If it s possible to set different bit depths with your camera click the Toggle Ect Bitdepth button to set the maximum bit depth Use the View gt Toolbars gt Calibrations command to have the Calibrations toolbar displayed Switch off the white balance and the shading correction To do that release these buttons H LJ If they are there and available Select the automatic exposure time In the Camera Control tool window click the Live button e Should the live image be too dark select a higher value in the Camera Control gt Exposure gt Exposure compensation list e Should the exposure time become longer than 300 ms reduce it by increasing the sensitivity or gain Bring the sample into focus e Inthe camera s black amp white mode you can reduce the diffused light Click the Online Deblur m button located in the Camera Control tool window s toolbar You can then decide whether or not
33. in the tree view From the Class Measurements list select the Object Count and Object Class entries 5 Click OK to close the Detection dialog box Setting threshold 6 In the Count and Measure tool window click the Manual Threshold button vaes to open the Manual Threshold dialog box Should the Manual Threshold button not yet be active you will have to first activate it To do that select the Manual Threshold entry in the Threshold button s context menu You open this menu by clicking the small arrow next to the button 7 Delete all but one of the phases by continuing to click the Remove Phase button until the button becomes inactive e By doing that you will make certain that no phases from earlier analyses are still defined 8 Doubleclick the Phase Name field and assign a name for the first phase 9 Click any position outside this field or press the Enter key to leave the field again e The first phase in the Phase thresholds for channel group will be automatically selected 10 Click on the New Threshold A phase s threshold value range button to set an initial value for the selected e As soon as you move your mouse pointer onto the image it will change its shape to that of a pipette 122 Measuring images 11 Click on one pixel or on the image area whose intensity value is to be utilized as the initial value for the threshold range e Once the initial value has been set your mouse pointer wil
34. is shown in the Acquisition Settings gt Saving gt Process Manager dialog box The preset file format is VSI Combining individual images into a stitched image 1 2 Load the images you want to combine or acquire a suitable set of images Open the Gallery tool window To do this use e g the View gt Tool Windows gt Gallery command Select all of the images you want to combine in the Gallery tool window Use the Process gt Multiple Image Alignment command This command is only active when more than one image of the same image type has been selected e The Multiple Image Alignment dialog box will open A description of this dialog box can be found in the online help e The dialog box s stitching area will display a preview of the individual images If necessary while keeping your left mouse button depressed drag on the bottom left hand corner of the dialog window to enlarge it Alternatively doubleclick the header of the dialog box to enlarge the dialog box to full screen size Check whether the images positions are correct You can change the arrangement of the individual images e g by exchanging two images in the stitching area by Drag amp Drop The illustration shows the stitching area with four individual images On the left the images 1 and 2 are not in the correct position Image 1 green frame will therefore be dragged onto image 2 red frame On the right you see the stitching area
35. lq oe i 3 Move the slide control slowly and by doing so display frames acquired at differing Z positions in the image window Search out a Z position at which the sample can be clearly recognized 4 Use the View gt Toolbars gt Life Science Application command to have the Life Science Application toolbar displayed You can find the functions for defining ROIs and for measuring the intensity profile on this toolbar Defining ROIs Region 5 Rotate the mouse wheel to change the zoom factor Enlarge the image until erieles you can see at least one enlarged segment of the sample in the image window that is fluorescing in red 6 Click the New ROI Polygon is button on the Life Science Applications toolbar 7 By clicking with your left mouse button define an area on the image that only includes red fluorescing sample positions 8 Rightclick to finish the definition of the ROI 9 Then define another ROI on an image segment that only includes green fluorescing sample positions 10 Click the New ROI Rectangle E button 11 Define a square in a dark image segment that shows no fluorescing objects This ROI will be used as a reference for the background correction Calculating an m 16 On the Life Science Applications toolbar click the Intensity Profile button profile 87 Viewing intensity profiles 20 21 22 23 24 20 26 Life Science Applications e The Intensity Profile lt
36. of positions on the sample at a variety of Z positions cco 3 I g The illustration shows an overview over the frames in a multi channel Z stack The multi channel image contains a red and a blue color channel For the acquisition of the Z stack a through focus series was taken of the sample The sample can only be seen clearly and sharply focused in the middle of the Z stack 86 Life Science Applications The following process flow chart displays the basic steps of the process Preparing the analysis Load the image that you want to measure Find a suitable frame on which to define the ROIs Defining ROIs Region Of Interest On your image define the areas whose intensity profile is to be measured Calculating and viewing an intensity profile Define the settings for the calculation of the intensity profile Specify how the intensity profiles should be displayed in the tool window hA Exporting and saving intensity profiles Preparing the analysis 1 Several example images were supplied together with your software You can follow these step by step instructions using the PeroxysomOrganelles tif example image This example image is a multi channel Z stack image e When you load a multi channel Z stack it will be automatically displayed in the Single Frame View in the image window Displaying a suitable 2 Use the navigation bar at the top of the image window image for the definition of the image segment
37. settings in the Camera Control tool window as you do for the acquisition of a snapshot Additionally in the Process Manager tool window you have to define the time sequence in which the images are to be acquired You want to acquire a time stack over a period of 10 seconds One image is to be acquired every second 1 Switch to the Acquisition layout To do this use e g the View gt Layout gt Acquisition command 2 On the Microscope Control toolbar click the button with the objective that you want to use for the image acquisition 3 Switch to the live mode and select the optimal settings for your acquisition in the Camera Control tool window Pay special attention to setting the 29 Selecting the acquisition process Selecting the acquisition parameters Acquiring a time stack o ee 10 11 12 13 14 Acquiring image series correct exposure time This exposure time will be used for all of the frames in the time stack Choose the resolution you want for the time stack s frames from the Resolution gt Snap Process list Find the segment of the sample that interests you and focus on it Activate the Process Manager tool window Select the Automatic Processes option Click the Time Lapse button e The button will appear clicked You can recognize this status by the button s colored background e The t group will be automatically displayed in the tool window Should another acqu
38. the Measurement and ROI tool window In it click the ROI s name to change it Should the tool window not be visible put it on display by using the View gt Tool Windows gt Measurement and ROI command Click the New ROI 3 Points Circle Q button once more Search out a dark area in the background of reference image 1 in which as far as possible no fluorochrome can be seen Define a circular ROI within this area with three mouse clicks e This ROI was defined for the image background It will be automatically assigned the name ROI 2 Using the same procedure define in reference image 2 an ROI for the fluorochrome 2 and an ROI for the image background Using the same procedure define in reference image 3 an ROI for the fluorochrome 3 and an ROI for the image background Activate reference image 1 in the document group In the Life Science Applications toolbar click the Fluorescence Unmixing E button to open the Fluorescene Unmixing dialog box Activate the Calibration tab Enter the label for fluorochrome 1 in the Name field This is at the same time the name for the calibration for color channel 1 Select reference image 1 in the Image list In the ROI list located immediately below the mage list select ROI 1 which was defined for the fluorochrome 1 In the Background subtraction group select the ROI option for the background correction of reference image 1 In the neighboring list to the right sele
39. the Acquire gt Devices gt Device Settings dialog box to configure the connected devices so that they can be correctly actuated by your software When all of the hardware components have been registered with your software and have been configured the functioning of the system is already ensured However it s only really easy to work with the system and to acquire top quality images when you have calibrated your software The detailed information that helps you to make optimal acquisitions will then be available Your software offers a wizard that will help you while you go through the 15 When do you have to configure the system Switching off your operating system s hibernation mode Configuring the system individual calibration processes Use the Acquire gt Calibrations command to start the software wizard You can find more information on the individual dialog boxes in the online help About the system configuration You will only need to completely configure and calibrate your system anew when you have installed the software on your PC for the first time and then start it When you later change the way your microscope is equipped you will only need to change the configuration of certain hardware components and possibly also recalibrate them When you use the MS Windows Vista operating system Switch the hibernation mode off 1 Todo so click the Start button located at the bottom left of the operating sys
40. the Automatic Threshold dialog box e All of the objects that have been detected will be displayed in color 8 Check whether the objects have been correctly recognized Should the objects not have been correctly recognized go to the Background group and enter whether the background is bright or dark Select e g for the image shown above the Background gt Dark option since the image shows bright objects against a dark background 120 Viewing the results Measuring images 9 Delete all but one of the phases by continuing to click the Remove Phase button until the button becomes inactive e By doing that you will make certain that no phases from earlier analyses are still defined 10 To obtain the results click the Count and Measure button in the Automatic Threshold dialog box e The Automatic Threshold dialog box will be closed Note You can also set up your software so that it doesn t automatically output the results of the analysis as soon as the threshold value settings have been made To do this use the Tools gt Options gt Count and Measure gt Detection command Clear the Execute the segmentation after setting parameters check box Then close the dialog box for setting the threshold values with OK and when you ve done that click the Count and Measure button in the Count and Measure tool window to have the results output th amp F my Z 2R il The number of objects detected will
41. the OEX file format 72 Task Adding a multi channel group T oa p ogi Running experiments Acquiring multi channel fluorescence images Your sample has been stained with the DAPI FITC and TRITC fluorochromes Define an experiment plan for acquiring a multi channel fluorescence image Run the experiment and acquire a multi channel image 1 Load an experiment plan in which three fluorescence images are acquired one after the other 2 Use the File gt Save As command to save the experiment plan with a new file name 3 Click the Multichannel Group button You can find the button on the toolbar at the top of the experiment plan 4 Draw a frame around the fluorescence image acquisition commands All the commands in the multi channel group will automatically be combined into a multi channel image after the acquisition a x 7 map sey se O O R The illustration shows an experiment plan for a multi channel image acquisition The name of the multi channel group 1 and the status of the online display 2 are displayed in the experiment plan The experiment will produce a multi channel image with three color channels 5 Test the experiment To do this click the Start button in the Experiment Manager tool window e The acquisition of the multi channel fluorescence image starts immediately e The acquisition has been completed when you can again see the Start button in the Experiment
42. the light path The reflected light shutter is closed 1 10 11 Use the Acquire gt Devices gt Device Customization command Activate the Observation Methods tab Click the New Observation Method ae button e A dialog box in which you can enter a name will be opened Give the new observation method a name then close the dialog box with OK You could name your observation method e g MyBF Select the mirror turret in the Available components list e Inthe central area of the dialog box the settings for the hardware components that have been selected will be displayed In transmission brightfield the mirror turret isn t allowed to contain a mirror cube Therefore select the Adjust entry in the Status list located in the middle of the dialog box and in the list that s below that one select the Free entry e For manual microscopes Where a manual microscope is concerned the mirror turret can t be automatically moved to the position you want For this reason when you use a manual microscope you ll receive a message that asks the user to make this setting manually This message will also appear when you are defining the observation method Confirm the message with OK Select the condenser Select the Adjust entry in the Status list located in the middle of the dialog box and in the list that s below that one the Free entry Select the hardware component Aperture Stop Select the Adjust entry in the Statu
43. the minimal time needed to carry out all the commands in the current time lapse loop This is the amount of time that will be needed when you select the As fast as possible check box e The Total loop duration field 2 displays the time needed to carry out the time lapse loop Carrying out an experiment plan p33 0 50h a 9 27 x 0 81 p 4 The illustration shows a completed experiment plan for the acquisition of a multi channel image It consists of three nested loops There is a multi channel group 1 a Z stack loop 2 and a time lapse loop 3 The result will be an image file 22 Click the Start gt button located in the Experiment Manager tool window to run the experiment e The experiment starts immediately Nested loops are carried out starting on the inside and working to the outside To be exact in this case that 78 Running experiments means First a multi channel fluorescence image is acquired After this has been done the stage s Z position changes and another multi channel fluorescence image is acquired at the new Z position The Z position keeps changing until all Z position have been used Your system waits for half an hour and then repeats the image acquisitions The acquisition has been completed when you can once more see the Start button in the Process Manager tool window and the progress bar has been faded out Your system s hardware components are now Set as specified for the
44. the monitor equals exactly one pixel in the image With a zoom factor of 50 one pixel on the monitor equals 2 x 2 pixels in the image When you make a measurement you should use the zoom factor 100 whenever possible Then you will achieve a maximum of measurement precision Should the zoom factor 100 not be possible because the image area you want to measure can t then be completely seen choose the largest possible zoom factor under 100 Information about changing an image s zoom factor can be found in the online help 00150 114 Measuring images 12 1 Measuring images Task Loading an image Setting the labeling color Measuring lengths Your software offers a wide range of measurement functions They enable you to quickly count objects and measure distances and areas on an image The following step by step instructions present the measurement functions to you by way of several examples Measuring image objects interactively You want to measure the diameter of some cells To do this load a suitable image or acquire one Subsequently edit the measurement Delete some of the measurements you ve made Enter the results in a MS Excel sheet 1 If necessary use the View gt Tool Windows gt Measurement and ROI command to have the Measurement and RO tool window displayed e You ll find the tool window at the lower edge of the user interface It s possible that it may be covered by the Count and Measure Results
45. the movies you have acquired e The Path field located in the Directory group shows the directory that will currently be used when your movies are automatically saved 6 Click the button next to the Path field to alter the directory e The AVI file format is preset in the File type list This is a fixed setting that cannot be changed 7 Click the Options button when you want to compress the AVI file in order to reduce the movie s file size 8 From the Compression list select the M JPEG entry and confirm with OK Please note Compressing the movie is only possible if the selected compression method codec has already been installed on your PC If the compression method has not been installed the AVI file will be saved uncompressed The selected compression method must also be available on the PC that is used for playing back the AVI Otherwise the quality of the AVI may be considerably worse when the AVI is played back 9 Close the Acquisition Settings dialog box with OK 28 Setting the image quality Switching to the Movie recording mode Starting movie recording Stopping movie recording Task Selecting an objective Setting the image quality Acquiring image series 10 Switch to the live mode and select the optimal settings for movie recording in the Camera Control tool window Pay special attention to setting the correct exposure time e This exposure time will not be changed during th
46. the weighting of individual color channels in relation to one another Combining fluorescence images with a transmission image 1 Load one or more fluorescence images and the transmission image that you want to lay over the color channels 2 Activate the transmission image in your software s document group 3 Use the Image gt Combine Channels command e The Combine Channels dialog box will open e lf you want to use a true color image as a transmission image that image will be automatically selected in the Transmission list When the transmission image is a gray value image it will be automatically entered in the Available Images table If necessary choose the transmission image in the Transmission list 5 Click once in the Images cell You get a picklist containing all of the loaded images that you can combine with the selected transmission image Select the fluorescence image you want 6 If necessary change the new fluorescence channel s properties Give the channel a name and assign it a color 7 Click the OK button to create the resulting image A new image document with the default name Image_ lt serial No gt is created e Inthe document group you can then see a superimposition of all of the images you ve combined e The resulting image is a multi layer image with two image layers Normally the two image layers are not of the same image type For this reason the image has this icon in the document g
47. their ratio value e The intensity of the ratio image is adjusted to the intensity of the source image Where there is a low intensity in the source image the ratio image Is also dark Image 1 is only the ratio image The colors are all equally bright Image 2 is the source image Your software hasn t applied color mapping to it In the image at the bottom the intensity in the ratio image corresponds to the intensity in the source image The intensity decreases noticeably towards the edges of the cell 00245 25072013 111 Measuring images 12 Measuring images Preconditions Measuring with help of the tool window Measuring with help of the toolbar Measuring with help of menu commands Working in the measurement mode Finishing the measurement mode Changing the default measurement mode Your software offers a wide range of measurement functions They enable you to quickly count objects and measure segments and areas All the results will be saved together with the image and can also be issued as a sheet For making measurements correctly calibrated images are an essential prerequisite Images that you have acquired with your software will have been automatically correctly calibrated when you have specified the objective you used Should the image not yet have been calibrated use the Image gt Calibrate Image command to carry out a calibration Selecting the measurement environment Switch to the C
48. tool window Click the Measurement and ROI tab at the bottom of the user interface to bring the tool window into the foreground 2 Acquire an image or load one EF vi fi over E pa 80 an a e During the installation of your software some sample images have been installed too You can follow these step by step instructions for measuring images when you use the exemplary image Neurons tif The measurement results will be written into the image according to the default settings in red font color and without a background This can be hard to read on some images Change the labeling settings 3 Use the Tools gt Options command 4 Click the Measurement and ROI gt Measurement Display entry in the tree view 5 Click in the Background Color field and choose e g the color Black 6 Select the Text color gt Fixed colors option then select the color White from the palette to see the measurements in white and the labeling in black in the image 7 Close the dialog box with OK 8 Click the Arbitrary Line s button located on the toolbar at the top of the tool window 9 Click with your left mouse button at the starting point and end point of the reference distance 10 If you have measured a reference distance you can immediately proceed with the next measurement 11 Click the Arbitrary Line button again to end the length measurement 115 Deleting measurements Exporting results to M
49. will be 76 Selecting the acquisition parameters for the Z stack loop Adding a time lapse loop Running experiments 13 If necessary release both of the buttons showing the lock icon a Set the acquisition parameters for the acquisition of a Z stack in the Experiment Manager tool window The acquisition parameters apply to the Z stack loop that is selected in the current experiment plan Use the fields and buttons with black numbers 1 4 for this The values in the fields with white numbers are automatically calculated and updated by your software 14 Click the Apply button 4 located next to the Recommended Step Size field e The Step Size field 5 adopts the value from the Recommended Step Size field e The Z Slices field 6 now displays how many Z positions will be moved to by the Z stack loop The number of Z positions will be automatically calculated on the basis of the Start and End values and the Z spacing 15 Finish the live mode The illustration shows an experiment plan for the acquisition of a multi channel Z stack image The experiment plan displays the number of Z positions that will be moved to and the Z spacing 1 In this example 27 individual images with a Z spacing of 0 86 um will be acquired for each channel Ag 16 Click the Time Lapse Loop button You can find the button on the toolbar at the top of the experiment plan 17 Draw a frame around the whole multi channe
50. you want to apply the deconvolution filter It s possible that you might have to lengthen the exposure time via the exposure time correction Finish the live mode To do so click the Live button in the Camera Control tool window Multi channel images will be saved by default as soon as the acquisition has been completed As file format the VSI file format will be used 1 2 Before you start the acquisition specify where the file is to be saved To do this click the Acquisition Settings i button located in the Process Manager tool window s toolbar Select the Saving gt Process Manager entry in the tree view e You will find the current directory in the Directory gt Path field Click the button next to the Path field to change the directory into which the image is to be saved after its acquisition 38 Viewing a multi channel image Properties tool window Saving multi channel images Acquiring fluorescence images After you ve acquired a fluorescence image A multi channel image is made up of the individual fluorescence images You can set which color channels resp combination of color channels will be displayed on your monitor To do this use the navigation bar in the image window Click a color field to make the channel appear or disappear All of the color channels that are at the moment displayed on your monitor will be identified by an eye icon The navigation bar also offers you additiona
51. 1 lets through also excites fluorochrome 2 a little and part of the light that fluorochrome 2 then emits can pass the emission filter 1 cell structure 2 will also be dimly visible in fluorescence image 1 One can then speak of an unwanted spectral mixing of the individual fluorescence images Spectral unmixing The spectral mixing can be subsequently removed from a digitally recorded multi channel fluorescence image by recalculation That s to say the image will be spectrally unmixed When you do this it improves the visual separation of the different cell structures in the image and improves the image quality To do that use the Process gt Enhancements gt Fluorescence Unmixing command 00360 93 Acquiring reference images Defining ROIs Life Science Applications Carrying out a fluorescence unmixing The spectral unmixing of a multi channel fluorescence image takes place in two steps The first step is the calibration of the color channels with the help of reference images When the experimental conditions don t change you will only need to carry out this step once In the second step the actual spectral unmixing takes place You require precisely one reference image for each color channel that is to be calibrated Each reference image must have exactly the same number of color channels as the image that is to be unmixed In the instructions that follow it will be assumed that you have acquired a three channel fl
52. 48 bit true color image as a transmission image Combining fluorescence images 1 Load the gray value images that you want to combine into a multi channel fluorescence image The sample was for example marked with the fluorochromes DAPI and Texas Red Activate the first image in your software s document group Use the Image gt Combine Channels command e The Combine Channels dialog box will open e Inthe Available Images table the active image will be automatically entered as the first color channel e When you have acquired the individual fluorescence images with a suitable observation method the name of the color channel and the fluorescence color will be read out of the image and automatically used in the Combine Channels dialog box In case you have to change the channel name or want to do so Click once in the Name cell Enter a description of the color channel e g the name of the fluorochrome used Texas Rea You can increase the width of the row so that the description will fit into it When the fluorescence color can t be read out of the image the first channel will by default be assigned the color Red To change the active color click this color field Select one of the colors from the palette on the Standard tab or activate the Custom tab to define a color of your choice Click the OK button to close the color palette and return to the Combine Channels dialog box In the next free row click the Im
53. 8 Click the Ratio Analysis es button located on the Life Science Applications toolbar 107 Life Science Applications e The Ratio Analysis dialog box opens 9 Make the settings for the background correction in the Background group Select the ROI option In both lists select the reference ROI for the background correction 10 In the Ratio group select the parameters for the calculation of the ratio image Select the Fura340 color channel from the Numerator list and the Fura380 color channel from the Denominator list e The preview image in the Ratio Analysis dialog box displays the ratio image that has been computed for the time point that is currently displayed in the image window The ratio image is the result of dividing the intensity of the Fura340 color channel by the intensity of the Fura380 color channel e The ratio image is a gray value image which automatically has a predefined pseudo color table applied to it in the preview window High ratio values are displayed in red with this pseudo color table and low ratio values are displayed in magenta e The ratio image has single pixels with a high intensity in the background This is image noise 11 In the Thresholds fields increase the value until the image noise disappears and only the cells remain visible 12 In the Scale list accept the value of 1000 that is given AGN a49 a On the left you can see the preview image before the threshold values were set
54. In the online help you can find detailed help for all elements of your software An individual help topic is available for every command every toolbar every tool window and every dialog box New users are advised to use the manual to introduce themselves to the product and to use the online help for more detailed questions at a later date In this documentation the term your software will be used for cellSens 00054 Sample images The DVD that comes with your software contains among a lot of other data also images that show different examples of use for your software You can load these so called example images from the DVD However in many cases installing the example images on your local hard disk or on a network drive is more helpful Then the example images will always be available no matter where the DVD with the software currently is Note Your software s user documentation often refers to these example images You can directly follow some step by step instructions when you load the corresponding example image You can open and view the example images with your software Additionally you can use the example images to test some of your software s functions for example the automatic image analysis the image processing or the report creation Due to the fact that the example images also contain multi dimensional images like Z stacks or time lapse images making use of them enables you to quickly load images that require m
55. Name of the active image gt dialog box opens e Your software recognizes the image type and automatically selects the corresponding option In this example the Z stack option is preset Select the Results gt Average check box Clear all the other check boxes e Inthe ROI data group all of the ROIs that have been defined on the active image will be listed In this example you ll find three ROIs there two on sample positions showing different fluorescence colors and one on the background Each ROI defines a specific image segment Now select the image segments for which intensity profiles are to be calculated In this example select both of the ROIs at fluorescing sample positions In the Background Subtraction group select the ROI option e In the list next to the ROI option all of the ROIs that have been defined on the active image will be listed In the list select the ROI that was defined on the image background Click the Execute button e The intensity profiles will be calculated and displayed in the ntensity Profile tool window If necessary use the View gt Tool Windows gt Intensity Profile command to show the tool window The tool window offers you several ways of displaying the intensity profile that has been measured In the ntensity Profile tool window s toolbar click the Arrange Charts and ao Layout button e The ntensity profile chart arrange dialog box opens Make the following s
56. S Excel Closing the image Measuring images 12 Take a look at the results in the tool window and in the image 13 14 e The illustration shows the image with three executed measurements The measurement 2 has been oC RS Sop 9 0 an ty PL Click one of the measurement results in the Measurement and ROI tool window e The corresponding line will be marked in the image Press the Del key e The measurement will be deleted both in the image and in the tool window e When a measurement has been deleted the tool window contains one measurement less The IDs of the remaining measurements won t be i by the deletion of a aie St i OR G XO When you ve completed the AN you should switch off the measurement mode since otherwise you might inadvertently select your measurements and move them 15 16 17 18 19 20 Check whether one of the buttons on the Measurement and ROI tool window s toolbar appears clicked Release this button To do this click the Export to Excel button In the In Output dialog box you set up the directory in which the data is to be saved and enter the name of the MS Excel sheet Adopt the file type Excel Sheet xls Click the Save button to have the MS Excel sheet with the measurement results saved Click the Close El button located at the top right of the document group e You have changed the image because you ve added interacti
57. Select the DAPI command in the experiment plan e Inthe Experiment Manager tool window the Image Acquisition Camera Settings and Display groups are now shown 8 Click the Set Current Device Settings button You can find the button in the Image Acquisition group in the Experiment Manager tool window e The DAPI observation method is specified on the microscope 9 Click the Live button in the toolbar at the top of the Experiment Manager tool window to switch to live mode In the live image check the exposure time and focus on the sample e Note The live image covers the experiment plan in the document group When you turn off live mode again the live image is closed by default and you see the experiment plan again If you ve selected a different setting for the live image you can activate the experiment plan after ending the live mode in the Gallery tool window for example 10 Set the resolution and the bit depth in the Experiment Manager tool window Set the exposure time and the sensitivity or gain for an optimal image e Note You can also use the Camera Control tool window to optimize the live image The acquisition settings in the Camera Control tool window are not however transferred automatically to the selected command in the experiment plan To do this click the Get Current Device Settings button in the Experiment Manager tool window 11 In the same way define the acquisition settings for the FITC and TRITC comman
58. Specifying which hardware is available Yy Configuring the interfaces Configuring the specified hardware Calibrating the system The first time you start your software after the installation has been made a quick configuration with some default settings will be made In this step you need only to specify the camera and microscope types in the Quick Device Setup dialog box The microscope will be configured with a selection of typical hardware components Your software has to know which hardware components your microscope is equipped with Only these hardware components can be configured and subsequently controlled by the software In the Acquire gt Devices gt Device List dialog box you select the hardware components that are available on your microscope You can find more information on this dialog box in the online help If you use a preset configuration that was offered in the Quick Device Setup dialog box check now whether your system is really equipped with the hardware components that are defined in the configuration Use the Acquire gt Devices gt Interfaces command to configure the interfaces between your microscope or other motorized components and the PC on which your software runs If you use a preset configuration that was offered in the Quick Device Setup dialog box you can skip this step Usually various different devices such as a camera a microscope and or a stage will belong to your system Use
59. T Ai xn ACQUIFING A NUorescence Image sanini nn O Me ee 45 FOS se OMONE GMAMINGIS Sieksccss epost a eek cat Geek eRe ed soe ae 46 7 6 Acquiring multi channel fluorescence IMAGES ccccceeeceeceeseeecaeeeeeeneeeeeseaeeesseeeeeaaeees 51 Creating sitehed IMagE siccceiicesicvsicsticesicwticesicsdieedicsteeetieaticstiesbinelicsteeeliwwticrnes 57 Running experiMentsS ssssnnsnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn mnnn 64 Ferec OVEMEW oran a ioe ee eee 64 9 2 Working with the Experiment Managet cccccccsssecccceeeccseseeecseseeecceseeseeuseeessaeesenaaeees 69 PROCESSING IMAG CS wesiczcscscecaveteiccsvees racer sestscasstecacetsnesscavstevsencetswecsveteneccuststeceuzea 84 Life Science Applications sicccvaicvewsieneresavereveuravevessverevesavereveusveravessseueseseceasveuctes 85 Ties DON Ozrena a a 86 Ti 22 s6rt OLE SCCNGCE UIMIMIXING resarcir a a aaa 92 Bcc e 01E Pase a E E EAEN EAO EE E A EE AE E A EEA 97 Vl 27 22 DECONVOU UON Srctecensoaute osetia txciodatiee veces uecdatsnabtewaince Sout wma a a a a nies 103 A TD secl Vall OPEL SISi vive fiw a tne manatee aaa uel dpamdin a manne emanden am eatecnna 105 12 IMGASUMING Mage S cne 112 Tata IMCASUMING IMMAGS S arn a a maderasuentaleneeravanumataas 115 12 2 Performing an automatic image ANALYSIS cccceeseeeeeeeeeseeeecsasseeeeeseasseeeesseaseeesenseaseees 120 13 WOrKINO WIIN FED OMS soson saaa aaa AEEA ERAEN NENE 129 TS A
60. Z positions Fluorescence Use fluorescence unmixing to remove spectral Unmixing mixing from a multi channel fluorescence image Use brightfield unmixing to break down a brighttfield Brightfield Unmixing image containing three different colors into its individual color components Intensity profile On a multi channel fluorescence image measure Colocalization the colocalization to identify image segments where the individual fluorescences overlap Measure how the calcium ions concentration is Ratio Analysis changing in a time lapse image For the active image check whether all of the parameters have been correctly defined which are necessary for successful deconvolution Verify Channel Parameters gt 2D deconvolution Pal Nearest Neighbor Use a deconvolution filter to remove disturbing diffused light from an image LERGA hs Constrained Iterative Filter 6619 16072013 85 Life Science Applications 11 1 Intensity profile Example of use Before using the command Supported image types Task With the Measure gt Intensity Profile command you can measure the intensity profile over the time time stack or over the different Z positions Z stack An image series can be a time stack or a Z stack As a result you will obtain an intensity profile that shows how the intensity within one or within several image segments changes over a period of time or over the different Z positions You can use inten
61. able in the live image You can therefore e g quickly measure a segment in the live image 113 PJE Measuring on multi dimensional images il Measuring on multi channel images Influence of the X Y calibration Influence of the zoom factor Measuring images Measuring on different image types You can combine a series of individual images into one image What results is e g a time stack in which all of the frames will have been acquired at different times You can make measurements on every separate image Display the required frame on your monitor To do this use the navigation bar in the image window Then carry out the measurement on this frame The measurement will be permanently linked to this frame i e the measurement will only be displayed on your monitor when the frame on which you made this measurement is also on display The measurement results will be shown in the Measurement and RO tool window You can give every measurement the number of the frame on which it was made To do so use e g the measurement parameter Index t for time stacks A multi channel image is made up of individual fluorescence images For multi channel images you can choose to measure on each fluorescence image separately or to define one measurement object for all color channels simultaneously Clear the Tools gt Options gt Measurement and ROI gt General gt Measure on all channels check box Now you will measure on e
62. ach fluorescence image separately To do so set up the color channel you want on your monitor To do this use the navigation bar in the image window Then carry out the measurement on this image The measurement will be permanently linked to this color channel i e the measurement will only be displayed on your monitor when the color channel on which you made this measurement is also on display The measurement results will be shown in the Measurement and RO tool window You can give every measurement the name of the color channel on which it was made To do this use the Channel measurement parameter Select the Tools gt Options gt Measurement and ROI gt General gt Measure on all channels check box Now each measurement object you define will be measured on each color channel All measurement results will be shown in the Measurement and ROI tool window Measurement precision How precise the measurement is depends on the X Y calibration and the image s current zoom factor The X Y calibration defines the width and height of the sample area that is represented by one pixel For example it could be that one pixel displays a sample area of 10 um x 10 um A pixel is the smallest image structure that can be measured For this reason the maximum measurement precision where this example is concerned is 10 um The zoom factor tells you how large the image will be displayed on your monitor With a zoom factor of 100 one pixel on
63. after the two images have been interchanged When the individual images overlap select the Correlation option in the Output gt Alignment list Then your software will search for the same image structures in neighboring 62 Checking a stitched image 1 Creating stitched images individual images The stitched image will be put together in such a way that image areas that are the same will be superimposed Click the OK button to carry out the automatic image alignment e The Multiple Image Alignment Manual Align dialog box opens e The stitched image will be displayed Check the stitched image on display Use the zoom buttons in the dialog box to zoom in the stitched image in the dialog box 100 H e E Should individual images have been incorrectly assembled you can manually shift one or more of them in respect to one another To do this click in the image you want to shift then drag it with your left mouse button depressed in the required direction e The currently selected image will be displayed semi transparently to make it easier for you to find the point of contact with the neighboring image Two images wer misalignment When the manual alignment has been made the two images fit together seamlessly Select the Cut Edges check box to clip the image in such a way that there are no longer any empty areas visible on its borders e Inthe preview the image edges that are to be clipped will be displa
64. age window Multi dimensional images have their own navigation bar directly in the image window Use this navigation bar to set or to change how a multi dimensional image is to be displayed on your monitor There are some other document types with their own navigation bar directly in the image window One example is a report instruction or an experiment plan 00139 02072013 2 4 Tool Windows Docked tool windows Tool windows combine functions into groups These may be very different functions For example in the Properties tool window you will find all the information available on the active document Which tool windows are shown by default depends on the layout you have chosen You can at any time make specific tool windows appear and disappear manually To do so use the View gt Tool Windows command Manually displayed or hidden tool windows will be saved together with the layout and are available again the next time you start the program A return to the original layout using the View gt Layout gt Reset Current Layout command will have the result that only the tool windows that are defined by default for this layout will be displayed Position of the tool windows The user interface is to a large degree configurable For this reason tool windows can be docked freely positioned or integrated in document groups Tool windows can be docked to the left or right of the document window or below it To save space several to
65. ages cell You will be presented with a picklist containing all of the images that you can combine with the active image Select your next image As soon as you click in another row the new image will be included in the sheet You can now change the characteristics of the new channel Give the new channel a name and assign a color It is possible to shift the individual images with respect to each other To do this if necessary use the arrow buttons in the Pixel Shift group You can set the weighting of the individual color channels Increase e g the value in the ntensity field to weight a channel more strongly Click the OK button to create your multi channel image A new image document with the default name Image_ lt serial No gt is created 47 Acquiring fluorescence images With the Combine Channels command you combine fluorescence images 1 and 2 into a multi channel image 3 this can be done with more than two images also Saving a multi channel 11 Use the File gt Save as command to save your new multi channel image image Always use the TIF or VSI file format when saving an image Viewing a multi channel 12 Use the Dimension Selector tool window to change the fluorescence color nae to choose another color mapping or to switch individual color channels off and back on 13 Use the Adjust Display tool window to change the display of a multi channel image on your monitor You can e g change
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67. and e You can find the Microscope Control 1 toolbar at the upper edge of the user interface right below the menu bar To the left of the document group you see the Camera Control 2 tool window On the Microscope Control toolbar click the button with the objective that you use for the image acquisition In the Camera Control tool window click the Live button e You can find more information in the online help e The live image 3 will now be shown in the document group Go to the required specimen position in the live image Bring the sample into focus The Focus ndicator toolbar is there for you to use when you are focusing on your sample Check the color reproduction If necessary carry out a white balance Check the exposure time You can either use the automatic exposure time function or enter the exposure time manually Select the resolution you want In the Camera Control tool window click the Snap button e The acquired image will be shown in the document group Use the File gt Save As command to save the image Use the recommended TIF file format 00027 18012012 17 Acquiring single images 4 2 Behavior of the live window Prerequisite The behavior of the live window depends on the acquisition settings in the Acquisition Settings gt Acquisition gt General dialog box For the following step by step instructions the Keep document when live is stopped option is selected and the Cre
68. are or the hardware For example you can specify an observation method that opens a shutter before the image acquisition and closes it again after the image acquisition When you are using streaming the shutter stays open for the whole duration of the time stack acquisition Without streaming the shutter would be opened before a single image acquisition and closed again 2 l x d 54 14 ms This is how an experiment plan for the fast acquisition of a fluorescence time stack with the TRITC observation method looks 80 Task Preconditions Running experiments Acquiring multi channel Z stack images at different positions on the sample Define an experiment plan with which you can acquire a multi channel Z stack image at different positions on the sample The experiment should always start at a specific stage position Your sample has been stained with the DAPI and FITC and fluorochromes e The system has been configured e You have defined suitable observation methods for your color channels e Your microscope has a motorized Z drive The Z drive has been set up and calibrated e Your microscope has a motorized XY stage The XY stage has been set up and calibrated 1 Load an experiment plan in which a multi channel Z stack image is acquired 2 Check the settings for each image acquisition command A 3 Add the Move XY iE command to your experiment plan Position the command behind the image acquisition c
69. asuring images a measurement you will automatically leave the measurement mode again This means you have to select the measurement function again before you start each interactive measurement Displaying and saving measurement results The measurement results will be displayed directly on the image and in the Measurement and ROI tool window Use the View gt Tool Windows gt Measurement and ROI command to have the tool window displayed The measurements will be saved along with the image if you save the image in the TIF or VSI file format You can however also export the measurement results in a results sheet and save this as a file To do this use the Export to Excel or Export to Workbook command The measurement results will be shown on the image in a special data layer the measurement layer On your monitor image and measurement layer are shown together The data of each however is individually stored if you use the TIF or VSI image file format Try and picture the measurement layer as a transparency which is placed over the image When you measure an image the image data will not be changed by having the measurement results displayed on it You can at any time hide or show the measurement layers To do so use the Layers tool window There you have access to all of an image s layers The eye icon identifies all of the layers that are currently on display on your monitor Click the eye icon in front of the measuremen
70. ate new document when live is started check box is cleared Switching the live image on and off without acquiring an image 1 Make the Camera Control tool window appear To do this use e g the View gt Tool Windows gt Camera Control command 2 Click the Live button in the Camera Control tool window e A temporary live window named Live active will be created in the document group e The live image will be shown in the live window e You can always recognize the live modus by the changed look of the Live button in the Camera Control tool window 3 Click the Live button again e The live mode will be switched off e The active live image will be stopped e The live window s header will change to Live stopped You can save the stopped live image located in the live window just as you can every other image The live window may look similar to an image window but it will be handled differently The next time you switch on the live mode the image will be overwritten Additionally it will be closed without a warning message when your software is closed Switching to the live image and acquiring an image 1 Make the Camera Control tool window appear To do this use e g the View gt Tool Windows gt Camera Control command 2 Click the Live button in the Camera Control tool window e Atemporary live window named Live active will be created in the document group e The live image will be shown in the l
71. before saving it e This step can be left out if your software is configured to import images into a database directly after acquisition 07500 27122011 22 Acquiring multi dimensional images 5 Acquiring multi dimensional images Image containing several dimensions Navigation bar in the image window What is a multi dimensional image You can combine a series of individual images into one image You can e g assemble separate images that belong to different color channels Depending on how the frames differ the multi dimensional image that results from their combination will also vary A standard image is two dimensional The position of every pixel will be determined by its X and Y values Fluorescence color time and Z position of the microscope stage are the possible additional dimensions of a multi dimensional image HA multi channel image as a rule shows a sample that has been marked with several different fluorochromes The multi channel image is made up of a combination of the individual fluorescence images 4n a time stack all frames have been acquired at different points of time A time stack shows you how an area of a sample changes with time You can play back a time stack just as you do a movie EJA Z stack contains frames acquired at different focus positions You need a Z stack for instance for the calculation of an EFI image The different multi dimensional images can be arbitrarily combined A multi
72. ch tab a small x button is located Click the button with the cross to close the document If it has not yet been saved the Unsaved Documents dialog box will open You can then decide whether or not you still need the data Button with a hand q p Arrow buttons User interface 3 Buttons in the document bar At the top right of the document bar you will see several buttons 4 Db Click the button with a hand on it to extract the document group from the user interface In this way you will create a document window that you can freely position or change in size If you would like to merge two document groups click the button with the hand in one of the two document groups With the left mouse button depressed drag the document group with all the files loaded in it onto an existing one Prerequisite You can only position document groups as you wish when you are in the expert mode In standard mode the button with the hand is not available The arrow buttons located at the top right of the document group are to begin with inactive when you start your software The arrow buttons will only become active when you have loaded so many documents that all of their names can no longer be displayed in the document group Then you can click one of the two arrows to make the fields with the document names scroll to the left or the right That will enable you to see the documents that were previously not shown 4 Navigation bar in the im
73. channel time stack that has 2 color channels The sample has been dyed with the Fura 2 fluorescence dye Between the frames that are framed in red in the illustration the image intensity decreases visibly The cause is a change in the calcium ion concentration Preparing the analysis 1 Several example images were supplied together with your software You can follow these step by step instructions using the Fura tif example image This example image is a multi channel time stack image 2 Use the View gt Toolbars gt Life Science Application command to have the Life Science Application toolbar displayed You can find the functions for defining ROIs and for Ratio Analysis on this toolbar Defining ROIs Region i a Of Interest 3 Click the New ROI Polygon button on the Life Science Applications toolbar 4 Draw a rectangle inside a cell 5 Define another ROI in a different cell 6 Define another ROI in a dark image segment that has no fluorescing objects This ROI will be used as a reference for the background correction 7 Rename the ROls you defined To do this open the Measurement and ROI tool window In the Measurement and ROI tool window double click on the first ROI s name Enter a descriptive name for the ROI Name the ROIs Cell01 Cell02 and Background for example Three ROIs have been defined on the image The red and yellow ROIs contain cells The white ROI is on the background Carrying out a Ratio Analysis
74. cks is not possible 00355 091032012 104 Life Science Applications 11 5 Ratio Analysis Certain multichannel fluorescence microscopy inspection modes allow you to monitor changes in ion concentration or pH value within cellular structures Fluorescence dyes whose excitation characteristics depend on the concentration of ions are used for this The Fura 2 fluorescence dye for example shifts its excitation level from 340 nm to 380 nm when the calcium ion concentration decreases At an excitation wavelength of 340nm the intensity increases when the calcium concentration increases At an excitation wavelength of 380nm it s the exact opposite The higher the calcium concentration is the less light is emitted The process flow of a ratio analysis on a multi channel fluorescence image 1 Acquiring a multi channel fluorescence image The fluorescence dye is excited with two different wavelengths one after the other The multi channel fluorescence image contains two color channels created with the same fluorescence dye but at different excitation wavelengths The excitation wavelengths 340 nm and 380 nm are typically used with the Fura 2 fluorescence dye y 2 Carrying out background correction A background correction is carried out on both color channels You can make settings for the background correction in the Ratio Analysis dialog box A description of this dialog box can be found in the online help yY 3 Calculating a
75. ct ROI 2 that was defined for the image background in reference image 1 Click the Save button e The calibration of color channel 1 has now been completed e The Fluorochrome 2 gt gt button will become available Click the Fluorochrome 2 gt gt button to skip to the calibration of color channel 2 e The name of the Fluorochrome 1 group will then change to Fluorochrome 2 Then using the same procedure calibrate color channel 2 with reference image 2 and fluorochrome 2 Then using the same procedure calibrate color channel 3 with reference image 3 and fluorochrome 3 Click the Cancel button to close the Fluorescence Unmixing dialog box 95 Life Science Applications Spectrally unmixing a three channel fluorescence image 1 10 11 12 13 In the document group activate the three channel fluorescence image you want to spectrally unmix e Should you have already acquired the image at an earlier point in time you can load it into the document group by using the File gt Open gt Image command In the Life Science Applications toolbar click the New ROI 3 Points Circle Q button Search out a dark area in the background of your image in which as far as possible no fluorochrome can be seen Define a circular ROI within this area with three mouse clicks e This ROI was defined for the image background It will be automatically assigned the name ROI 1 In the Life Scienc
76. ctivate the Observation Methods tab In the Name list you will find all of the observation methods that have been predefined Should no observation methods be available click the Select Predefined akg Observation Methods ime button Click the Select All button Click OK As soon as filter cubes have been entered for the mirror turret in your device settings the appropriate observation methods will be automatically set up You will always find the observation method beneath the filter cube s name 2 Select a fluorescence channel e g U MNU in the Name list 3 Click the Rename Observation Method 2 button The Enter a New Observation Method Name dialog box opens 4 Enter amore general name e g Blue or DAPI then click OK The Selected components list contains the following hardware components There can be more or fewer components depending on what you have chosen in the way of hardware components in the device list In the mirror turret the corresponding mirror cube e g U MNU will be selected The transmission lamp will be switched off The transmission light path s shutter will have the Use for acquisition status This means that it will be open when the image is acquired 5 Assign a fluorochrome such as Blue or DAPI to the fluorescence channel 6 Click the OK button to save the new observation method The Microscope Control tool window will then contain a new button with this observation meth
77. cument group is empty While you use your software it gets filled e g wnen you load or acquire images or perform various image processing operations to change the source image and create a new one 3 Toolbars Commands you use frequently are linked to a button providing you with quick and easy access to these functions Please note that there are many functions which are only accessible via a toolbar e g the drawing functions required for annotating an image Use the Tools gt Customization gt Start Customize Mode command to modify a toolbar s appearance to suit your requirements 4 Tool windows Tool windows combine functions into groups These may be very different functions For example in the Properties tool window you will find all the information available on the active document In contrast to dialog boxes tool windows remain visible on the user interface as long as they are switched on That gives you access to the settings in the tool windows at any time 5 Status bar The status bar contains a large amount of information e g a brief description of each function Simply move the mouse pointer over the command or button for this information 00108 6 User interface 2 2 Layouts Which predefined layouts are there What is a layout Which elements of the user interface belong to the layout To switch backwards and forwards between different layouts click on the right hand side in the menu bar on th
78. d to specify the minimum object size By doing this you will make certain that individual pixels that belong to the phase will be taken into account in the area ratio 4 Select the Measurements entry in the tree view From the Class Measurements list select the ROI and Area Fraction ROI entries 5 Click OK to close the Detection dialog box Defining an RO 6 In the Count and Measure tool window click the Count and Measure button s small black arrow to open a context menu In it use the New ROI gt Polygon command 7 Move your mouse pointer onto the image e The mouse pointer will then take on the form of a cross 8 With your left mouse button define the segment in the image that is to be used for the analysis Rightclick to confirm the ROI Setting threshold 9 In the Count and Measure tool window click the Manual Threshold button VALES to open the Manual Threshold dialog box 10 Delete all but one of the phases by continuing to click the Remove Phase button until the button becomes inactive 11 Define only one phase within the ROI The ROI itself must not be defined as a phase 12 Set suitable threshold values that include the objects 13 Make sure that only one phase has been defined Should additional phases have been defined during an earlier analysis delete the superfluous phases Viewing the results 14 To obtain the results click the Count and Measure button in the Manual Threshold dialog box e
79. databases had to be opened for this command they will be closed as well Your software supports the handling of workbooks A workbook is created for example when you open the Measurement and ROI tool window and export a results sheet You can find more information on workbooks in the online help 138 Working with reports Note If you want to use workbooks in your reports MS Excel must be installed on your PC The minimum MS Excel version required is MS Excel 2003 Apart from the image and chart document type reports can also contain workbooks A workbook is imported as an Excel object in MS Word You can further edit it in the report 1 Inthe report doubleclick on the workbook e You will change into the edit mode You can recognize it by the fact that now the column headers and the row numbers are shown In edit mode as well as that you can see all of the workbook s worksheets If need be select the worksheet that you want to edit 3 Doubleclick the workbook in order to switch to edit mode Make the required change e When you want to format individual cells differently select the cell and use the Format Cells command in the context menu e When you want to format the complete worksheet differently e g other font or other background color select the complete worksheet e g with the keyboard shortcut Ctrl A then select the Format Cells command in the context menu e When you want to hide a
80. documents are arranged in the report instruction at any time 1 Load the report instruction that you want to edit 2 Select an image and with the left mouse button depressed drag it to another position Drag amp Drop 00153 30012012 133 Working with reports 13 3 Working with the Olympus MS Word add in Inserting documents and fields from the software in any MS Word document you want When your software is installed an add in from Olympus is added to the MS Word application program When you start MS Word you can recognize this because the Olympus menu is displayed Depending on the MS Word version used the Olympus MS Word add in looks slightly differently In MS Word 2003 the Olympus menu and the Olympus toolbar are available In MS Word 2007 and MS Word 2010 only the Olympus menu is available When it is selected the commands are available in a ribbon This add in will help you to carry out important tasks that are necessary when working with reports It will support you with tasks that prepare the report creation as well as tasks that are for editing the already created reports Preparatory tasks 1 You define page templates that you want to use for your reports For each page template you define placeholders for the document types you want Note During the installation of your software some predefined page templates have been installed too Please check first whether these predefined page templates are al
81. documents in a database That enables you to store all manner of data that belongs together in one location Search and filter functions make it quick and easy to locate saved documents 1 When you exit your software all data that has not yet been saved will be listed in the Unsaved Documents dialog box This gives you the chance to decide which document you still want to save 2 With some acquisition processes the acquired images will be automatically saved after the acquisition has finished 3 You can also configure your software in such a way that all images are saved automatically after image acquisition To do so use the Acquisition Settings gt Saving dialog box Here you can also configure your software in such a way that all images are automatically saved in a database after the image acquisition Closing documents There are a number of ways in which you can close documents 1 Use the Documents tool window Select the desired documents and use the Close command in the context menu For the selection of documents the standard MS Windows conventions for multiple selection are valid 2 To close a single document activate the document in the document group and use the File gt Close command Alternatively you can click the button with the cross x You can find this button at the top right of the document tab located next to the document name 3 Use the Gallery tool window Select the desired documents and use the
82. ds You can make different settings for each image acquisition command For example expose the different fluorescence images differently to even out the differences in the light intensity T1 Running the experiment Saving the experiment plan Running experiments A geO ys P O P k o sae The experiment plan contains three individual image acquisition commands Because the image acquisition commands are linked to an observation method before the image acquisition your system automatically takes on the settings you defined in the observation method For the acquisition of fluorescence images the required mirror turrets are brought into the light path for example The experiment will produce three fluorescence images Specify some general acquisition settings for the running of the experiment These acquisition settings are not saved together with the experiment plan 12 Click the Acquisition Settings fas button located in the Experiment Manager tool window s toolbar 13 Select the Saving gt Process Experiment entry in the tree view Here you specify whether and how the acquired images are to be automatically saved You can have the acquired images saved in a database or in a directory of your choice Or you can turn automatic save off In this case the acquired images stay open in your software s document group after the experiment has finished 14 Select the Document Name gt Process Experiment entry in t
83. e a chart or a workbook Select the Olympus gt Templates command and choose one of the following options nsert Image Placeholder Insert Chart Placeholder Insert Workbook Placeholder You can find more information on workbooks in the online help e The placeholder you ve selected will be inserted 4 If necessary you can change the size of the placeholder To do so move your mouse over a handle then with the left mouse button depressed drag it in the required direction The length width ratio remains unchanged so that the objects won t be distorted by this action 5 Doubleclick a placeholder for an image to change the default settings for its appearance e The mage properties dialog box opens You can find more information in the online help 6 If required insert additional placeholders for images charts or workbooks e Make sure that your page template isn t longer than a page It s better to set up two page templates each of one page than it is to set up one page template that is made up of two pages 7 If you want to you can insert a placeholder for a field Additional information about a placeholder can be shown in this field for example the name or the date it was set up You will find additional information on inserting placeholders for fields further down 8 Save your page template in DOC or DOCX for MS Word 2007 2010 file format You can choose any storage place you like 9 If required add a docum
84. e A picklist with the different methods for setting threshold values will open 8 Select the Automatic Threshold method e This method requires the user to make the smallest number of settings Therefore you should only use the other methods for setting threshold values when the Automatic Threshold method doesn t lead to the result you wanted You can find an overview on the threshold values in the online help e The Separation Channel Threshold dialog box opens Your software will carry out an automatic setting of threshold values In the image window you will now see the image structures that are detected by the automatic threshold settings 9 Inthe Channel group select the required channel again in this case DAPI 10 Check in the image window whether the automatic threshold setting has correctly found the image structures that are to be analyzed e Inthe Separation Channel Threshold dialog box select the Dark or Bright option in the Background group should the Automatic option not lead to the results you want 11 When the image structure that is to be analyzed has been correctly found click the OK button 101 12 13 14 15 Life Science Applications e You will then once more see the Colocalization dialog box In the preview the image structures found via the Channel segmentation will now be displayed with a yellow outline The colocalized pixels shown lie exclusively within these image s
85. e Applications toolbar click the Fluorescence Unmixing i button to open the Fluorescene Unmixing dialog box Activate the Linear Unmixing tab In the Fluorochrome 1 list select the calibration with which the fluorescence image in your multi channel fluorescence image s color channel 1 is to be corrected e The name of this calibration is identical with the label you gave the fluorochrome 1 while you were performing the calibration In the Fluorochrome 2 list select the calibration with which the fluorescence image in your multi channel fluorescence image s color channel 2 is to be corrected In the Fluorochrome 3 list select the calibration with which the fluorescence image in your multi channel fluorescence image s color channel 3 is to be corrected In the Background subtraction group select the ROI option for the background correction of your image In the neighboring list to the right select ROI 1 which was defined in your image for the image background Click the OK button to carry out the spectral unmixing and to close the dialog box e Anew image document will be created for the spectrally unmixed image The source image will not be changed e ltcan occur that immediately after the spectral unmixing the image will not be optimally displayed on your monitor In this case click the Apply button in the Adjust Display tool window When you do this the image contrast on your monitor will be automatically optimized
86. e Options button to open this dialog box 3 Inthe Size group select one of the options that specify how large the image is to be displayed in the report You can find more information on these options in the online help 4 If your settings should apply for all future images click the Set as Default button 5 Click OK e The mage Properties dialog box closes The changed image properties will be shown in the MS Word report now 137 Adjusting an image Editing a workbook in the report Working with reports Adjusting documents In the MS Word report you can select a document of the image or chart type and select the Olympus gt Adjust Document command You will then change over to the image analysis software where you can edit the document and then automatically change back to the report Note Basically the Adjust Document command is similar to the Update Placeholders command The difference is that the Adjust Document command is meant for users who mainly use MS Word for creating reports and who only change over to the image analysis software in order to make certain changes to some documents Example In the MS Word application program you edit a report that contains a lot of images With a certain image you notice that an important measurement is missing Using the Adjust Document command you change over to the image analysis software add the missing measurement and then change back to MS Word in order to c
87. e how a Z stack image is to be displayed on your monitor or to change this Please note Z stacks can only be saved in the TIF or VSI file format Otherwise they loose a great deal of their image information during saving Use the Image gt Separate gt Z Slices menu command to have a Z stack broken down into selected frames 31 Processing a Z stack Selecting an objective Setting the image quality Acquiring image series It is possible that within a Z stack only a short Z range interests you Use the Extract command to create a new Z stack that only contains selected frames from an existing Z stack In this way you will reduce the number of frames within a Z stack to only those that interest you You will find this command in the context menu in the tile view for Z stacks If you save a Z stack in another file format as TIF or VSI the Z stack will automatically be converted The Z stack will then be turned into a standard true color image This image shows the frame that is at that moment displayed on the monitor Image processing operations e g a sharpen filter affect either the whole image or only a selection of individual images You will find most of the image processing operations in the Process menu You can find more information on working with image processing functions in the online help The dialog box that is opened when you use an image processing operation is made up in the same way for every operation
88. e is to be arranged For example click this button gt when the next image is to be laid to the right of the current image 57 Creating stitched images e Your system now acquires an image at the current position on the sample In the image window you now see on the left 1 the acquired image and on the right 2 the live image is displayed Since you haven t moved the sample the live image still shows the current sample position too which means that you now see the current image twice The two images overlap Since the live image is shown transparent you see both images in the overlap area simultaneously Make a note of a significant structure on the live image s right border You will find the same sample structure in the overlap area On the illustration a significant structure has been indicated by a circle Now move the stage very slowly to make the structure on the live image move to the left Keep moving the stage until the image structures in the overlap area lie as exactly over each other as possible The image structures need not lie precisely over each other since your software will match the individual images with each other e Inthe overlap area 3 the same image segments are shown now This enables your software to seamlessly combine the two images i s ire i T sian F ET 3 l x a Ser L e You can reverse the direction in which your stage moves in the Device Settings gt Stage dialog box De
89. e movie recording 11 Find the segment of the sample that interests you and focus on it 12 Select the Movie recording check box 1 The check box can be found below the Live button in the Camera Control tool window _ T AfMmeaasa z e The Snap button will be replaced by the Movie button 13 Click the Movie button to start the movie recording e The live image will be shown and the recording of the movie will start immediately e Inthe status bar a progress indicator is displayed At the left of the slash the number of already acquired images will be indicated At the right of the slash an estimation of the maximum possible number of images will be shown This number depends on your camera s image size and cannot exceed 2GB 71 3633 e This icon amp on the Movie button indicates that a movie is being recorded at the moment 14 Click the Movie button again to end the movie recording e The first image of the movie will be displayed e The navigation bar for time stacks will be shown in the document group Use this navigation bar to play the movie e The software will remain in the Movie recording mode until you clear the Movie recording check box once more Acquiring a time stack In a time stack all frames have been acquired at different points of time With a time stack you can document the way the position on the sample changes with time To begin with for the acquisition of a time stack make the same
90. e name of the layout you want or use the View gt Layout command For important tasks several layouts have already been defined The following layouts are available Acquiring images Acquisition layout Viewing and processing images Processing layout Measuring images Count and Measure layout Generating a report Reporting layout In contrast to your own layouts predefined layouts can t be deleted Therefore you can always restore a predefined layout back to its originally defined form To do this select the predefined layout and use the View gt Layout gt Reset Current Layout command Your software s user interface is to a great extent configurable so that it can easily be adapted to meet the requirements of individual users or of different tasks You can define a so called layout that is suitable for the task on hand A layout is an arrangement of the control elements on your monitor that is optimal for the task on hand In any layout only the software functions that are important in respect to this layout will be available Example The Camera Control tool window is only of importance when you acquire images When instead of that you want to measure images you don t need that tool window That s why the Acquisition layout contains the Camera Control tool window while in the Count and Measure layout it s hidden The illustration shows you the elements of the user interface that belong to the
91. e optimized across all the frames in the time stack 4 Select the Histogram of all frames check box Your software now takes the smallest and largest values in all the frames and assigns the colors black and red to these values 5 Click the Apply button to make the changed settings visible in the image window a J lox Hah Jat B fe J i ae Bet B jii Eoo 1 e W The illustration shows the same ratio image where different settings have been made in the Adjust Display tool window On the left the contrast has been optimized for the first frame The Histogram of all frames check box was clear Because the values in the ratio image increase over time the color shifts increasingly towards red On the right the contrast was optimized across all frames The Histogram of all frames check box was selected With this setting differences in the ratio image can be seen in all frames 110 Life Science Applications Viewing the ratio image 1 Display all the image layers in the image window In the Layers tool window and the source image you can see an eye icon next to each image layer at the same time 2 Select the ratio image in the Layers tool window and click your right mouse button to open a context menu 3 Select the Mode gt Intensity Modulation command from the context menu If this mode is already set keep it e The colors in the ratio image remain unchanged and their color value reflects
92. easurement and ROI tool window on the Measurement and ROI toolbar or in the Measure menu As soon as you have clicked a measurement function your software will automatically switch to a measurement mode In the measurement mode your mouse pointer will take on the shape of a cross on the image You can make as many measurements as you like with the measurement function that has been selected The continuous measurement mode is valid for all loaded images You can therefore easily measure numerous images one after the other The selected measurement function s button will keep its clicked appearance and in this way show you the current measurement function You can recognize this status by the button s background color You will remain in this measurement mode until you explicitly switch it off To do so click the Select Measurement Objects iF button You can find the button either in the Measurement and ROI tool window or on the toolbar The continuous measurement mode described above is preset by default You can change this default setting To do this use the Tools gt Options command Select the Measurement and ROI gt General entry in the tree view Select the Switch to Select mode after creation check box Then when you have completed 112 Saving the measurement results Showing and hiding measurement results in an image Setting the unit for the measurement results Outputting measurement results in a sheet Me
93. ecessary additional settings in the Select AVI Save Options dialog box Use the Movie acquisition process in the following cases e Use the Movie acquisition process when processes that run very quickly are to be documented the number of acquisitions per second is considerably higher with movies than with time stacks e Use the Movie acquisition process when you want to give the movie to third persons who do not have this software AVI files can also be played back with the MS Media Player e Use the Movie acquisition process when keeping file sizes small is of great importance 00107 Recording a movie You can use your software to record a movie When you do this your camera will acquire aS many images as it can within an arbitrary period of time The movie will be saved as a file in the AVI format You can use your software to play it back 1 Switch to the Acquisition layout To do this use e g the View gt Layout gt Acquisition command 2 On the Microscope Control toolbar click the button with the objective that you want to use for the movie acquisition 3 Inthe Camera Control tool window s toolbar click the Acquisition Settings button e The Acquisition Settings dialog box opens Select the Saving gt Movie entry in the tree structure 5 You have to decide how a movie is to be saved after the acquisition Select theFilesystem entry in the Automatic save gt Destination list to automatically save
94. ect the optimal settings for your acquisition in the Camera Control tool window Pay special attention to setting the correct exposure time This exposure time will be used for all of the stitched image s individual images 2 Find the position on the sample at which you want to start acquiring the stitched image 3 Finish the live mode 1 Activate the Process Manager tool window 2 Select the Manual Processes option 3 Click the Manual MIA button e The button will appear clicked You can recognize this status by the button s colored background e The Manual MIA group will be automatically displayed in the tool window e Should the nstant EFI acquisition process have been active it will be automatically switched off You can however use images with extended depth of focus for the stitched image To do this before you acquire each of the individual images click the Instant EFI button located in the Manual MIA group 1 Make quite certain that the Auto Align button appears clicked It should then look like this e Then your software will search for the same image structures in neighboring individual images The stitched image will be put together in such a way that image areas that are the same will be superimposed 1 Click the Start button e Your software switches into the live mode 2 Bring the sample into focus 3 Click on one of the arrow buttons to set the side of the current image at which the next imag
95. ed e Inthe image window you will now see the fluorescence image that has been acquired The fluorescence image has the image type Multi channel image even if it consists only of one single channel You can immediately recognize a multi channel image by this icon which appears in front of the image name in the document group or in the Documents tool window e The fluorescence image will be displayed using the fluorescence color that you have defined together with the observation method 11 You can use the Dimension Selector tool window to define how the fluorescence image is to be displayed on your monitor or to change this There you can for example change the fluorescence color 12 Use the File gt Save As command afterwards to save the new multi channel image Use the TIF file format 00395 12032012 7 5 Combine Channels Gray value images a Multi channel images Multi dimensional images Transmission image The Image gt Combine Channels command creates a new multi channel image from several separate images A description of this dialog box can be found in the online help Which images can you combine You can combine a series of gray value images into a multi channel image These can be either 8 bit gray value images or 16 bit gray value images The prerequisite therefore is that all of the separate images have the same bit depth image size and image calibration Multi channel images don t
96. ee multi channel fluorescence images Each of them contains three channels These will in what follows be designated as reference image 1 reference image 2 and reference image 3 Reference image 1 is to be used to calibrate color channel 1 that belongs to fluorochrome 1 With the reference images 2 and 3 the method is analogical e Each of the reference images will be displayed in its own window in the document group The reference image that was last acquired will be the currently displayed active image e Should you have already acquired the three reference images at an earlier point in time you can load them into the document group by using the File gt Open gt Image command 1 Activate reference image 1 in the document group 2 Inthe Life Science Applications toolbar click the New ROI 3 Points Circle button e Should the toolbar not be visible put it on display by using the View gt Toolbars gt Life Science Applications command 94 Finishing the calibration Life Science Applications Search out an area in reference image 1 in which fluorochrome 1 is especially bright and glows as evenly as possible Define a circular ROI within this area with three mouse clicks e This ROI was defined for the fluorochrome 1 It will be automatically assigned the name ROI 1 e You can still subsequently change the size and position of this ROI e You can change this automatically created name To do so use
97. eground or the objects should be optically clearly separated from the image s background In the example image the background is dark and the foreground bright 1 Use the View gt Tool Windows gt Count and Measure command to have the Count and Measure tool window displayed 2 Acquire an image or load one e During the installation of your software some sample images have been installed too You can immediately follow these step by step instructions when you use the exemplary image WoodVessels tif 3 Open the Options dialog box by clicking the Count and Measure Options ir Select the Detection entry in the tree view button located in the Count and Measure tool window 5 In the Options group enter the value 5 in the Minimum object size field to specify the minimum object size By doing that you will rule out the possibility that individual pixels that may well belong to the phase but not to an object are counted as objects which would then falsify the results 6 Click OK to close the Detection dialog box 7 Inthe Count and Measure tool window click the Automatic Threshold button to open the Automatic Threshold dialog box Should the Automatic Threshold button not yet be active you will have to first activate it To do so select the Automatic Threshold entry in the Threshold button s menu You open this menu by clicking the small arrow next to the button e The threshold values are set automatically in
98. ent plan for acquiring several fluorescence images and run the experiment Preconditions e The system has been configured e You have defined suitable observation methods for your color channels The following process flow chart displays the basic steps of the process Setting up the new experiment plan Defining and configuring the experiment plan Define the experiment plan Specify settings for each command in the experiment plan Save the completed experiment plan Running the experiment Specify general acquisition settings These are only valid for the current experiment Run the experiment Yy Saving the experiment plan Setting up thenew 1 If necessary use the View gt Tool Windows gt Experiment Manager command experiment plan to show the Experiment Manager tool window Be 2 Inthe Experiment Manager tool window click the New button to create a new experiment e This automatically creates a new document of the experiment plan type in the document group e The experiment plan contains its own toolbar with all the commands that you can use in an experiment plan Exactly which commands appear on the toolbar depends on your system configuration e Inthe document group experiment plans are identified by this icon E in the header e The default name for the experiment plan is Experiment _ lt sequential No gt You can change the experiment plan s name to anything you want when saving it A small asterisk next to the
99. ent title In this case your software shows this title in the Report Composer tool window If you don t enter a document title the file name is shown instead in the Report Composer tool window To define a document title do the following e In MS Word 2003 use the File gt Properties command and switch to the Summary tab Enter the document title in the Title field Close the dialog box with OK 135 Working with reports e In MS Word 2007 2010 use the File gt Save as command In the Save as dialog box enter the document title in the Title field Close the dialog box with OK 10 Close the file 11 Register the page template in your software You can find more information on this topic in the online help Adjusting the insertion order The placeholders are numbered in the order in which they were inserted Should you have initially set up placeholders for two images have then decided to put a placeholder for a chart right at the top of the page the insertion order would be that shown in the example on the left 1 In this case use the Olympus gt Templates gt Adjust Insertion Order command to have the insertion order numbered serially from top to bottom see the example on the right WN IN Inserting a placeholder for a field 1 Inthe page template select the placeholder into which you want to insert a field 2 Use the Olympus gt Templates gt Insert Field Placeholder command e The nsert Fie
100. ettings in the dialog box Select the Select all check box in the Show charts group In the Layout group specify a grid size of 2x1 Close the dialog box with OK In the Intensity Profile tool window s toolbar click the Arrange Data a button e The Intensity profile data arrange dialog box opens Make the following settings in the dialog box Select the Separate chart per channel check box Clear the other check boxes Close the dialog box with OK e You can see two charts each with two curves Along the X axis the Z position that s to say the height has been plotted The intensity range has been plotted along the Y axis 88 Exporting and saving intensity profiles Task Life Science Applications 0 s000 10000 0 s000 10000 For each of the image s color channels an individual chart will be created The name of the corresponding color channel will be displayed in the chart s header On the left you see the results for the green color channel on the right those for the red one In each chart you will see a curve for each ROI that has been defined You can display a legend with the name of the ROls in the chart The green curve was measured on the ROI on the green fluorescing position on the sample the red on the red fluorescing position 25 In the ntensity Profile tool window s toolbar click the Export to Workbook D button e Anew workbook will be created in the document window This workbook contai
101. ew observation method a name then close the dialog box with OK Name the observation method e g Blue Assign the fluorescence channel a fluorochrome e g Blue or DAPI and a color e g Blue at 470 nm To do so select the Fluorochromes a entry in the Available components list Select the Use entry in the Status list In the Fluorochrome list located below that one select the fluorochrome to be used e g the entry Blue or DAPI You can change the fluorescence color in the Color list should that be necessary e tis important in all cases to define the fluorochrome for a fluorescence observation method even if you don t automatically change any device settings When the fluorescence color is linked to the observation method every image you acquire with this observation method will be automatically colored in the corresponding color This is valid irrespective of whether you work with a manual or a motorized microscope 42 Ez For motorized microscopes Setting up the mirror turret amp For motorized microscopes Setting the shutter for the fluorescence light path For motorized microscopes Switching off the transmission lamp fic Including camera settings Saving the observation method Acquiring fluorescence images It can make sense to use this setting and the additional settings for motorized microscopes also for manual microscopes When you use a manual microscope you ll receive a message that prompts
102. ewing a multi channel image 3 6 Define a process for the acquisition of a multi channel fluorescence image or load acquisition parameters that have already been saved To do this click the Load Process Definition l button located in the Process Manager tool window s toolbar If you are disturbed by the light of your monitor you can switch your software to a dark user interface To do so use the View gt Dark Application Skin command In the Process Manager tool window click the Start button e lf you use a manual microscope you ll receive several different messages about switching the mirror cube and opening and closing the shutter For microscopes that aren t motorized Follow the instructions and make the necessary settings on your microscope e The acquisition of the multi channel fluorescence image starts immediately The order of the color channels in the Process Manager tool window corresponds to the order in which the color channels were acquired e The acquisition has been completed when you can again see the Start button in the Process Manager tool window e The multi channel fluorescence image will be automatically saved You can set the storage directory in the Acquisition Settings gt Saving gt Process Manager dialog box The preset file format is VSI e Inthe image window the superimposed fluorescence image of all channels is displayed The navigation bar will be displayed at the top of
103. focus In the Process Manager tool window click the Autofocus button e The button will appear clicked You can recognize this status by the button s colored background e The Autofocus group will be automatically displayed in the tool window In the Autofocus group select the Multiposition MIA check box If the sample surface is not plane or if it is inclined to the objective choose the Every MIA frame option Now the software autofocus will be performed before every image acquisition In the Process Manager tool window click this button aj e The Stage Navigator tool window is shown When you have acquired an overview image of your sample you will see this area of the image in the stage navigator s image segment Set the magnification for the image segment in the Stage Navigator tool window To do this use the zoom buttons at the bottom left of the tool window 2 The current stage position will be shown by a yellow rectangle in the image segment 1 You should choose a magnification that enables you to see this 60 Defining the MIA scan area ts Creating stitched images rectangle clearly rs Cyt M me 1 4 a Me nmin a 2 af e PIE Pee TS 2 1 e You can find more information on the Stage Navigator tool window in the online help In the Process Manager tool window click this button E e The system will automatically switch into the live mode e The Define MIA Scanning Area dialog box
104. ge The stitched image will by default be automatically saved The storage directory is shown in the Acquisition Settings gt Saving gt Process Manager dialog box The preset file format is VSI By default in the overlap area the intensity values of two adjoining individual images will be matched with each other to make the image s overall impression homogeneous Stitched images are calibrated This means that you can measure distances and objects on a stitched image 59 Preconditions Selecting the acquisition process Using the software autofocus Putting the stage navigator on display Creating stitched images Acquiring a stitched image with a motorized XY Positions MIA Example You want to acquire an image of a large sample area Use the automatic MIA acquisition process to scan a rectangular area of the sample and to have adjoining images combined into one stitched image e The stage has been set up and initialized i e its stage limits have been defined e The camera is aligned parallel to the XY stage The angle between camera and stage should be smaller than 1 e The shading correction has been set up Activate the Process Manager tool window Select the Automatic Processes option Click the XY Positions MIA button e The XY group will be automatically displayed in the tool window If your microscope is equipped with a motorized Z drive you can switch ona software auto
105. ge acquisition command is inside the multi channel group The experiment will produce a multi layer image with two image layers One image layer is the multi channel image and the second layer is the transmitted light image 14 Running experiments Haaa Alternatively you can also use this experiment plan to acquire a multi channel image with a transmitted light image The transmitted light image acquisition command is in this case outside the multi channel group The experiment then produces two images one multi channel image and the brightfield image In this case you can t place one images on top of the other to view the superimposition Acquiring multi dimensional images Task Define an experiment plan for acquiring a multi channel Z stack You want the acquisition of the multi channel Z stack image to be repeated several times at intervals of one hour Run the experiment Preconditions e The system has been configured e You have defined suitable observation methods for your color channels e Your microscope has a motorized Z drive The Z drive has been set up and calibrated The following process flow chart displays the basic steps of the process Preparing acquisition Yy Adding a Z stack loop Add a Z stack loop Specify the acquisition parameters for it Adding a time lapse loop Add a time lapse loop Specify the acquisition parameters for it Yy Carrying out an experiment plan 1 Load an ex
106. hat contains the colocalization channel will be created e Atthe same time a workbook that contains the results of the colocalization measurement will be displayed If required use the File gt Save as menu command to save the new image and the workbook 98 Life Science Applications Measuring the colocalization on a part of the image ROI Frequently a colocalization of fluorescence signals occurs only in a small image segment In this case it makes sense to define a ROI Region of Interest then determine the colocalization only within this ROI You can also define several ROIs ROIs can have any shape you wish You can find more information on working with ROIs in the online help 1 Load the multi channel image you want to use for the colocalization measurement 2 Onthe Life Science Applications toolbar click the Colocalization m button If this toolbar is not displayed use the View gt Toolbars gt Live Science Applications command e The Colocalization dialog box opens 3 Inthe Channels field choose the two color channels for which the measurement of colocalization is to be carried out 4 When you work with multi channel time stacks or multi channel Z stacks Determine in the Apply on group whether the colocalization measurement is to be carried out on all frames or only on selected frames Should you want to limit the image selection select the Selected frames entry then click the Dimension Selector but
107. he parameters that are shown in the Settings group Change the image processing operation s parameters After every change that is made in a parameter the operation will be immediately applied to the source image and the resulting image will be shown in the preview window Click the Default button to readopt the preset parameters in the Settings group when the current parameter doesn t make sense to you When you have found the optimal parameters click the OK button to have the active image processing operation applied to the image with the active parameters e The image processing dialog box will closed e Please note that the image processing operation changes the source image No new image document will be created You can however use the Edit gt Undo command to restore the source image 00175 22022011 84 Life Science Applications 11 Life Science Applications The Life Science Application toolbar offers you various evaluation methods for your images If this toolbar is not displayed use the View gt Toolbars gt Live Science Applications command OOD RRB mygg The following table lists the buttons which are available by default on the toolbar Use one of several options to define an image let ass New ROls segment in the active image as a region of interest ROI An intensity profile shows how the intensity within one or within several image segments ROls changes over a period of time or over the different
108. he Z Stack acquisition process with other acquisition processes If you software supports the Multi Channel acquisition process you can combine the Z Stack acquisition process with the multi channel acquisition to acquire a multi channel Z stack You can only use this acquisition process when your microscope is equipped with a motorized XY stage With this acquisition process you can carry out one or more automatic acquisition processes at different positions on the sample or acquire a stitched image of a larger sample position If your microscope stage is equipped with a motorized Z drive you can use the autofocus for this acquisition process You ll find a description of the individual settings along with the description of the acquisition process 24 Acquisition process Acquisition process Instant EFI Acquisition process Manual MIA Pa Foa Examples Acquiring multi dimensional images With the automatic acquisition process Multi Channel you acquire a multi channel fluorescence image You can combine e g the Multi Channel acquisition process with the Z stack acquisition process to acquire a multi channel Z stack Note When you use the DP80 camera please note the following restriction When you acquire a transmitted light image simultaneously with a multi channel fluorescence image the Multi Channel acquisition process can t be combined with another acquisition process for example the Z stack acquisit
109. he list Close the dialog box with OK Take a look at the result for the circle s diameter in the image Note The measurement display in the image has to be updated once so that the settings that have been changed are also taken into account You update the measurement display for instance by adding another measurement or by once selecting an existing measurement in the image Measuring several images You want to measure cells on multiple images To do so acquire some images and measure them one after another Have the results from all images displayed simultaneously Take a look at the mean value for all of the measurements 1 2 6 NA hi LEER iss z R LL ee i e During the installation of your software some sample images have been installed You can carry these step by step instructions out directly with the example images Clematis04 tif and Clematis05 tif Activate the first image in the document group Click the Arbitrary Line s button located on the toolbar at the top of the Measurement and ROI tool window Measure the diameter of several cells Activate the next image Measure the diameter of several cells on this image too Click the Arbitrary Line button again and switch off the length measurement In the Measurement and ROI tool window click the Measurement and ROI Options h button Select the Measurement and ROI gt Results entry in the tree view Clear the Show measurement objects
110. he tree view Here you specify how the acquired images should be named 15 You set some camera settings like Online Deblur for example globally for the whole experiment in the Camera Control tool window Just like the acquisition settings they re not saved together with the experiment 16 Click the Start gt button located in the Experiment Manager tool window to run the experiment e Note You can only start the experiment when the experiment plan is active in the document group If another document is active an image for example use the Gallery tool window to activate the experiment plan in the document group e The experiment starts immediately Three individual fluorescence image will be acquired e When the images acquired by your experiment are automatically saved the experiment plan will be automatically saved too The automatically saved experiment plan is named like the first image that has been acquired When you run an experiment the experiment plan will normally be automatically saved to ensure an optimal documentation of your experiment You are recommended to explicitly save the experiment plan in addition to the automatic saving so you Can use it later to run similar experiments 17 Activate the experiment plan in the document group 18 Use the File gt Save As command to save the experiment plan Save the experiment plan under the name 3_FL_ mages for example e An experiment plan will be saved in
111. help e lf you want to use workbooks in your reports MS Excel must be installed on your PC The minimum MS Excel version required is MS Excel 2003 e The placeholder for a workbook can also be used for a MS Excel file To do so select the MS Excel file in the File Explorer tool window and drag it onto the report instruction In the report instruction MS Excel files are shown with this icon z meee 5 Drag the documents you want onto the lower part of the report instruction 3 e Inthe Reporting layout the Database Gallery and File Explorer tool windows are arranged to the left to the document window In each of the tool windows you can select one or more documents and drag them onto the report instruction If you use the File Explorer tool window the documents do not need to be open for this If you use the Database tool window the documents don t have to be open either It is sufficient to open the database However the Gallery tool window only allows you to select documents that are currently open in your software e You can also integrate MS Word files e g background information regarding the project into your MS Word reports MS Word file don t need a placeholder in the report instruction Select the MS Word file in the File Explorer tool window and drag it directly onto the report instruction In the report instruction MS Word files are shown with this icon w e The documents must have been saved because unsaved
112. image With the Ctrl Alt T shortcut you can generate a test image that is made up of 256 gray values Activating documents in the document group There are several ways to activate one of the documents that has been loaded into the document group and thus display it on your monitor Use the Documents tool window Click the desired document there Use the Gallery tool window Click the desired document there Click the title of the desired document in the document group Pea D a To open a list with all currently loaded documents use the Ctrl Tab shortcut Left click the document that you want to have displayed on your monitor 5 Use the keyboard shortcut Ctrl F6 or Ctrl Shift F6 to have the next document in the document group displayed With this keyboard shortcut you can display all of the loaded documents one after the other 6 Inthe Window menu you will find a list of all of the documents that have been loaded Select the document you want from this list 13 User interface Attaching a document to an e mail Load the documents you want to attach to your e mail Use the File gt Send E mail command Check whether all documents you want to attach are selected oe M Click the Send button to generate an e mail with the selected documents included as attachments e You will receive a warning message if the sum of file sizes of all documents exceeds the maximum permitted size e Anew e mail fo
113. ion process This restriction protects the camera from being damaged by permanently toggling between the two CCDs the camera provides Use the manual acquisition process nstant EFI to acquire an EFI image at the camera s current position that is sharply focused all over When you use the Manual MIA acquisition process you move the stage manually in such a way that different adjoining sample areas are shown With this acquisition process you combine all of the images that are acquired directly during the acquisition just like a puzzle into a stitched image The stitched image will display a large sample segment in a higher X Y resolution than would be possible with a single acquisition Combination of several acquisition processes You can combine several automatic acquisition processes To do so click the corresponding button for each acquisition process you require Note Which automatic acquisition processes you can combine with each other depends on your software The order of the automatic acquisition processes in the Process Manager tool window from left to right corresponds to the order in which the acquisition processes were carried out complete multi channel image will be acquired at every focus position The Multi Channel acquisition process will then be carried out first Only after this has been done will the stage s Z position be changed and another multi channel image acquired When you combine the two ac
114. is WENVIEW arnon ag ict ie es el anc ead ea ape ea Naa 129 13 2 Working with The report COMPOSE scesi cvctcvicancdeaed dadhandcedsdavasdbetieicantiaaiaits ieteadietselaatiiatlanks 130 13 3 Working with the Olympus MS Word Add in ccccccccccccsssssssessseeeeeeeeeeeeeeseeseaaaaaaseseeseees 134 13 4 Creating and editing a page template eeececccccesssessseeeeeeeeeeeeseeeeeeeeseeeaasseeeeeesseaaeees 135 ToD 22 EANG A TO DOM ii cwasuceianaumatesstuanrauaunat we a Aclawiucrene eanvesd a 137 About the documentation for your software 1 About the documentation for your software Where do you find which information Writing convention used in the documentation Installing example images Note cellSens is available in a variety of versions For this reason it can happen that your cellSens version doesn t have some of the functions described there The documentation for your software consists of several parts the installation manual the online help and PDF manuals which were installed together with your software The installation manual is delivered with your software There you can find the system requirements Additionally you can find out how to install and configure your software In the manual you will find both an introduction to the product and an explanation of the user interface By using the extensive step by step instructions you can quickly learn the most important procedures for using this software
115. isition process be active e g Z Stack click the button to switch off the acquisition process e The group with the various acquisition processes should now look like this ome Clear the check boxes Start delay and As fast as possible Specify the time that the complete acquisition is to take e g 10 seconds Enter the value 00000 00 10 for 10 seconds in the Recording time field You can directly edit every number in the field To do so simply click in front of the number you want to edit Select the radio button on the right hand side of the field to specify that the acquisition time is no longer to be changed The lock icon will automatically appear beside the selected radio button Specify how many frames are to be acquired Enter e g 10 in the Cycles field e The nterval field will be updated It shows you the time that will elapse between two consecutive frames Click the Start button e The acquisition of the time stack will start immediately e The Start Process button changes into the Pause button A click on this button will interrupt the acquisition process e The Stop button will become active A click on this button will stop the acquisition process The images of the time stack acquired until this moment will be preserved e Atthe bottom left in the status bar the progress bar will appear It informs you about the number of images that are still to be acquired e The acquisition has been comp
116. it back Complex acquisition processes Use the Process Manager tool window to handle complex acquisition processes With the automatic acquisition process Time Lapse you acquire a series of frames one after the other This series of individual images makes up a time stack A time stack shows you how an area of a sample changes with time You can play back a time stack just as you do a movie You can combine the Time Lapse acquisition process with other acquisition processes If you software supports the Multi Channel acquisition process use e g the Time Lapse acquisition process to acquire a multi channel time stack If your microscope stage is equipped with a motorized Z drive when you acquire a time stack you can use the autofocus You ll find a description of the individual settings along with the description of the acquisition process Use the automatic acquisition process Z Stack to acquire a Z stack A Z stack contains frames acquired at different focus positions That is to say the microscope stage was located in a different Z position for the acquisition of each frame Alternatively you can also acquire an EFI image with the Z Stack acquisition process Then a resulting image EFI image with a practically unlimited depth of focus is automatically calculated from the Z stack that has been acquired Such an image is focused throughout all of its segments EFI is the abbreviation for Extended Focal Imaging You can combine t
117. ith regards to the minimum and maximum exposure time 1 Switch to the Acquisition layout To do this use e g the View gt Layout gt Acquisition command 2 On the Microscope Control toolbar click the button with the objective that you want to use for the acquisition of the HDR image 3 Switch to the live mode and select the optimal settings for your acquisition in the Camera Control tool window Carry out a white balance Choose an approximate exposure time 4 Search for the part of the sample which you want to acquire an HDR image of This should be a position which has such significant differences in brightness that not all segments can be shown with optimal lighting 5 Finish the live mode 20 Acquiring an HDR image 6 10 11 12 Acquiring single images In the Camera Control tool window select the Activate HDR check box e Inthe upper part of the tool window the Snap button changes to the HDR button In the Determine exposure range group click the Manual button to define the exposure range for this acquisition anew e The Determine exposure range message box appears It prompts you to reduce the exposure time so far that enough image details can be recognized in the bright image segments and no segments are overexposed Change the exposure time in the Exposure group which is part of the Camera Control tool window Make sure that the Manual option is chosen You can change the value b
118. ive window e You can always recognize the live modus by the changed look of the Live button in the Camera Control tool window 3 Click the Snap button e The live mode will be switched off The live window s header will change to Live stopped e Atthe same time a new image document will be created and displayed in the document group You can rename and save this image If you have not already saved it when you end your software you will be asked if you want to do so 18 Task Acquiring single images Displaying the live image and the acquired images simultaneously You want to view the live image and the acquired images simultaneously When you do this it should also be possible to look through the acquired images without having to end the live mode 1 2 Close all open documents Open the Acquisition Settings gt Acquisition gt General dialog box To do so click e g the Acquisition Settings tes button on the Camera Control tool window There make the following settings e Choose the Keep document when live is stopped option e Clear the Create new document when live is started check box e Select the Continue live after acquisition check box Switch to the live mode Acquire an image then switch the live mode off again e Both of the image windows Live stopped and Image_ lt Serial No gt are now in the document group e The Live stopped image window is active That s to say right now y
119. k is to be displayed on your monitor or to change this You can also hide the navigation bar To do this use the Tools gt Options command Select the Images gt General entry in the tree view Clear the Show image navigation toolbar check box 26 Saving time stacks Converting time stacks Processing time stacks When is it better for me to acquire a time stack Acquiring image series When you save time stacks you will as a rule use the VSI file format Only when you use this file format is there no limit to the size a time stack can be When you save smaller time stacks you can also use the TIF or the AVI file format With any other file format you will lose most of the image information during saving To do so use the File gt Save As command Use the Image gt Separate gt Time Frames menu command to have a time stack broken down into selected individual images lt is possible that within a time stack only a short period of time interests you Use the Extract command to create a new time stack that only contains a selection of frames from an existing time stack In this way you will reduce the number of frames within a time stack to only those that interest you You will find this command in the context menu in the tile view for time stacks You can find more information on this command in the online help When you save a time stack in another file format as TIF or VSI the time stack will also be converted
120. k the New Report Instruction button You find this button in the Report Composer tool window e Anew document of the report instruction type will be created in the document group This document is at the same time the workspace in which you put the report together 3 If no default document template has been defined Drag the document template you want onto the upper part 1 of the report instruction You find a list of the available document templates in the upper part 2 of the Report Composer tool window e Ifa default document template has been defined it will be automatically inserted in the upper part of the new report instruction e Creating a report is also possible when you leave the upper part of the report instruction empty In this case the default MS Word document template is used 130 Working with reports 4 Drag the page templates you want onto the lower part of the report instruction 3 You find a list of the available page templates in the lower part 4 of the Report Composer tool window e Every report has to contain at least one page template e Make sure that the page templates contain the correct placeholders for the document types that you want to drag onto the report instruction Accordingly if your report is to contain an image and a chart select a page template that contains one placeholder for an image and another for a chart You can find more information on page templates in the online
121. l Z stack image The acquisition of the multi channel Z stack image will now be repeated All acquired images will be combined into a single multi dimensional image a multi channel Z stack image T7 Running experiments e The Time Lapse group is displayed in the Experiment Manager tool window Set the acquisition parameters for the time lapse loop here 18 Define the acquisition parameters for the Z stack loop in the Experiment Manager tool window Selecting the acquisition parameters a for the time lapse loop 5 S SiO mo Set the acquisition parameters for the acquisition of a time lapse loop in the Experiment Manager tool window The acquisition parameters apply to the time lapse loop that is selected in the current experiment plan Use the fields with black numbers and the check box 1 3 for this The values in the fields with white numbers are automatically calculated and updated by your software 19 In the Cycles field 1 enter how often the command should be repeated in the time lapse loop Enter the value 5 to acquire five multi channel Z stack images for example 20 Clear the As fast as possible check box 2 21 In the nterval field 3 enter the time interval you want between two cycles Enter the value 0 5 h for example Now the acquisition of a new multi channel Z stack image will begin half an hour after the start of the previous acquisition e The Approximate minimum interval field 1 displays
122. l automatically F 12 Then continue clicking pixels that are typical of the first phase until the required structures in the image are a part of the phase change into a pipette with plus icon 13 Should too many pixels have been selected click the Shrink Threshold button to have these pixels excluded from the phase again e The threshold value range will continue to be reduced until it no longer contains the pixels you have selected 14 Click the Add Phase 3 button to add the second phase then proceed exactly as you did for the first phase Viewing the results 15 To obtain the results click the Count and Measure button in the Manual Threshold dialog box e The Manual Threshold dialog box will be closed 16 Open the Count and Measure Results tool window by using the View gt Tool Windows gt Count and Measure Results command The total number of objects detected in all of the phases will be shown below in the Count and Measure tool window in the Object Count group The results for the Object Class and Object Count measurement parameters that s to say the sum of the objects per phase will be displayed in the results sheet Furthermore you will recognize the phases by the colors that have been assigned to them You can compare the results for both of the phases directly with each other 123 Measuring images Determining the area ratios of several phases Task You have a multi channel image with 3 chan
123. l possibilities for changing the appearance of the multi channel image You can find more information on the navigation bar in the online help Numerous acquisition parameters will be saved together with the image Use the View gt Tool Windows gt Properties command to make the Properties tool window appear In the Properties tool window you will find that every color channel has its own Channel information group This contains the channel name the emission wavelength the name of the observation method and the exposure time The multi channel image will be automatically saved You can set the storage directory in the Acquisition Settings gt Saving gt Process Manager dialog box The file format used is VSI For the VSI format a JPEG compression of 90 is preset You can change the compression in the Acquisition Settings dialog box under Saving gt Process Manager gt Automatic save gt Options 00363 39 Acquiring fluorescence images 7 3 Defining observation methods for the fluorescence acquisition Before you acquire a fluorescence image you have to define observation methods for your color channels Usually observation methods that you can adapt for your microscope configuration have already been predefined Prerequisite The system has already been configured and calibrated For this purpose you have to enter your hardware components in the Acquire gt Devices gt Device List dialog box and configure them i
124. lamp for this observation method will be switched off It s usually better to use a black amp white camera for acquiring fluorescence images Should you use a camera that can be toggled between a color mode and a grayscale mode you can integrate the grayscale mode into the observation mode This setting is not necessary if you acquire fluorescence images with the Multi Channel acquisition process Before the Multi Channel acquisition process starts your software checks whether or not your camera is working in the gray scale mode You will then receive a corresponding message and can reset the camera before the image acquisition is made 11 Select your camera in the Available components list 12 Select the Use entry in the Status list 13 Some cameras offer gray scale modes in different bit depths Select the gray scale mode with the highest bit depth from the Image type list 14 Click the OK button to save the new observation method e The Microscope Control tool window will then contain a new button with this observation method s name e You can now use this color channel for the Multi Channel acquisition process 43 Acquiring fluorescence images Using predefined observation methods As a rule you don t have to completely redefine the observation method required Use one of the predefined observation methods and customize it for your microscope 1 Use the Acquire gt Devices gt Device Customization command A
125. last observation method that was used 79 Running experiments Acquiring fast fluorescence time stacks You can use the Experiment Manager to acquire very fast fluorescence time stacks In this way you can acquire kinetic processes with your system s highest possible time resolution 1 Define an experiment plan with a fluorescence image and a time lapse loop 2 Select the command for acquiring fluorescence images in the experiment plan Select the Streaming check box in the Experiment Manager tool window e Pay attention to the experiment s total duration shown at the top of the Experiment Manager tool window The total duration display gets smaller which directly shows the effect the streaming is having e The element for the fluorescence image acquisition in the experiment plan now looks a little different On the left you can see the element for a fluorescence image acquisition using the DAPI observation method On the right the Streaming check box is selected A slightly different icon indicates the status of the check box in the experiment plan 3 Select the command for the time lapse loop in the experiment plan Select the As fast as possible check box in the Experiment Manager tool window 4 Click the Start button located in the Experiment Manager tool window to run the experiment e All images in the time stack are now acquired at the maximum possible frame rate without waiting to be triggered by the softw
126. layers transmission image 2 and multi channel image 3 The icon h at the left side of the multi channel image means that it is not possible to move the multi channel image Select the transmission image in the Layers tool window Activate the Toolbox toolbar To do this use e g the View gt Toolbars gt Toolbox command Click the Move Tool he button on the Toolbox toolbar e The mouse pointer will change its shape when you move it on the image window Move the whole image with the left mouse button depressed Click e g the Selection Tool button on the Toolbox toolbar to leave the move mode 00369 56 Creating stitched images 8 Creating stitched images Prerequisite Selecting an objective Setting the image quality Selecting the acquisition process Selecting the acquisition parameters Acquiring a stitched image Acquiring a stitched image without a motorized XY stage Manual MIA Example You want to acquire an image of a large sample area Use the Manual MIA acquisition process to acquire several individual images of adjoining positions on the sample and to have them combined into a stitched image The camera is aligned parallel to the XY stage The angle between camera and stage should be smaller than 1 1 On the Microscope Control toolbar click the button with the objective that you want to use for the acquisition of the stitched image 1 Switch to the live mode and sel
127. ld dialog box opens A description of this dialog box can be found in the online help 3 Click the Use Selected Placeholder button e Inthe Placeholder list the name of the placeholder into which you want to insert a field appears 4 Inthe Available fields list select the field that is to be inserted The entries in this list are arranged hierarchically Click the plus sign to expand the list e Two types of field are available The Document Properties list contains fields that are by default in your software managed for this document type The Database fields list contains all of the fields that are available in the database for the selected placeholder For this purpose a database must have been opened 5 Inthe nsert Field dialog box click the nsert button e The placeholder for a field will then be displayed You can recognize it by the curly blue bracket and by the field name shown 6 If required move the field to another position in the MS Word file By default field values are shown to the left of or above the selected placeholder 7 lf necessary add placeholders for further fields 8 Save the page template 00402 30012012 136 Working with reports 13 5 Editing a report Preliminary considerations When you have created a report and want to make some changes in it before doing so you should decide whether it will be better to make the changes in the report i e in MS Word or in the report in
128. lder s check box if your MS Word report contains fields which should also be updated e You can find more information on this topic in the online help 5 Click OK e The placeholders will be updated Inserting a document In a report or in any other MS Word document you can insert a document at any position When you have for example created a report and while you are viewing it notice that you ve forgotten an image you can retroactively insert it into the report 1 Use the Olympus gt Insert Document command e The nsert Document dialog box opens 2 Inthe area on the left select the source the document comes from You have the following possibilities e Select the Open Documents entry if you want to insert a document that is currently opened in your software e Select the Database entry if you want to insert a document that is part of the currently selected database folder For this purpose the database must be opened in your software Should you work with a version of the software that doesn t support databases the Database entry is hidden e Select the File Explorer entry if you want to insert a document that is stored on your PC or in your network 3 Select the required document in the document preview Click the nsert button e The required document will be inserted into the MS Word report e The nsert Document dialog box remains open 4 Insert further documents now or close the dialog box e The pa
129. lect the Detection entry in the tree view 3 Inthe Options group select the All frames and channels option Now the analysis will be carried out for all of the frames 4 Inthe Options group enter the value 200 in the Minimum object size field to specify the minimum object size By doing that you will rule out the possibility that individual pixels that may well belong to the phase but not to an object are counted as objects which would then falsify the results 5 Select the Measurements entry in the tree view From the Class Measurements list select the Object Count and t Value entries 6 Then switch to the Results tab Make sure that the Show results only of the active frames check box has been cleared Make sure that the Accumulate class results over time check box has been cleared Click OK to close the Detection dialog box Setting threshold 8 In the Count and Measure tool window click the Automatic values Threshold outton to open the Automatic Threshold dialog box e The threshold values are automatically set 9 Delete all but one of the phases by continuing to click the Remove Phase button until the button becomes inactive Viewing the results 10 To obtain the results click the Count and Measure button in the Automatic Threshold dialog box e The Automatic Threshold dialog box will be closed 125 Measuring images 11 Open the Count and Measure Results tool window by using the View gt Tool Windows
130. leted when you can once more see the Start button in the Process Manager tool window and the progress bar has been faded out e You will see the time stack you ve acquired in the image window Use the navigation bar located in the image window to view the time stack You can find more information on the navigation bar in the online help 30 Acquiring image series e The time stack that has been acquired will be automatically saved The storage directory is shown in the Acquisition Settings gt Saving gt Process Manager dialog box The preset file format is VSI Note When other programs are running on your PC for instance a virus scanning program it can interfere with the performance when a time stack is being acquired 00304 12012011 6 2 Acquiring a Z stack What is a Z stack How do I recognize a Z stack Creating a Z stack Displaying a Z stack Saving a Z stack Converting a Z stack Fj You can combine a series of separate images into one image file A Z stack contains frames acquired at different focus positions A Z stack is needed e g for calculating an EFI image by the Process gt Enhancement gt EFI Processing command A standard image is two dimensional The position of every pixel will be determined by its X and Y values With a Z stack the focus position or the height of the sample is an additional item of information for every pixel The frames making up a Z stack can be 8 bit gray val
131. licks define a small rectangle around the paramecium at the top left border of the image 10 Move the mouse pointer over the ROI you just defined Click the right mouse button to open a context menu Select the Convert to dynamic ROI over t ie command from the context menu to turn the static ROI into a dynamic ROI e Inthe Measurement and ROI tool window the keyword ROI in the Type column changes into the new keyword dROI t Following the 11 In the image window display the frame in which the paramecium changes its movement of the object direction using the dynamic ROI e The position of the ROI that has been defined is the same on all frames 12 Move the ROI on this frame so that it contains the paramecium again e The system will now automatically reposition the ROI on each frame between the first and the current frame The positions are calculated as a linear interpolation of the ROI positions in the first and the current frames Check whether the paramecium is completely within the ROIs on these frames 13 In the image window display the last frame in which the paramecium is still completely visible in the image 90 Calculating an intensity profile Life Science Applications 14 Move the ROI on this frame again so that it contains the paramecium again Make sure that the ROI doesn t include any other paramecium 15 Check whether the ROls position is correct for all of the frames up till now e Inthe follo
132. m all frames only see the pixels with the highest intensity values EFI Projection For Z stacks an EFI projection is available The EFI projection uses a series of differently focused separate images Focus series to calculate a resulting image EFI image that is focused in all of its parts 00354 11 User interface 2 6 Working with documents Autosave and close You can choose from a number of possibilities when you want to open save or close documents As a rule these documents will be images In addition your software supports some other document types You will find a list of supported documents in the online help Saving documents You should always save important documents immediately following their acquisition You can recognize documents that have not been saved by the star icon after the document s name There are a number of ways in which you can save documents 1 To save a single document activate the document in the document group and use the File gt Save As command 2 Use the Documents tool window Select the desired document and use the Save command in the context menu For the selection of documents the standard MS Windows conventions for multiple selection are valid 3 Use the Gallery tool window Select the desired document and use the Save command in the context menu For the selection of documents the standard MS Windows conventions for multiple selection are valid 4 Save your
133. mission image and a multi channel image Saving a multi layer image 12 13 14 15 16 Acquiring fluorescence images multi channel image means that it is not possible to move the multi channel image Select the transmission image in the Layers tool window Activate the Toolbox toolbar To do this use e g the View gt Toolbars gt Toolbox command Click the Move Tool he button on the Toolbox toolbar e On the image window the mouse pointer will change its shape Move the whole image with the left mouse button depressed Click e g the Selection Too button on the Toolbox toolbar to leave the move mode When you display the transmission image and the multi channel image simultaneously in the image window the transmission image will cover the multi channel image and for this reason you can t see the multi channel image You can display both images transparently and in that way be able to see parts of both images 17 18 19 20 21 To start with select the image layer in the Layers tool window To do so simply click the layer s name e The layer you have selected will then be shown with a colored background in the tool window Then click the Set Layer Opacity ie button You can find this button in the tool window s toolbar e Inthe tool window a slide control will then appear with which you can set the degree of transparency Use the slide control to set the degree of tra
134. motorized Z i drive a focus regulator will be at your disposal in the a Microscope Control tool window A 20 Click the Read Zoftset button in the Process Manager tool window to 21 22 23 24 adopt the current Z position of your microscope stage 5 Activate the other channels focus the sample and transfer the current Z position of the microscope stage to the acquisition process e The first color channel is always used as a reference for the Z offset Below the other color channels you can find the Z offset value It shows how the focus positions of the individual color channels differ from each other Select the Use Z offset check box 6 Finish the live mode e The reflected light shutter will be closed Click the Save Process Definition kK button in the toolbar at the top of the Process Manager tool window to save the acquisition parameters for the process that has just been defined e A channel contains an observation method an exposure time a gain value and where necessary a focus position All of these settings will be saved together with the process definition e You can now use the acquisition parameter for this acquisition process again at any time 53 Acquiring fluorescence images Acquiring and viewing a multi channel fluorescence image Defining the acquisition 1 process Switching to a dark 2 user interface Starting the acquisition process 3 4 Vi
135. n the area marked in blue cell nucleus is to be measured All other positions on the image where pixels colocalize are to be ignored 1 Load the multi channel image for which you want to carry out a colocalization measurement 2 On the Life Science Applications toolbar click the Colocalization m button e The Colocalization dialog box opens 3 When you work with multi channel time stacks or multi channel Z stacks Determine in the Apply on group whether the colocalization measurement is to be carried out on all frames or only on selected frames Should you want to limit the image selection select the Selected frames entry then click the Dimension Selector button e Then you can limit the image selection in the Dimension Selector tool window You can find more information on this tool window in the online help 100 Life Science Applications 4 Click the Options button then select the Colocalization channel Image and Measurement results Workbook check boxes 5 Inthe Target area group select the Channel segmentation entry in the Area field e Inthe Area field a picklist with all of the available channels will open TRITC 1 FITC 2 DAPI 3 6 Select the channel of the fluorochrome with which the image structure that is to be analyzed has been stained In the example shown above this is the DAPI channel 7 Click the button located next to the Area field on its right hand side
136. n the Camera Control tool window In order to set the sensitivity to the lowest ISO value set the gain to the value of 0 Optimize the exposure time In the Process Manager tool window click the Read settings button e The exposure time will be adopted for the channel Finish the live mode In the Process Manager tool window click the Start button e Then together with your fluorescence images a transmitted light image will also be acquired and saved together with the multi channel fluorescence image The result of this acquisition process is a multi layer image that you can view with the Layers tool window Take a look at the multi channel fluorescence image with the superimposed transmitted light image in the image window T can find a button for showing and hiding the transmission image next to the button for the individual color channels The eye icon indicates that the transmission image is currently visible Click this button a in the navigation bar to hide the transmission image e Now you will only see the multi channel fluorescence image Click this button fe e Now you see a superimposition of the transmission image and the multi channel fluorescence image and show the transmission image again Use the View gt Tool Windows gt Layers command to make the Layers tool window appear In the Layers tool window click the sign 1 and open the image s layers e You can now see the image s individual
137. n the Device Settings dialog box To finalize this action calibrate your system by using the Acquire gt Calibrations command The tables that follow contain example configurations for both motorized and not motorized microscopes Only the hardware components that are relevant for the acquisition of multi channel fluorescence images are listed Example entries Device List Non motorized Motorized microscope microscope Nosepiece Motorized UCB Z axis lt Name a ai controller for the Not Motorized Motorized UCB stage s Z drive gt Fluorescence reflected light path Shutter Some OLNE TENECIEE NGNE Manual Shutter Motorized UCB shutter gt Transmission light path Lamp veld of your transmission UCB Halogen Lamp amp gt Condenser lt Name of your condenser gt Manual Condenser U UCD8A Nosepiece lt Your objectives gt Mirror turret U MNU U MWB U MWG For a position where there is no mirror cube select the Free entry The hardware components Aperture Stop and Top Lens are Condenser additionally listed under the device settings 40 Pa Setting up the mirror turret a For motorized microscopes Setting up the condenser 2 For motorized microscopes Switching on the transmission lamp Acquiring fluorescence images Defining the observation method for transmission brightfield Example Hardware components in transmission brightfield In transmission brightfield there is no fluorescence mirror cube in
138. name indicates that the document hasn t been saved yet Please note that the name of the experiment plan isn t linked to the experiment name that you enter in the Experiment Manager tool window The entry in the Experiment name field is by default incorporated into the names of the images that you acquire with the experiment plan 69 Running experiments E a Ji JH E daal W B j o 1 ET of Click the New button 1 to create a new empty experiment plan 2 Defining the experiment 3 Define the first image acquisition command plan Click the small arrow next to the Image Acquisition button to open a menu g Select the observation method that you want to use for the first image acquisition DAPI for example e All of the observation methods that have been defined in your software are listed in the menu 4 Click in the canvas on the position where you want to place the image acquisition command with the DAPI observation method in the experiment plan The experiment plan already contains the image acquisition command with the DAPI observation method 1 Click the Image Acquisition button to add another image acquisition command to the experiment plan 2 The button corresponding to the selected command becomes active You can recognize this status by the button s colored background You can now move the image acquisition command on the experiment plan with your mouse 3 5 Add the other two
139. ncerning image acquisition as a rule Each command is graphically displayed in the experiment plan with its own icon The example displayed above contains the icons for a fluorescence image acquisition 2 the icons for the creation of a multi channel image 3 and the icon for the Wait command 4 Two commands are connected with a line while an arrow unambiguously defines the order of the commands Experiment plans have their own toolbar in the document window itself 5 You can find all the commands that you can use in the experiment on this toolbar The toolbar is described in the online help Note When you re running an experiment the acquired images are displayed in the document group as a rule In doing so they cover up the experiment plan Click on the experiment plan s header in the document group to reactivate the experiment plan and to carry on working When a lot of images have been acquired you can use the Documents tool window to reactivate the experiment plan after the experiment has finished Switch to tree view to find the experiment plan quickly Experiment plans are displayed under the Experiment entry 66 Running experiments General process flow You can use the Experiment Manager for two different types of tasks 1 Defining and running a new experiment 2 Using an existing experiment plan 1 Defining and running a new experiment The following process flow chart displays the basic steps of the proces
140. necessarily have to be made up of several color channels There can also be multi channel images that only contain one fluorescence channel You can also combine these images into a new multi channel image that then contains several fluorescence channels The prerequisite therefore is that all of the separate images have the same bit depth image size and image calibration You can combine several multi dimensional images into one image Prerequisite for such an operation is that all of the individual images only differ in one dimension color channel focus position or time point and are of the same image size One example of this is two single color time stacks that are each made up of 50 separate images Each time stack was acquired with a different color channel In this case you can create one multi channel time stack Sometimes another image that shows the same position on the sample in the transmitted light mode belongs to a series of fluorescence images You can combine such a transmission image with the multi channel image 46 Combine Channels Acquiring fluorescence images The transmission image doesn t have to be of the same image type as the separate images However the image size image calibration and the bit depth have to be the same as the fluorescence images For example You can use a true color image as a transmission image When the individual fluorescence images have a bit depth of 16 bit you can only use a
141. nels You want to know how large the area ratio of the individual fluorescence channels is Preconditions The analysis is performed on a fluorescence multi channel image In the previously installed image directory you will find a multi channel image that you can use for this under the file name HER2 3x16 bit tif Setting options l l l Ap 1 Inthe Count and Measure tool window click this Options dialog box button to open the Select the Detection entry in the tree view In the Options group select the All frames and channels option Now the analysis will be carried out for all of the color channels simultaneously 4 Inthe Options group enter the value 1 in the Minimum object size field to specify the minimum object size By doing this you will make certain that individual pixels that belong to the phase will be taken into account in the area ratio 5 Select the Measurements entry in the tree view From the Class measurements list select the Object Class and Sum Area entries 6 Click OK to close the Options dialog box Setting threshold 7 In the Count and Measure tool window click the Automatic Threshold VAES button to open the Automatic Threshold dialog box e The three channels will be automatically displayed in the dialog box under their names The threshold values are automatically set 8 Make sure that only one phase has been defined for each channel To do this you will have to select each cha
142. nnel image combines a series of monochrome images into one image The multi channel image usually shows a sample that has been stained with several different fluorochromes The multi channel image is made up of a combination of the individual fluorescence images You can have the individual fluorescence images displayed separately or also as a superimposition of all of the fluorescence images At the top of the illustration you can see the individual fluorescence images 1 Below you can see the superimposition 2 of the separate fluorescence images The separate images making up a multi channel image can be 8 bit gray value images or 16 bit gray value images A multi channel image can be combined with a time stack or a Z stack A multi channel time stack for instance then incorporates several color channels Every color channel incorporated in the image is reproduced with its own time stack How do I recognize a multi channel image You can immediately recognize a multi channel image by this icon which appears in front of the image name in the document group or in the Documents tool window In the Properties tool window you can use the Channels entry to find out how many channels are contained in any given image A multi channel image will automatically have its own navigation bar directly in the image window Use this navigation bar to set how a multi channel image is to be displayed in the image window or to change this
143. nnel once in the Channel group Should more than one phase per channel have been defined from an earlier analysis delete the superfluous phases by clicking the Remove Phase button Viewing the results 9 To obtain the results click the Count and Measure button in the Manual Threshold dialog box e The Manual Threshold dialog box will be closed 10 Open the Count and Measure Results tool window by using the View gt Tool Windows gt Count and Measure Results command e The results will be shown in the results sheet for every color channel You can identify the channels in the Object Class column You then have 124 Measuring images a direct comparison of how much area is taken up by which channel or phase Determining how the number of objects changes over a period of time Task You have atime stack and want to know how the number of objects changes over a period of time Preconditions The analysis must be performed on a time stack The objects that you want to count must not be connected but must be clearly separated from one another In the previously installed image directory you will find a time stack that you can use for this under the file name ParameciumTimeSeries tif eu The illustration shows a time stack that comprises several images The number of objects changes over the time period Setting options 3 In the Count and Measure tool window click this _ button to open the Options dialog box 2 Se
144. ns results sheets with all of the results When you ve measured a multi channel image you ll find an individual work sheet for each of the color channels 26 Use the File gt Save as command to save a workbook e A workbook will be saved in the file format OWB This format is an exclusive file format and can only be opened with your software Workbooks are obviously therefore not suitable for using to exchange data with other application programs If you would like to use the results in a different application use the File gt Export to gt Excel command Measuring the intensity profile of moving objects You ve acquired a time stack of moving paramecia Define a dynamic ROI that contains a paramecium and move the ROI so that it follows the paramecium through all of the frames in the time stack Measure the intensity profile 89 Life Science Applications The illustration shows an overview over the frames in a Z stack The time points associated with the frames are shown under the images The red circle shows the movement of a single paramecium 1 Several example images were supplied together with your software You can follow these step by step instructions using the ParameciumTimeSeries tif example image Specifying the user 2 Use the View gt Toolbars gt Life Science Application command to have the interface
145. nsparency you want At a value of 100 the image layer is opaque At a value of 0 the image layer will be completely faded out When you re satisfied with the transparency setting click once on any place on the user interface Use the File gt Save as command to save your new multi layer image Always use the TIF or VSI file format when saving an image 00067 25072011 50 Acquiring fluorescence images 7 6 Acquiring multi channel fluorescence images Use the Multi Channel automatic acquisition process to acquire a multi channel fluorescence image Example Define a process for acquiring a multi channel fluorescence image When you set up the fluorescence sample illuminate it as little as possible to minimize the bleaching effect At the top of the illustration you can see the individual fluorescence images 1 Below you can see the superimposition 2 of the separate fluorescence images Defining the Multi Channel acquisition process Selecting the 1 Use the View gt Tool Windows gt Process Manager command to make the acquisition process Process Manager tool window appear Select the Automatic Processes option 3 Click the Multi Channel button e The button will appear clicked You can recognize this status by the button s colored background e The C group will be automatically displayed in the tool window 4 Should another acquisition process be active e g Z Stack click the button to swi
146. nt group or in the Documents tool window When it is a time stack this icon will be supplemented by a small clock A time stack that is made up of true color images has e g this icon e In the Properties tool window you can use the Frame Count entry to find out how many individual images are contained in any given image A time stack will automatically have its own navigation bar directly in the image window Use this navigation bar to browse through the frames making up a time stack or to play back the time stack like a movie You can find more information on the navigation bar in the online help There are different ways in which you can generate a time stack To acquire a time stack use one of the two acquisition processes Time Lapse or Movie Use the Image gt Combine Frames command to have several individual images combined into a time stack A description of the Combine Frames dialog box can be found in the online help A time stack contains much more data than can be displayed on your monitor A time stack will automatically have its own navigation bar directly in the image window Use this navigation bar to determine which of the frames from a time stack is to be displayed on your monitor You can also play back a time stack just as you would a movie You can find more information on the navigation bar in the online help Alternatively you can also use the Dimension Selector tool window to determine how a time stac
147. nt parameters that are currently calculated for all objects e A detailed description of this dialog box can be found in the online help Go to the list of all of the available parameters then click the Diameter measurement parameter e On the right an illustration shows you how the parameter is calculated You can see that there are different ways in which the diameter of a 2D object can be calculated Click the Mean entry in the list under the illustration to select the Mean Diameter measurement parameter When you do this the mean value of all of the possible diameters is determined Click the Add Mean Diameter button e This measurement parameter will be added to the list of measurement parameters to be calculated All of these measurement parameters will be displayed in the tool window Close the dialog box with OK Take a look at the result for the circle s diameter in the Measurement and ROT tool window 117 Outputting measurement parameters in the image Task Loading images Measuring cells Displaying the measurement results of all of the images 13 14 19 16 17 Measuring images Open the Select Measurements dialog box At the bottom of the list of all of the calculated measurement parameters click the Mean Diameter measurement parameter To the right of this list you ll see a button with a blue arrow Click this button to move the measurement parameter to the top of t
148. o a different Z position when all the commands in the Z stack loop have been carried out e The Z stack group is displayed in the Experiment Manager tool window Set the acquisition parameters for the Z stack loop here 9 Define the acquisition parameters for the Z stack loop in the Experiment Manager tool window 10 Select the Top and bottom entry in the Define list 1 e The stage s current Z position will be shown in the Position 1 field Because you ve already focused this is the focus position 11 Using the arrow buttons 2 move the microscope s Z drive up to the Z position at which the structures that are directly under the surface of the sample are displayed in sharp focus The buttons with a double arrow move the stage in larger steps Now move the Z drive the same distance again in the same direction Click the upper Set button 3 e The current Z position will be adopted in the Start field 3 12 Now define the final position in exactly the same way e The Recommended Step Size field 4 displays the distance required between two Z positions in the Z stack The required distance depends on the objective s Numerical Aperture among other things and is automatically calculated using the Nyquist theorem This makes sure that no positions on the sample between two individual images remain out of focus The higher the objective magnification and your objective s Numerical Aperture is the smaller the required distance
149. o color table of your choice or also display the original images 3 You can also hide the navigation bar To do this use the Tools gt Options command Select the Images gt General entry in the tree view Clear the Show image navigation toolbar check box Alternatively you can also use the Dimension Selector tool window to stipulate how a multi channel image is to be displayed on your monitor or to change this There you can for example change the fluorescence colors for individual color channels Saving multi channel images Please note Multi channel images can only be saved in the TIF or VSI file format Otherwise they loose a great deal of their image information during saving Converting multi channel images Use the Separate command to have a multi channel image broken down into chosen color channels The resulting images are still of the multi channel type contain though only one color channel There are several ways of accessing this command e Click the Separate Channels button in the Dimension Selector tool window e Use the Separate command from the Dimension Selector tool window s context menu e Use the Image gt Separate gt Channels menu command Use the Extract command to create a new multi channel image that is made up of fewer color channels than the source image 36 Converting multi channel images while saving them Acquiring fluorescence images Select all of the color channels you
150. ocuments and fields from your software in any MS Word document that hasn t been set up via the Report Composer tool window In this case you work mainly in the MS Word application program and you switch to your software only when you want to open the documents that you want to insert into the MS Word document 10404 30012012 134 Working with reports 13 4 Creating and editing a page template In a page template placeholders are set up for the documents that the report is to contain There are placeholders for images charts workbooks and fields When for instance the report is to contain pages that have an image at the top and below it a chart you should then set up a page template which has a placeholder for an image and a placeholder for a chart The Olympus gt Templates menu contains all of the commands that you need for setting up or changing a page template Creating a page template and adding a placeholder for a document Note Page templates have the DOC or DOCX for MS Word 2007 2010 file format To be able to carry out the following step by step instructions you must therefore have opened MS Word and have the Olympus menu on display 1 In the MS Word application program select the File gt New command 2 Select the Empty Document option if you don t want to use an existing page template as template but instead want to start from scratch 3 Decide whether you want to insert a placeholder for an imag
151. od s name 00370 29122011 44 Acquiring fluorescence images 7 4 Acquiring a fluorescence image Switching to a dark user interface Selecting the fluorescence observation method Selecting the exposure time for the acquisition Focusing a fluorescence sample You software supports several image types The multi channel image usually shows a sample that has been stained with several different fluorochromes However it is also possible to acquire a multi channel image that consists only of one single channel The illustration shows three fluorescence images of the same sample position Each image shows another fluorochrome 1 If you are disturbed by the light of your monitor you can switch your software to a dark user interface To do so use the View gt Dark Application Skin command 2 Use the View gt Toolbars gt Observations Methods command to have the Observation Methods toolbar displayed 3 To load an observation method click the button with the name of the fluorescence observation method you want e For manual microscopes Loading a fluorescence observation method defines that a fluorescence image is to be acquired For your software all observation methods using the Fluorochromes component are automatically identified as fluorescence observation methods e For motorized microscopes When you load an observation method this leads to the microscope being brought into a defined condition To do so
152. of the same number of separate images as the source image 00010 7 2 Before and after you ve acquired a fluorescence image Defining observation methods Setting up the microscope for the acquisition of a fluorescence image Setting up the camera for the acquisition of a fluorescence image Before you acquire a fluorescence image Define observation methods for your color channels 1 Use the View gt Tool Windows gt Microscope Control command to make the Microscope Control tool window appear e Inthe Objectives group you will find the buttons you use to change objectives 10 Ale e Inthe Observation method group you will find a button for every observation method that has been defined Observation methods should have been defined at least for brightfield and for every color channel Click the required objective s button 3 Click the button for the observation method with the excitation that has the longest excitation wavelength e g Red Ke 1 Use the View gt Tool Windows gt Camera Control command to make the Camera Control tool window appear 37 Switching off the corrections for brightfield acquisitions Focusing a fluorescence sample Setting the storage location Acquiring fluorescence images Set the image resolution for the acquisition With a high objective magnification you require a lower resolution For this purpose select the required resolution from the Snap Process list located
153. ol windows may lie on top of each other They are then arranged as tabs In this case activate the required tool window by clicking the title of the corresponding tab below the window Freely positioned tool windows Integrating a tool window into a document group User interface You can only position tool windows as you wish when you are in the expert mode You can at any time float a tool window The tool window then behaves exactly the way a dialog box does To release a tool window from its docked position click on its header with your left mouse button Then while keeping the left mouse button depressed drag the tool window to wherever you want it You can only add tool windows to a document group when you are in the expert mode You can integrate certain tool windows in the document group for example the File Explorer tool window To do this use the Document Mode command To open a context menu containing this command rightclick any tool window s header The tool window will then act similarly to a document window e g like an image window Use the Tool Window Mode command to float a tool window back out of the document group To open a context menu containing this command rightclick any tool window s header Buttons in the header In the header of every tool window you will find the three buttons Help Auto Hide and Close Click the Help button to open the online help for the tool window Click
154. olor to the individual color channels you have to define observation methods 64 Running experiments Experiment Manager s user interface The Experiment Manager is composed of the Experiment Manager tool window and an experiment plan The Experiment Manager is composed of the Experiment Manager tool window 1 and an experiment plan 2 When you run an experiment a progress bar is displayed on the left of the status bar at the bottom of the monitor 3 When an experiment is running over a long period of time you can also display the Task tool window 4 to get more information about the progress of the experiment Use the Experiment Manager tool window to create an experiment plan to edit an existing experiment plan or to start an experiment plan To keep a good overview of the flow chart in the experiment plan specify the settings for the commands you use in the Experiment Manager tool window 65 Activating experiment plans in the document group Running experiments The experiment plan il j The illustration shows the elements on the user interface that belong to the experiment plan The experiment plan is a document that is displayed in exactly the same way as images and other documents in your software s document group A document of the Experiment plan type is essentially a canvas on which you define the experiment by creating a flow chart 1 An experiment plan contains particular commands co
155. ommands and add a connector as well You can find the command either as a button on the experiment plan s toolbar or in the stage control menu on the experiment plan s toolbar e he Move XY group is displayed in the Experiment Manager tool window Here you specify how the stage should move 4 Select the Relative move option In the X and Y fields enter the distance the stage should move 5 Add the commands for the acquisition of another multi channel Z stack image to your experiment plan You can select the whole Z stack loop and copy it to the clipboard using the Ctrl C keyboard shortcut Use the Ctrl V shortcut to paste the copied commands to a different position in the experiment plan e Your experiment plan now contains the acquisition of two multi channel Z stack images at two different positions on the sample Eg 22 x 0 10 pm Eg 22 x 0 10 pm i parn i a t The experiment plan contains the acquisition of two multi channel Z stack images 1 The XY Stage is moved to a different position between the two acquisitions 2 The experiment will produce two multi channel Z stack images 6 If necessary expand your experiment plan The following illustrations show several examples 81 Running experiments Acquire further multi channel Z stack images at other positions on the sample The illustrated experiment plan moves the stage three times The experiment produces four multi channel Z stack images
156. ompleted experiment plan as an OEX file 67 Running experiments 2 Using an existing experiment plan You can load an existing experiment plan at any time and run the experiment again The following process flow chart displays the basic steps of the process 1 Defining the acquisition settings In the Acquisition Settings gt Saving gt Process Experiment dialog box specify whether and where you want the resulting images to be saved 2 Loading the experiment plan a Load a OEX file in the document group 3 Adjusting the experiment plan Select each element of your experiment plan one after the other and make the necessary settings nt Manager tool window Change the exposure time for the acquisition of fluorescence images for example y 4 Running the experiment In the Experiment Manager tool window click the Start button hA 5 Saving the experiment plan H Decide whether you want to save the changed experiment plan 00800 17072013 68 Running experiments 9 2 Working with the Experiment Manager Use the Experiment Manager to define and run complex experiments involving image acquisition with your software The following instructions guide you step by step through the definition of a typical experiment The complexity of the experiments described increases from example to example Acquiring fluorescence images Task Your sample has been stained with the DAPI FITC and TRITC fluorochromes Define an experim
157. ontinue editing the report 1 Open the report in MS Word and select the image that you want to adjust 2 Select the Olympus gt Adjust Document command e You switch to the image analysis software If it was closed it will be started and displayed in the foreground e The image that you want to adjust is also opened In case it is from a database that is currently closed the database will be opened in the background Note The image analysis software is now in a special adjust document mode In this mode you can only make certain adjustments to the image This is why a lot of other functions are hidden 3 Make the required change 4 Ifthe image information was changed Save the image in the image analysis software e Some changes made to an image don t have to be saved e g when you select another frame in a multi dimensional image Other changes have to be saved e g adding a measurement The fact that a change has to be saved will be indicated by an asterisk shown behind the file name in the document group 5 Click the Update Report button You find this button in the Adjust Document message box that is shown in the foreground e The MS Word application program will now be shown in the foreground again The edited image will be displayed You can now continue to edit the report e lf your image analysis software was closed before you selected the Adjust Document command it is closed again If any images or
158. onvolution filter on an image use the Process gt Deconvolution gt Verify Channel Parameters command to check the relevant parameters for the image changing them if necessary A description of this dialog box can be found in the online help What is deconvolution In fluorescence and brightfield microscopy diffused light from areas above or below the focal plane leads to over exposure distortion and blurring A suitable mathematical model to describe this problem is a convolution operation Q x f x A x n x x Point in xy space g x Observed image f x Ideal image h x Point spread function n x Noise function Convolution To be able to reconstruct the ideal image f x from the observed image g x you must know the noise function n x and the point spread function h x While an estimation of the noise function n x is highly possible the point spread function h x depends normally so strongly on the optical properties of the microscope and the sample that an experimental determining of this function is not directly possible For this reason mathematical algorithms become necessary to even approximately determine the point spread function h x and to subsequently make the best possible reconstruction of the ideal image f x by means of deconvolution A perfect unambiguous reconstruction is generally not possible since information can be lost during a convolution The deconvolution filters The individual
159. opens Move the XY stage to the top left hand corner of the MIA scan area you want 3 Focus then select the optimal settings for your acquisition in the Camera Control tool window Pay special attention to setting the correct exposure time This exposure time will be used for all of the stitched image s individual images Confirm the starting position in the Define MIA Scanning Area dialog box with OK Move the XY stage to the bottom right hand corner of the MIA scan area 4 Confirm this position in the Define MIA Scanning Area dialog box with OK e Inthe Stage Navigator tool window the MIA scan areas that have been defined are displayed Here you can immediately see how many individual images are required for the acquisition of the stitched image when the current magnification is used Eic ied H R T ailas 214 216 Tt o a kE oj a Lal rA Har i ae riai P 4 a al z ae a HERE AF ar y 61 Acquiring a stitched image Loading and selecting images Assembling images 1 Creating stitched images Click the Start gt button e The acquisition begins immediately The individual images are acquired then immediately assembled You can watch how the stitched image grows in the image window e You see the completed stitched image in the image window The individual images won t be saved separately e The stitched image will by default be automatically saved The storage directory
160. ore complex acquisition settings You can install the example images after you ve installed the software or at any later point in time To do so insert the DVD that contains the software into the DVD drive If the installation wizard starts browse to the directory that contains the example images and install them 07005 04072013 User interface 2 User interface 2 1 Overview The graphical user interface determines your software s appearance It specifies which menus there are how the individual functions can be called up how and where data e g images is displayed and much more In the following the basic elements of the user interface are described Note Your software s user interface can be adapted to suit the requirements of individual users and tasks You can e g configure the toolbars create new layouts or modify the document group in such a way that several images can be displayed at the same time Appearance of the user interface The illustration shows the schematic user interface with its basic elements 1 Menubar You can call up many commands by using the corresponding menu Your software s menu bar can be configured to suit your requirements Use the Tools gt Customization gt Start Customize Mode command to add menus modify or delete them 2 Document group The document group contains all loaded documents These can be of all supported document types When you start your software the do
161. ou see the stopped live image in the document group In the document bar the Name Live stopped is highlighted in color Split the document group to have two images displayed next to each other That s only possible when at least two images have been loaded That s why you created two images in the first step Use the Window gt Split Unsplit gt Split Unsplit Document Group Left command e This command creates a new document group to the left of the current document group In the newly set up document group the active document will be automatically displayed Since in this case the active document is the stopped live image you will now see the live window on the left and the acquired image on the right Start the live mode e Inthe document group the left window will become the live window Live active Here you see the live image Activate the document group on the right To do so click for example the image displayed there Click the Snap button e The acquired image will be displayed in the active document group In this case it s the document group on the right e After the image acquisition has been made the live image will automatically start once more so that you ll then see the live image again on the left e While the live image is being shown on the left you can switch as often as you want between the images that have up till then been acquired 19 Acquiring single images
162. ount and Measure layout when you want to measure images You can find the Measurement and RO tool window in the bottom section of this layout In this tool window you have fast access to all measurement functions and settings which relate to the measurement This tool window is at the same time the measurement display and contains all of the values that have been measured on the active image Note Should right at the bottom of the user interface several tool windows lie one over the other activate the Measurement and ROI tool window by clicking on the header of the r Measurement and ROI tab The tabs can be found under the tool windows Should you need more room for displaying the image you can also use the Measurement and ROI toolbar instead of the tool window All the measurement functions can also be found on the Measurement and ROI toolbar To have this toolbar displayed use the View gt Toolbars gt Measurement and ROI command Begin a measurement by simply clicking the appropriate button In this case you will only see the measurements that you have carried out in the image The Measure menu also contains all of the measurement functions To start a measurement simply use the corresponding menu command In this case you will only see the measurements that you have carried out in the image Starting a measurement Begin a measurement by selecting the measurement function you want You will find the measurement function in the M
163. pending on how you can best orient yourself the live image will then move to the left or to the right when you move your stage to the right Check whether both images have been correctly combined Otherwise you can undo the last step by using the Undo last frame button You can then move the stage again and match the structures better e During the acquisition you can change the current stitched image s zoom factor e g to see certain parts in the overlap area better You will find an overview on the possibilities of changing an image s zoom factor in the online help 58 Properties of the stitched image Creating stitched images 7 Define your way through the sample with the arrow buttons and follow that with the stage In this manner you can display a sample in any form you like in the stitched image The illustration shows a stitched image that is made up of 9 individual images and the stage path TEE g d ih z if I rn F i od a me ES I a oom pi g E i ZER E eae Ti y E be ifa n P P a at ae arr 8 Click the Stop m button when you want to end the acquisition of the stitched image You see the completed stitched image 4 in the image window Since the individual images can lie a little askew of each other the stitched image isn t as a rule rectangular but contains empty areas on its borders 5 These areas will as a rule be cut off in the stitched ima
164. periment plan for acquiring a multi channel fluorescence image Or specify a new experiment plan 2 Use the File gt Save As command to save the experiment plan with a new file name Preparing acquisition 3 Select one of the commands in the experiment plan DAPI for example Click the Set Current Device Settings button in the Experiment Manager tool window to have the DAPI observation method set on the microscope 4 Click the Live button located in the toolbar at the top of the Experiment Manager tool window 15 Running experiments Terreo ae je wy a I fg oe sas Some of the settings for the Z stack acquisition can best be checked in the live image You can display the live image 2 and the experiment plan 1 in the document window at the same time The Z stack loop has been selected in the experiment plan In the Experiment Manager tool window the Z stack group 3 is now visible Set the acquisition parameters here 5 Arrange the live window and the experiment plan in the document window so that you can see both documents at the same time To do this use the Window gt Split Unsplit gt Document Group Right command for example 6 Bring the image into focus Adding a Z stack EE Click the Z stack loop button You can find the button on the toolbar at the top of the experiment plan 8 Draw a frame around the multi channel group Your microscope s Z drive will now be automatically moved t
165. quisition processes Z Stack and XY Positions MIA to acquire a Z stack at several positions on your sample to begin with the complete Z stack at the first position will be acquired When that has been done your system will move to the next position and will acquire the next Z stack etc 00442 13012011 25 Acquiring image series 6 Acquiring image series With your software you can acquire time stacks movies and Z stacks 6 1 Acquiring time stacks What is a time stack How do I recognize a time stack Creating time stacks Displaying time stacks Hiding the navigation bar t N You can combine a series of individual images into one image In a time stack all frames have been acquired at different points of time A time stack shows you how an area of a sample changes with time You can play back a time stack just as you do a movie A standard image is two dimensional The position of every pixel will be determined by its X and Y values With a time stack the time when the image was acquired is an additional piece of information or dimension for each frame The frames making up a time stack can be 8 bit gray value images 16 bit gray value images or 24 bit true color images Note A time stack can also be an AVI video You can load and play back the AVI file format with your software You can immediately recognize the different image types by the icon which appears in front of the image name in the docume
166. r to make it available to other users 16 Click the Export to Chart iz button to export the histogram to a chart e You ll find this button in the Count and Measure Results tool window in the Class histogram results view The histogram will be saved in an exclusive file format which can only be opened by your software e The active chart will be shown in the document group The chart s name will be Class histogram lt serial No gt 17 Select the File gt Save as command and save the chart in the Save Chart As dialog box under a significant name e As file type OCT will be automatically chosen this is short for the exclusive file format 126 Measuring images Performing an automatic image analysis on an ROI Task You have selected a specific segment of the image You define one phase You want to know what percentage of the complete area of the image segment is taken up by the phase In illustration on the left there is a tissue with two small bright objects The task is to calculate which percentage of the area is taken up by the objects In the second illustration the piece of tissue has been defined as an ROI framed in red and both of the two small objects as a phase Setting options a 1 Inthe Count and Measure tool window click this Options dialog box button to open the 2 Select the Detection entry in the tree view 3 In the Options group enter the value 1 in the Minimum object size fiel
167. ready sufficient for your needs before creating your own page templates 2 Incase you did not insert the placeholders in a chronological order You adjust the insertion order of the placeholders so that they are numbered consecutively from the first placeholder to the last one Tasks for editing the already created reports in MS Word 1 You add a document that is currently open in your software to a report or to any other MS Word file 2 You add afield to your report so that certain information that is saved in your software is also displayed in MS Word This makes sense for example when exceptionally you want to see the acquisition date of a certain image in the report 3 You change the image properties and set for example whether or not the info stamp and the scale bar should be shown You add one or more detail zooms to an image 5 You change the resolution of one or all images of the report If you want to share the report it may be sensible to reduce the resolution thereby also reducing the file size 6 You update all placeholders in your report This makes sense for example when you change the documents in your software after the report creation and when you now want to see them in the report 7 You insert an MS Word report into the database of your software This command is only available if your software supports the database functionality With the commands from the Olympus add in you can also insert d
168. riment Manager command to make it appear Load a saved experiment plan Experiment plans have the OEX file name extension Select the element in the experiment plan that you want to edit an image acquisition for example e You can view and change all current settings for the selected element in the Experiment Manager tool window Save the experiment plan with the changed settings 00801 17072013 83 Processing images 10 Processing images The Process menu offers numerous image processing functions with which you can change an acquired image e g increase the image contrast or the image sharpness 1 Load the image you want to process or activate the image in the document group e Please note that the Process menu will only be visible when an image window is active in the document group Use one of the commands in the Process menu e g Process gt Enhancement gt Adjust Intensity e The image processing dialog box will open The image processing operation that is active will be shown in the dialog boxes header Click the small arrow next to the Preview Ql 7 button to open a list of all of the preview functions Select the Original and Preview entry e This preview function displays the same image segment twice in the dialog box The first one shown is the source image The second is the image that results when the current parameters are used e Most of the image processing operations need one or two of t
169. rm will be opened by your e mail program Your e mail program does not have to be already running for this to happen The e mail contains all of the selected image and document files as attachments As long as the e mail form remains open you cannot use your software or your e mail program The e mail form cannot be minimized no can other e mails be generated nor can you read any incoming e mails You can t close the Send E mail dialog box nor continue working 5 Enter the recipient s address and your message and then send off your e mail 00143 02072013 14 Configuring the system 3 Configuring the system Why do you have to configure the system Selecting the camera and the microscope Specifying which hardware is available Configuring the interfaces Configuring the specified hardware Calibrating the system After successfully installing your software you will need to first configure your image analysis system then calibrate it Only when you have done this will you have made the preparations that are necessary to ensure that you will be able to acquire high quality images that are correctly calibrated When you work with a motorized microscope you will also need to configure the existing hardware to enable the program to control the motorized parts of your microscope Process flow of the configuration To set up your system the following steps are necessary Selecting the camera and the microscope hA
170. rom the sample only light from a narrow range within the emission wavelength range 1b reaches the camera The problem with this procedure is that the wavelength ranges of the different fluorochromes overlap If this overlapping didn t occur the aspired visual separation of the different cell structures in the resulting multi channel fluorescence image would be perfect Neither the excitation wavelength ranges nor the emission wavelength ranges have sharp limits and they lie very close to one another where numerous fluorochromes are concerned Therefore the excitation wavelength ranges 1a 2a 3a of the fluorochromes 1 2 3 normally overlap The same applies to the emission wavelength ranges 1b 2b 3b As well as that there are also 92 Excitation and Emissions spectra Spectral unmixing Life Science Applications overlappings between excitation wavelength ranges and emission wavelength ranges In the spectra that follow you can see a graphical demonstration of the way in which the excitation intensities and the emission intensities of several fluorochromes that are often used depend on the wavelength The way in which the different wavelength ranges overlap can clearly be seen in these spectra ja 2a sa 4a 5a 1h 2b 3b4b 50 nm nr Owing to the spectral overlapping the aspired visual separation of the different cell structures only succeeds partially When for example the light that excitation filter
171. roup 48 Acquiring fluorescence images With the Combine Channels command you combine several fluorescence images into a multi channel image 1 If you ve acquired the transmission image at the same place on the sample 2 you can combine it with the multi channel image to make a multi layer image 3 Viewing a multi layer fa es es image gt The navigation bar will be displayed at the top of the image window You can find a button for showing and hiding the transmission image next to the button for the individual color channels The eye icon indicates that the transmission image is currently visible 8 Click this button a in the navigation bar to hide the transmission image e Now you will only see the multi channel fluorescence image 9 Click this button and show the transmission image again e Now you see a superimposition of the transmission image and the multi channel fluorescence image Moving the 10 Use the View gt Tool Windows gt Layers command to make the Layers tool transmission image WINQOW r with respect to the do appea multi channel image 11 In the Layers tool window click the sign 1 and open the image s layers e You can now see the image s individual layers transmission image 2 and multi channel image 3 The height map can t be seen because it s absolutely transparent at the moment The icon at the left side of the 49 Changing the weighting between a trans
172. ruction and the report Editing a report instruction You can make the changes described below to a report instruction These changes do not apply to reports the have already been created on the basis of this report instruction Therefore you must create a new report in order to see the changes you made This will generate a new MS Word document Any changes that you may have made in the first version of the report will not be contained in the newly created DOC file 1 Load the report instruction that you want to edit e Report instructions have the file extension RCI To delete a document template select it and press the Del key on your keyboard Drag the new document template onto the upper part of the report instruction e By doing so the document template is exchanged Please note that a report instruction can only contain one document template e A report instruction must not contain a document template at all When you leave the upper part of the report instruction empty the MS Word default document template will be taken 132 Changing the page templates Shifting the page templates Deleting documents Adding documents Moving documents Working with reports 1 Load the report instruction that you want to edit In the report instruction select the page template you want to exchange 3 Use the Del key on your keyboard to delete the selected page template from the report instruction e By doing so
173. s 1 Preparing the experiment Think through the experiment e Make sure that all hardware components you want to use are registered in your software s device list and configured in the device settings e Check whether the observation methods that you want to use have been defined e Select the exposure time and optimize the image quality in the live image 2 Defining the acquisition settings i In the Acquisition Settings gt Saving gt Process Experiment dialog box specify whether and where you want the resulting images to be saved vy 3 Setting up the new experiment plan In the Experiment Manager tool window click the New button Y 4 Defining the experiment plan Add the commands for the image acquisition and the control of the hardware components to the experiment plan Each command is represented by a graphic element that you can arrange on the canvas however you like Connect all commands unambiguously with each other v 5 Making settings for the image acquisition and hardware control Select each element of your experiment plan one after the other and make the necessary settings in the Experiment Manager tool window y 6 Running the experiment Test your experiment while you re defining the experiment plan To do this click the Start button in the Experiment Manager tool window Yy 7 Saving the experiment plan H In case you want to run the same experiment again with a different sample for example save the c
174. s list located in the middle of the dialog box Enter 75 in the field below that Select the hardware component Top Lens Select the Adjust per objective entry in the Status list e Inthe middle of the Device Customization dialog box you can now set the top lens for each objective separately e The top lens is only used for objectives with higher magnifications upwards of 10x and is swung out for lower magnifications Specify for which objectives the top lens should be brought into the light path and for which objectives it should be removed from the light path To do so select the Use with this objective check box for all objectives In the Selected components list each objective with a lower magnification than 10x needs to show the Out status If that isn t the case click in the middle of the dialog box on the Out button In the Selected components list each objective with a higher magnification than 10x or exactly 10x needs to show the n status If that isn t the case click in the middle of the dialog box on the n button Select the transmission lamp You will find this lamp in the Available components list under the Transmission entry Select the Adjust entry in the Status list located in the middle of the dialog box Set 9 V for the lamp Use the button showing a small lamp to switch on the lamp 41 Saving the observation method Task Summary H Defining the fluorochrome Acquiring fluorescence images
175. scatterplot e Then only the colocalization of the pixels that lie within the chosen intensity range are shown in the preview 13 Click the OK button to finish the measurement of colocalization e If you haven t changed the default settings for colocalization a new image that contains the colocalization channel will be created e Atthe same time a workbook that contains the results of the colocalization measurement will be displayed The columns in the workbook contain the supplement ROI 14 If required use the File gt Save as menu command to save the new image and the workbook 15 The multi channel image will also have been changed when the ROI was defined Therefore if you want to keep the ROI save it also Measuring the colocalization on a color channel You can also measure the colocalization on image structures Where images are concerned on which the image structures that are to be analyzed are numerous and are spread over the whole image this procedure is quicker than setting a lot of ROIs If for example you want to measure the colocalization on image structures that have been stained with the blue fluorescent fluorochrome DAPI select the blue channel Then define the threshold values for this channel The colocalization measurement on a color channel only makes sense on a multi channel image with at least three color channels Example On the image the colocalization of the red and green pixels withi
176. sets in the microscope Overlapping of the wavelength ranges Multi channel fluorescence microscopy In the multi channel fluorescence microscopy different cell structures will be visually separated by acquiring them separately then displaying them in different colors To achieve this one stains the sample with several suitably chosen fluorochromes Each of these labels a special cell structure The fluorescence images will then be acquired The fluorescence image 1 created with fluorochrome 1 shows cell structure 1 the fluorescence image 2 created with fluorochrome 2 shows cell structure 2 etc The individual images will be combined into a multi channel fluorescence image that shows the different cell structures in different colors When for example three fluorochromes are used a three channel fluorescence image will be created Problem with the visual separation of the structures Your microscope has an appropriate filter set for each fluorochrome this set comprises an excitation filter a dichromatic mirror and an emission filter When the fluorochrome 1 is excited by light from the wavelength range 1a it emits light in the wavelength range 1b When fluorescence image 1 is acquired the excitation filter 1 takes care that only light from a narrow range within the wavelength range 1a reaches the sample from the microscope s illumination source At the same time the dichromatic mirror 1 and the emission filter 1 take care that f
177. sity profiles to measure how concentrations change with time For example when you make experiments with triggering the calcium flow with ATR and use suitable fluorescence stains To calculate an intensity profile all of the pixels within a specific image segment will be evaluated You software will e g determine the mean intensity of all of the pixels Before you can measure an intensity profile you have to define this image segment To do this define one or more ROIs Regions Of Interest on the image To define these ROls you can for example use the buttons on the Life Science Applications toolbar You can find more information on working with ROls in the online help With the Measure gt Intensity Profile command you can measure the following image types time stacks whose frames are gray value images ay Z Stacks whose frames are gray value images T Multi channel Z stacks multi channel time stacks The command is only available for gray value images Use the Image gt Mode gt Grayscale command to convert the an image into a gray value image 00324 Measuring intensity profiles With the Measure gt Intensity Profile command you can measure the intensity profile over the time time stack or over the different Z positions Z stack Measuring an intensity profile on a multi channel Z stack You have acquired a focus series for several fluorescences You want to know how the intensity develops at a variety
178. struction i e in your software Often it is advisable to change the report instruction first and then create a new report Changes you make in the report instruction are valid for every subsequent report that you create with this report instruction There are numerous changes that you can anyway only make in the report instruction for example the selection of other page templates Changes that you make in a report are only valid for that particular report There is also no possible way in which changes that are made in a report can be automatically adopted in the report instruction However there are some cases when it makes sense to make a change only in the report for example when you ve created a very large report with a large number of documents and only want to change the order of two images in it If you didn t use a report instruction you can only edit the report anyway Changing the image properties When images are transferred to an MS Word report the image link is transferred as well This makes it possible to change the image display in an MS Word report e g to scroll the image segment 1 Doubleclick the placeholder for the image to open the Image Properties dialog box 2 Inthe Display group select the Scale bar if calibrated Info Stamp and Border check boxes if these elements are to be displayed e The properties of these elements can be defined in the Options gt Image Information dialog box Click th
179. switch to the image window view that is currently shown as an icon on the button Every image window view has its own icon The button always shows the image window view that was previously selected For example when you switch from the single frame view to the Slice View image window view the button will automatically change its appearance to show the icon for the single frame view making it possible for you to immediately switch back to that view Single Frame View By default you will find yourself in the single view In the single frame view only one image will be shown in the image window mm Tile View Use the tile view to attain an overview of all of the individual images that make up a multi dimensional image In this view you can also select individual images Wi l Slice View Use the Slice View image window view to look at any cross sections of an image series you want The Slice View tool window offers you numerous possibilities for configuring this view Voxel View You can display a Z stack as a 3D object To do so use the Voxel View image window view and the Voxel View tool window Projection Views For image series e g Z stacks and time stacks a single projection image can be calculated from all of the frames that is representative for the whole multi dimensional image The available projection images differ in the calculation algorithm For example if you use the maximum intensity projection you will fro
180. t layer to hide the measurements Click an empty cell without an eye icon to make the corresponding layer reappear The unit in which the measurement results will be issued is determined by the measurement function that has been selected and the image s calibration But you can choose whether the length e g is shown in mm or um Use the Tools gt Options command Select the Measurement and ROI gt Results entry in the tree view Select the unit you want to use from the Prefix of the unit list You can export the measurement results from the Measurement and ROI tool window as a sheet for example to be able to save the measurement results in their own file independently of the image You will find the functions e g as a button next to the measurement functions on the Measurement and RO tool window s toolbar Click the Export to Workbook fe button to export measurement results from the Measurement and ROI tool window s results sheet to a workbook Use this export possibility to save the measurement results in a file format that you can at any time load and edit with your software Click the Export to Excel E button to export the results to a MS Excel sheet Use this export possibility for example when you want to evaluate the measurement results still further This will also enable you to supply the results to other users who don t have your software Measuring in the live mode All of the measurement functions are also avail
181. t the field hasn t been filled out in your software In this case select another field that has been filled out instead Click the nsert button e The field contents will be displayed in the MS Word document If required move the field to another position in the MS Word file By default field values are shown to the left of or above the selected placeholder If necessary add further fields Close the nsert Field dialog box 10 Save the MS Word report Note Should you want to have the contents of a specific field regularly shown in your reports you can already insert this field that is to say a placeholder for this field into the page template Then this field will be automatically filled out in every report 00403 17022012 141 OLYMPUS Manufactured by OLYMPUS CORPORATION Shinjuku Monolith 3 1 Nishi Shinjuku 2 chrome Shinjuku ku Tokyo Japan Distributed by OLYMPUS EUROPA SE amp CO KG Wendenstrasse 14 18 20097 Hamburg Germany OLYMPUS AMERICA INC 3500 Corporate Parkway Center Valley Pennsylvania 18034 0610 U S A OLYMPUS SINGAPORE PTE LTD 491B River Valley Road 12 01 04 Valley Point Office Tower Singapore 248373 OLYMPUS AUSTRALIA PTY LTD 31 Gilby Road Mount Waverley VIC 3149 Melbourne Australia OLYMPUS LATIN AMERICA INC 5301 Blue Lagoon Drive Suite 290 Miami FL 33126 U S A OLYMPUS CHINA CO LTD biological A8F Ping An International Financial Center No 1 3 Xinyua
182. tch off the acquisition process e The group with the various acquisition processes should for example now look like this Adding color channels 5 Click the Add Channel button 1 CI e A context menu will open The context menu will list all of the observation methods that are currently defined 6 Select the color channel that is to be acquired first e g Red 51 Selecting the exposure times for the individual color channels Focusing a fluorescence sample 8 10 11 12 13 14 19 16 17 Acquiring fluorescence images Select the other channels e g Green and Blue in the same manner Note that the fluorescence images are later on acquired in the same order as they are listed there Use the View gt Tool Windows gt Camera Control command to make the Camera Control tool window appear Click the small plus sign 2 next to the first color channel Bg e The channel has now been activated 3 The active color channel will be shown highlighted in color in the tool window e he color channel entries in the Process Manager tool window are organized like a tree view Expand an entry to open a list with additional information about the selected color channel e When you activate the color channel you also automatically select the corresponding observation method You can recognize which observation method is active by the microscope icon E At the same time this means
183. tem s task bar 2 Use the Control Panel command 3 Open the System and Maintenance gt Power Options gt Change when the computer sleeps window e Here you can switch off your PC s hibernation mode When you use the MS Windows 7 operating system Switch off your PC s power saving options and make sure that your PC does not automatically goes to hibernation mode 1 Todo so click the Start button located at the bottom left of the operating system s task bar Click Control Panel System and Security and then click Power Options 3 On the Select a power plan page click Change plan settings On the Change settings for the plan page click Change advanced power settings e Here you can switch off your PC s power saving and hibernation mode 00159 16 Acquiring single images 4 Acquiring single images 4 1 Acquiring a single image You can use your software to acquire high resolution images in a very short period of time For your first acquisition you should carry out these instructions step for step Then when you later make other acquisitions you will notice that for similar types of sample many of the settings you made for the first acquisition can be adopted without change Selecting an objective Switching on the live image Ia Setting the image quality Acquiring and saving an image 1 10 Switch to the Acquisition layout To do this use e g the View gt Layout gt Acquisition comm
184. th of all documents that you inserted will be saved That enables you to later update the inserted documents by using the Olympus gt 140 Working with reports Update Placeholders command in case the documents were changed after they have been inserted into the report Inserting a field You can insert a field into an MS Word report that describes the image in more detail All of the values that have been saved in your software for this document can be displayed in this field 1 2 8 9 Select the image in the report to which you want to insert a field Select the Olympus gt Insert Field command e The nsert Field dialog box opens Click the Use Selected Placeholder button e Inthe Placeholder list the name of the placeholder into which you want to insert a field appears In the Available fields list select the field that is to be inserted The entries in this list are arranged hierarchically Click the plus sign to expand the list e Two types of field are available The Document Properties list contains document specific fields just as they are shown in your software s Properties tool window The Database fields list contains all of the fields that are available in the database for the selected image For this purpose a database must have been opened Check at the bottom of the dialog box whether a field value is displayed in the Field value group Should the Field value group remain empty this means tha
185. that the microscope is now set up correctly for the acquisition of the fluorescence image for the first color channel Switch to the live mode To do so click the Live button in the Camera Control tool window e The reflected light shutter will be automatically opened This behavior will be specified when the observation method is defined For the shutter the Use for acquisition status should have been selected In the Camera Control tool window select the Exposure gt Manual option Some cameras offer the SFL mode for fluorescence acquisitions e g the DP72 Switch this mode on Optimize the exposure time Should the exposure time become longer than 500 ms reduce it by increasing gain To do this change the value in the Exposure gt Sensitivity field or use the Gain slide control In the Process Manager tool window click the Read settings button x x e This exposure time will be automatically adopted for the active channel and will be shown in the Process Manager tool window 4 Then activate the other channels and set the exposure time for each of them Finish the live mode To do so click the Live button in the Camera Control tool window e The reflected light shutter will be closed Activate the first channel 52 Finishing the process definition Acquiring fluorescence images 18 Switch to the live mode 19 Bring the image into focus gt F If your microscope stage is equipped with a
186. the Auto Hide button to minimize the tool window Click the Close button to hide the tool window You can make it reappear at any time for example with the View gt Tool Windows command Context menu of the header To open a context menu rightclick a tool window s header The context menu can contain the Enable Auto Hide Document Mode and Transparency commands Which commands will be shown depends on the tool window Additionally the context menu contains a list of all of the tool windows that are available Every tool window is identified by its own icon The icons of the currently displayed tool windows will appear clicked You can recognize this status by the icon s background color Use this list to make tool windows appear 00037 10 User interface 2 5 Image window views The button s appearance Overview Image window views When working with multi dimensional images you can choose between different views of the image in the image window Multi dimensional images have their own navigation bar directly in the image window Click the small arrow next to the last button on this navigation bar to open a menu with commands you can use with image window views In it you can select the image window view you want and also edit the settings for some views This button is configured in such a way that you can switch backwards and forwards exactly between two different views simply by clicking it once Click the button to
187. the display of the ratio image 1 Carry out a ratio analysis e The image resulting from a ratio analysis is a multi layer image One image layer is the source image and the other image layer is the ratio image There are several ways of displaying the resulting image on the monitor 2 Use the View gt Tool Windows gt Layers command to make the Layers tool window appear You have access to the individual image layers in the Layers tool window 3 Select the View gt Tool Windows gt Adjust Display command to make the Adjust Display tool window appear In the Adjust Display tool window you can specify how an image is displayed on the monitor 4 Activate the image resulting from the ratio analysis in the document group 1 Take a look at the peak in the intensity profile in this time stack To do this use the navigation bar at the top of the image window lq CT 2 Ifthe info stamp isn t displayed in the image window use the View gt Info Stamp command to display it e The info stamp should display the time for each frame e lf this time is not on display select the Tools gt Options command In the tree view select the nfo Stamp gt Properties entry In the list of available properties select the mage gt t check box Close the dialog box with OK You can view only the ratio image or only the source image in the image window whenever you want In the Layers tool window click once on the source
188. the image window It contains a button for each channel to enable you to display or hide that channel The eye icon indicates that the channel is currently visible Click the color channel button in the navigation bar to have a color channel displayed or hidden Take a look at all of the color channels one by one When you ve finished superimpose all of the channels again Click the Tile View Eja button located in the navigation bar to change the image window view 54 Viewing information on the individual color channels Task Defining the acquisition process Adding the acquisition of a transmitted light image to the acquisition process Defining the transmitted light image acquisition 10 11 Acquiring fluorescence images e You will then see all of the color channels that have been acquired FITC Usammentuhren Compare the color channels Click the Single Frame View E button on the navigation bar e You will then once more see the superimposition of all of the color channels in the image window Use the View gt Tool Windows gt Properties command to make the Properties tool window appear e Inthe Properties tool window you will find that every color channel has its own Channel information group Should an information group has been reduced Click the plus sign H to have all of the information displayed again e The color channel s name the corresponding wavelength the observation method
189. the live mode and select the optimal settings for your acquisition in the Camera Control tool window Pay special attention to setting the correct exposure time This exposure time will be used for all of the frames in the Z stack 4 Search out the required position in the sample 32 Acquiring image series Selecting the 5 Activate the Process Manager tool window Select the Automatic Processes option 7 Click the Z Stack button e The button will appear clicked You can recognize this status by the button s colored background e The Z group will be automatically displayed in the tool window 8 Should another acquisition process be active e g Multi Channel click the button to switch off the acquisition process 9 Select the Range entry in the Define list 1 10 Enter the Z range you want in the Range field 2 In this example enter a little more than the sample s thickness 50 um e g the value 60 11 In the Step Size 3 field enter the required Z distance e g the value 2 for a Z distance of 2 um The value should roughly correspond to your objective s depth of focus e Inthe Z Slices field 4 you will then be shown how many frames are to be acquired In this example 31 frames will be acquired 12 Find the segment of the sample that interests you and focus on it To do this use the arrow buttons 5 The buttons with a double arrow move the stage in larger steps Selecting the acquisi
190. ti channel image you want to use for the colocalization measurement On the Life Science Applications toolbar click the Colocalization m button If this toolbar is not displayed use the View gt Toolbars gt Live Science Applications command e The Colocalization dialog box opens In the Channels field choose the two color channels for which the measurement of colocalization is to be carried out When you work with multi channel time stacks or multi channel Z stacks Determine in the Apply on group whether the colocalization measurement is to be carried out on all frames or only on selected frames Should you want to limit the image selection select the Selected frames entry then click the Dimension Selector button e Then you can limit the image selection in the Dimension Selector tool window You can find more information on this tool window in the online help In the Target area group select the Channel segmentation entry in the Area field Click the Options button then select the Colocalization channel Image and Measurement results Workbook check boxes Pay attention to the displayed results in the preview and in the Results group If necessary change the position and size of the intensity range in the scatterplot e Then only the colocalization of the pixels that lie within the chosen intensity range are shown in the preview Click the OK button to finish the measurement of colocalization e Anew image t
191. tion parameters Acquiring an image 13 Click the Start button e Your software now moves the Z drive of the microscope stage to the start position The starting positions lies half of the Z range deeper than the stage s current Z position e The acquisition of the Z stack will begin as soon as the starting position has been reached The microscope stage moves upwards step by step and acquires an image at each new Z position 33 Acquiring image series e The acquisition has been completed when you can once more see the Start button in the Process Manager tool window and the progress bar has been faded out e You can see the acquired Z stack in the image window Use the navigation bar located in the image window to view the Z stack You can find more information on the navigation bar in the online help e The Z stack that has been acquired will be automatically saved You can set the storage directory in the Acquisition Settings gt Saving gt Process Experiment dialog box The preset file format is VSI Note When other programs are running in the background on your PC for instance a virus scanning program it can interfere with the performance when a Z stack is being acquired 00367 10072013 34 Acquiring fluorescence images 7 Acquiring fluorescence images 7 1 Multi channel image The separate images image type Combination with a times stack or Z stack What is a multi channel image A multi cha
192. ton e Then you can limit the image selection in the Dimension Selector tool window You can find more information on this tool window in the online help 5 Click the Options button then select the Colocalization channel Image and Measurement results Workbook check boxes 6 Inthe Target area group click once in the Area field to open the picklist Select the ROI entry e Next to the field to the right the buttons with the various ROI forms are displayed 7 Click the button for the required ROI form that you want to set up You have the choice between a rectangle a circle and a polygon e The mouse pointer will appear in the image window The Colocalization dialog box is hidden 8 Define the first ROI with clicks of your left mouse button When you have completed the definition of your ROI click your right mouse button then select the Confirm Inout command in the context menu e You will then once more see the Colocalization dialog box The ROI you have defined will now be shown in the preview image 9 If required define further ROIs 10 Select the required ROIs To do this click once in the box to the left of the ROI s name oon ROI 1 ROI 2 ROT 3 11 Pay attention to the displayed results in the preview and in the Results group 99 Measuring the colocalization on a color channel Life Science Applications 12 If necessary change the position and size of the intensity range in the
193. tructures If required change the position of the white rectangle gate in the scatterplot By doing this you ll change the observed intensity range You can now e g have pixels with a lower colocalization shown Pay attention to the display in the Results group Click the OK button to finish the measurement of colocalization e Anew image that contains the colocalization channel will be created e Atthe same time a workbook that contains the results of the colocalization measurement will be displayed The columns in the workbook contain the supplement Separation channel If required use the File gt Save as menu command to save the new image and the workbook The multi channel image will also have been changed when the Channel segmentation was defined Therefore if you want to keep these settings save it also 00709 26072011 102 Life Science Applications 11 4 Deconvolution The Process gt Deconvolution submenu offers you deconvolution filters with which you can remove disturbing diffused light from an individual image or a multi dimensional image With a suitable parameter selection the image will become sharper and more clear Before usinga The result of a deconvolution process largely depends upon whether certain deconvolution fiter Parameters are known with which the image was acquired These parameters include for example the objective s numerical aperture and refraction index Before using a dec
194. ucted from the image under examination With single images and simple time stacks the nearest neighbor filter works like a no neighbor filter In this case only the observed image s data are used in the calculation of the point spread function The Wiener filter approximates the point spread function by a linear function with the mean square deviation being minimized The actual filter is calculated from the linear inverse function With Z stacks the complete Z stack s data are used in the calculation with individual images only the observed image s data The Constrained Iterative filter does not make any presumptions about the point spread function but rather extracts it directly from the Z stack This occurs iteratively An estimated point spread function is used as a starting point Then an assumption is made as to which ideal image via this point spread function would have led to the observed image Then an estimation has to be made as to which point spread function caused the original image to be transformed into the observed image This alternating assessment can be repeated as often as wished Special mathematical processes are used to ensure that these iterations converge to reasonable values The Constrained Iterative filter promises the best results of all of the deconvolution filters requires though the most calculation time Since the filter works iteratively on the complete Z stack a use on individual images or simple time sta
195. ue images 16 bit gray value images or 24 bit true color images You can immediately recognize a multi dimensional image by its icon which appears in front of the image name in the document group or in the Documents tool window When it is a Z stack this icon will be supplemented by a small Z 38 In the Properties tool window you can use the Frame Count entry to find out how many individual images are contained in any given image A Z stack image will automatically have its own navigation bar directly in the image window Use this navigation bar to browse through the frames making up a Z stack or to play back the Z stack like a movie You can find more information on the navigation bar in the online help There are different ways in which you can generate a Z stack e To acquire a Z stack use the Z Stack acquisition process e Use the Image gt Combine Frames command to have several separate images combined into a Z stack A Z stack contains much more data than can be displayed on your monitor A Z stack image will automatically have its own navigation bar directly in the image window Use this navigation bar to determine which of the frames from a Z stack is to be displayed on your monitor You can also play back the Z stack just as you would a movie A detailed description of the navigation bar can be found in the online help B im p Cf fe Sle Alternatively you can also use the Dimension Selector tool window to defin
196. uorescence image and want to carry out a spectral unmixing with it For a two channel fluorescence image the procedure is analogical Calibrating color channels When the experimental conditions don t change you will only need to carry out the calibration once When you ve done that you can spectrally unmix all of the three channel fluorescence images that are acquired later on the basis of this calibration 1 Set up three samples that in each case have only been stained with one of the three fluorochromes Alternatively you can use a single sample that has been stained with all three fluorochromes In this case there must be three areas on the sample that have each been stained with only one fluorochrome 2 Acquire a three channel fluorescence image of each of the three samples alternatively of each of the three areas on your sample When you do this use either the excitation filter appropriate for each of them and a multiband emission filter or a multiband excitation filter and the emissions filter appropriate for each of them The experimental conditions must be the same as they were when the image that is to be spectrally unmixed was acquired e Differences in the exposure times of the individual color channels will be automatically linearly corrected when a spectral unmixing is carried out Nevertheless as a rule it makes sense not to change the exposure times when the reference images are acquired e The result will be thr
197. ve measurements For this reason you ll receive a query whether you wish to save the image or not Save the image in the TIF or VSI file format The measurements will then also be saved in the image file They can at any time be edited deleted or augmented 116 Task Measuring areas Viewing the list of measurement parameters Outputting additional measurement parameters Measuring images Outputting various measurement parameters You want to measure some cells Have a variety of measurement parameters such as the area the perimeter and the diameter output Have the diameter shown in the image 1 10 11 12 Acquire an image or use the example image BadTissue tif In the Measurement and ROI tool window click the 2 Points Circle 9 button Leftclick the center point of a cell that you want to measure Move your mouse and in the process drag out the circle Match the circular object as well as possible to the cell Click the left mouse button Click the 2 Point Circle o button again and switch off the measurement mode Take a look at the result in the Measurement and ROI tool window e The illustration shows the image with a circle measured P c gt s m fi s In the Measurement and ROI tool window click the Select Measurements T button e Inthe dialog box you ll see a list with all of the available measurement parameters At the bottom of the dialog box you ll see a list of the measureme
198. wing frames the paramecium disappears at the left border of the image and so can t be measured any more The dynamic ROI is still defined on all following frames in the image series You can t delete a dynamic ROI only for particular frames 16 On the Life Science Applications toolbar click the Intensity Profile button e The Intensity Profile lt Name of the active image gt dialog box opens e Your software will recognize the image type and will select the appropriate option in the Method group In this example the t stack option is preset 17 Make the following settings in the ntensity Profile dialog box Select the Results gt Average check box Clear the other check boxes Select the dynamic ROI in the ROI data group In the Background subtraction group select the none option Close the dialog box with OK 18 Click the Execute button e The intensity profile for the paramecium will be calculated and displayed in the Intensity Profile tool window 1 0 1000 2000 3000 4000 The intensity profile displays how the average intensity in the ROI changes over time The ROI contains the paramecium until about 3000 ms The intensity is relatively constant At time point 1 the paramecium begins to leave the image The intensity then increases to the level of the light image background The time point is at about 3300 ms 00379 12072013 91 Life Science Applications 11 2 Fluorescence Unmixing Filter
199. wish to retain in the Dimension Selector tool window Then use the Extract command in the tool window s context menu When you save a multi channel image in another file format as TIF or VSI the multi channel image will also be converted The multi channel image then becomes a standard 24 bit true color image This image will always show exactly what is currently displayed on your monitor that is to say e g the superimposition of all of the channels or possibly only one channel Processing multi channel images Image processing operations e g a sharpen filter affect either the whole image or only a selection of individual images You will find most of the image processing operations in the Process menu You can find more information on working with image processing functions in the online help Select the frames that you want to process in the Dimension Selector tool window The channels you have selected will be highlighted in color in the tool window The dialog box that is opened when you use an image processing operation is made up in the same way for every operation In this dialog box select the Apply on gt Selected frames and channels option to determine that the function only affects the selected frames Select the Apply on gt All frames and channels option to process all of the individual images An image processing operation does not change the source image s dimensions The resulting image is therefore comprised
200. wo processes Use the Time Lapse acquisition process in the following cases e Use the Time Lapse acquisition process when processes that run slowly are to be documented e g where an acquisition is to be made only every 15 minutes e Use the Time Lapse acquisition process when while the acquisition is in progress you want to see the frames that have already been acquired for example to check on how an experiment is progressing To do this click the Tile View L button in the navigation bar in the image window e Use the Time Lapse acquisition process when you want to use those of your software s additional functions that can only be saved in the VSI or TIF file format For example to measure objects to insert drawing elements such as arrows or a text or to have the acquisition parameters for the camera and microscope that you ve used available at any time in the future 27 Saving a time stack as an AVI When is it better for me to acquire a movie Selecting an objective Selecting the storage location Selecting the compression method Acquiring image series e Use the Time Lapse acquisition process when the important thing is to achieve an optimal image quality and the size of the file is no problem You can also save a time stack as an AVI file at a later date To do this load the time stack into the document group select the File gt Save as command and select the AVI file type Make if n
201. y using the slide control or by entering an exposure time with the keyboard and pressing the Enter key Check the live image on display Once the bright image segments are no longer overexposed click the OK button in the Determine exposure range message box e By doing so you have determined the lower limit of the exposure range the shortest exposure time Now the Determine exposure range message box prompts you to raise the exposure time so high that the dark image segments are no longer underexposed Change the exposure time in the Exposure group which is part of the Camera Control tool window Check the live image on display Once the dark image segments are bright enough click the OK button in the Determine exposure range message box e By doing so you have determined the upper limit of the exposure range the longest exposure time Click the HDR button in the Camera Control tool window to start the image acquisition e The image acquisition will begin Pay attention to the progress bar located in the status bar 3 18 55 It shows how long the acquisition has taken and the total acquisition time The progress bar contains the Cancel button which you can use to stop the current image acquisition e After the acquisition has been completed the HDR image will be shown in the document group Check the image If you want to change the settings to use a different algorithm for the output rendering for example
202. yed semi transparently Select the Equalize check box if the images aren t homogeneously illuminated Then the intensity values of the individual images will be matched with one another which will make the background appear more homogeneous Click OK e Anew image with the name Image_ lt consecutive No gt will be created 00383 63 Running experiments 9 Running experiments 9 1 Overview What exactly is the Experiment Manager The Experiment Manager is a graphic Process Manager How the Experiment Manager differs from the Process Manager The system has been _ configured The observation methods have been defined You can use your software to implement complex acquisition processes Use the Experiment Manager to define and run complex experiments involving image acquisition with your software You can always re use existing experiment plans or adapt them to new conditions You can acquire multi dimensional images during the experiment If your microscope has motorized hardware components you can control these with the software during the experiment Or you can use an RTC Real Time Controller to use external devices in your experiments The idea behind the Experiment Manager You can create a graphic experiment plan with the Experiment Manager tool window This experiment plan contains a series of commands the acquisition of images for example that are carried out one after the other Example Use the
203. you to make this setting manually As well as this the device settings are saved together with the acquired image 5 Select the mirror turret in the Available components list e Inthe central area of the dialog box the settings for the hardware components that have been selected will be displayed 6 For the acquisition of a fluorescence image a specific mirror cube has to be used Therefore select the Adjust entry in the Status list located in the middle of the dialog box and in the list that s below that one select the mirror cube you want 7 Inthe Available components list select the shutter that is located below the Fluorescence reflected entry 8 When the fluorescence acquisition is made this shutter must be open Therefore select the Use for acquisition entry in the Status list located in the middle of the dialog box e Then the shutter will be opened before the image acquisition is made and closed when this has been done to avoid bleaching of the sample 9 Select the transmission lamp You will find this lamp in the Available components list under the Transmission entry 10 For the acquisition of a fluorescence image the lamp must be switched off Select the Adjust entry in the Status list located in the middle of the dialog box Use the button showing a small lamp to switch off the lamp e The button looks like this if the lamp is switched off e Inthe Selected components list you can also see that the
204. you only deselect the page template no file will be deleted 4 Drag the new page template to the position in the report instruction where the deleted page template had been located e Every report has to contain at least one page template 1 To shift a page template to another place in the report instruction select it and with the left mouse button depressed drag it to a new position Drag amp Drop e n certain cases this may change the appearance of the report considerably All documents that come after this page template in the report instruction will use this page template in the report 1 Load the report instruction that you want to edit 2 Inthe report instruction select the documents that you want to delete e The standard MS Windows conventions apply to the multiple selection 3 Use the Del key on your keyboard to delete all of the selected documents in the report instruction e By doing so you only undo the document selection no file will be deleted You can add new documents to an existing report instruction at any time 1 Load the report instruction that you want to edit 2 Simply drag the new documents onto the position you want in the report instruction e Dragging amp dropping images onto the report instruction is possible from the Database Gallery and File Explorer tool windows e Please note the page templates must be placed before the images You can change the order in which the selected
205. your PC when you work with reports Note Two programs are involved in the creation of reports Your software and the MS Word application program You can use the versions MS Word 2008 2007 or 2010 for working with reports 1 New report instruction Create or open a report instruction and fill it with the documents you want hA 2 Generating a report Create a report hA 3 Working with the Olympus add in In Microsoft Word Finish the report Vv 4 Saving the report and the report instruction Save the report instruction and the report 00112 12022012 129 Working with reports 13 2 Working with the report composer The Report Composer tool window supports you when you are creating and updating report instructions In this tool window you also find the Create button that is used to start the report creation Note Two programs are involved in the creation of reports Your software and the MS Word application program Should the Report Composer tool window be hidden use the View gt Tool Windows gt Report Composer command to make it appear Creating a new report instruction To create a report first create a new report instruction in your software You can also use a Saved report instruction Note The report instruction has to contain at least one registered page template You can find more information on registering page templates in the online help 1 Switch to the Reporting layout 2 Clic
206. ysis dialog box A description of this dialog box can be found in the online help You can measure the intensity profile of several image segments at the same time F 4 Viewing a ratio image The result is a multi layer image One image layer is the source image and the other image layer is the ratio image The ratio image displays the concentration of calcium ions using pseudo colors The pseudo color image is superimposed on the source image so that you can see the structures in your sample and the concentration of calcium ions at the same time You can change the display of the resulting image in the image window in order to view only the ratio image for example 00244 25072013 Carrying out a Ratio Analysis Use the Measure gt Ratio Analysis command to measure the concentration of calcium ions in a time stack This command is also available as a button on the Life Science Applications toolbar 106 Life Science Applications Measuring changes in the concentration of calcium ions in a time stack Task The Fura 2 fluorescence dye makes it possible to measure the concentration of free calcium ions because its excitation level shifts from 340 nm to 380 nm as the calcium ion concentration decreases Use the ratio analysis to compute the ratio image over time and the intensity profile in two cells The image displays an overview of the frames in a multi

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