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1. Save Load Settings Apply Protocol to Selected Images Defaut C 10ng ml mimotope loaded Des cropped Ni oy Es Path Creation ka Fath Statistics Object Statistics Apply to Batch gt Select All Clear All Send Intermediate Results ToScreen ToDisk Choose Directory Apple and Close Apply Apples the protocol to a group of images and allows the user to select how intermediate results like PO cy statistics are displayed ote Menu lt no menu location ext gt Close 9 If you have more than one image in the slide you may choose to apply the protocol to other images in the slide Simply check the checkboxes of the images you wish to analyze and either select Apply and Close to finish the protocol or select Apply to perform the batch analysis You may select whether you would like to send results to the screen or straight to disk 10 If you would like to save the particle tracking parameters for processing another slide select Save As from the Save Load Settings dropdown menu Enter a name for the settings and select OK You may now select those settings the next time you open the Particle Tracking Protocol dialog You may also delete settings or modify the settings and then choose Save to save the settings with the same name 10 2 6 2 Performing Manual Particle Tracking 1 Open a main view of the image containing objects you wish to track 2 Goto Mask gt Particle Trackin
2. Center Of Area m Mask Scope O Major Axis Coordinates Entire Mask Object Major Axis Length Minor Axis Length Export Longest Chord Length i i IXY Shape Factor i C l decia Mask 1 hd 7 Subresolution Position anes 0 Intensity none Center Of Intensity Mean Adj Center Of Intensity Micror Pixe Mean Intensity Some advanced features require a secondary mask C Sum Intensity Minimum Intensity Secondary Mask None v A Meri intensiy Ci MI Median Intensiti Remove objects smaller than 0 Remove objects smaller than 10 Select Area pixels and Mean Intensity then select Display to generate statistics The following table will appear 48 Chapter 2 Quick Tour 8 QuickTourWIN2 HeLa Cells Image HeLa Cells Mask Mask 1 Capture Date 10 20 2001 175244 0 132 81 792 94 531 53 1 74 31 720 95 310 68 2 146 63 714 09 332 74 3 284 70 201 26 343 21 1 4 103 71 736 57 373 06 gt 5 141 99 704 45 357 33 E 0 51 587 60 281 40 7 2 55 587 56 285 64 a 2 04 601 80 587 20 3 168 33 732 54 423 53 10 204 88 879 43 407 58 11 182 11 842 91 407 78 12 21712 776 80 269 23 13 157 92 737 40 389 16 14 189 97 826 57 345 61 15 73 60 638 46 338 21 16 07 599 43 647 57 17 1 74 595 76 673 76 1A 180 NA AIA 09 477 Ad Es coe 11 Expand the column headers by clicking and dragging the edge of the header to the right Click on any objec
3. se xa l Current X Offset Y Offset Chi Default mod White Balance E lo Full Chip Advanced Timelapse Capture Available 60 83 GB Required 2 76 MB Capture Type of Time Points 1 3D Capture Range um iz y a i i m Use Planes 2 5 Interval 1000 ms Siep se je o Timelapse Ejs s te oii Offset um E Multiple XY Location Capture Stereology f CurrentLocation C P Filter Set Widefield Adi FENT ND Current 34 Aux ND Current Gain Current Intensify Current Move Up Move Down 0 Optics Image Information Optional Mag Changer 0 y Comment Cancel 5 Click OK to begin capture The resulting images will be flat field corrected 9 5 2 2 Applying Flat Field Correction after Image Capture 1 Select the image that you would like to correct by clicking on 1t You may wish to copy the image before applying the flat field as it is an irreversible operation see Copying an Image on page 159 You may also start from an open view of the image 2 Select Image gt Apply Flat Field The flat field correction will be applied to the image NOTE If you do not have an entry in the flat field database that match the optics used to capture an image SlideBook will alert you that it is unable to perform a flat field correction 211 SlideBook 5 0 User Manual 9 6 Performing Photobleach Correction Photobleach correction can be used to corr
4. EULA is a legal agreement between you either an individual or a single entity and Intelligent Imaging Innovations Inc 31 for the 31 software product identified in the software section of the system registration sheet which includes computer software and a hardware component 1 e the hardware key and may include associated media printed materials and online or electronic documentation SOFTWARE PRODUCT The SOFTWARE PRODUCT also includes any updates and supplements to the original SOFTWARE PRODUCT provided to you by 31 Any software provided along with the SOFTWARE PRODUCT that is associated with a separate end user license agreement is licensed to you under the terms of that license agreement By installing copying downloading accessing or otherwise using the SOFTWARE PRODUCT you agree to be bound by the terms of this EULA If you do not agree to the terms of this EULA do not install or use the SOFTWARE PRODUCT you may however return it to your place of purchase for a full refund SOFTWARE PRODUCT LICENSE The SOFTWARE PRODUCT is protected by copyright laws and international copyright treaties as well as other intellectual property laws and treaties The SOFTWARE PRODUCT is licensed not sold 1 GRANT OF LICENSE This EULA grants you the following rights e Applications Software You may install use access display run or otherwise interact with RUN one copy of the SOFTWARE PRODUCT or an
5. Main View Ctrl click on a region in a 2D timelapse image to display a graph of intensity vs time No function in Tile View or Channel View Measures a desired distance in 2D and 3D images and velocity in 2D timelapse images See Making Measurements with the Ruler Tool below Measures a desired angle in 2D and 3D images Click and drag to draw a single line then make a second click at the angle you wish to measure The angle measurement appears next to the zoom factor 185 SlideBook 5 0 User Manual Now we will explore several data view manipulations that require features of the tool menu 8 4 1 Making a 2D or 3D x y z or x y t Selection As discussed above the ability to make a 3D selection 1s useful when spawning a Tile View You may also want to make a 3D or 2D selection in order to perform volume rendering see page 189 crop an image see page 177 or generate statistics for analysis see page 22 7 3D selections will be visible in all three panes of the Three View and when scrolling through the invisible axis in the Main and Channel Views 8 4 1 1 Making a 2D Selection in any Data View 1 Select the Marquee tool from the tool menu This 1s the default tool in Main Tile and Channel Views 2 Click and drag to designate a 2D selection There are three ways to make 3D selections 3D selections may be for 3D volumes x y z or 2D timelapse x y t images One of the methods does not apply in Tile View
6. ccssseeeeeeeeeeeeeeeeeeseeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 241 10 6 GENERATING AND EXPORTING MASK AND OBJECT STATISTICS cccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 243 10 6 1 Count Ob COIN E MOSK 0 A e 243 10 6 2 Generating Statistics for Masks and ODO CtS 0 A A WO Dla ese 243 X1 SlideBook 5 0 User Manual LOST AIDISPEAVING ORADOR ista 248 10 7 1 VICWING a Line TOTES AO O da Macrae atts aces 248 10 7 2 Viewing Timelapse Intensity Er Olesa dl 249 a a AR cam eh cece cele ot eta eta th ce ents te ett stan er cnc ce 249 10 722 sWethod 2 FOr aC CK fis aia os ware desire ida 250 IOTZ Method gt WSS RO Sita lili li es 251 APPENDIX A SMOOTH CURVE ANALYSIS KYMOGRAPH occccccccccccccccccncncnononcncnnnononcncnononcncccccccccccccnss 253 xii Technical Support Technical Support If you have any questions or experience any problems with SlideBook M please contact 31 support at support intelligent imaging com or 303 607 9429 xiii SlideBook 5 0 User Manual Manual Conventions 1 1 Typographic Conventions Menu commands are written in bold and follow the order of menu navigation For instance choosing Open from the File menu is written as File gt Open Dialog fields and other interface items are written in bold as in Initial Offset References to other sections of the manual are underlined as in Image Capture New terms are italicized NOTES will be written in bold and small cap
7. ie SlideBook 5 0 InstallShield Wizard Click Install to begin the installation If you want to review or change any of your installation settings cick Back Click Cancel to exit the wizard InstallShield 6 Select Install and the setup utility will display status of the installation SlideBook 5 0 InstallShield Wizard Please wait while the InstallShield Wizard installs SlideBook 5 0 This may take several minutes otatus InstallShield 7 When the installation is complete the following message will confirm installation 21 SlideBook 5 0 User Manual ie SlideBook 5 0 InstallShield Wizard Please wait while the InstallShield Wizard installs SlideBook 5 0 This may take several minutes Status _ TT 8 Click Finish to complete the setup You may now launch SlideBook from either the Start Menu gt Programs or from the Shortcut to SlideBook exe NOTE When you start SlideBook for the first time it will notice that a preferences file which is stored along with the application does not exist SlideBook will create a default preferences file As you configure your hardware and define optics discussed in Chapter 4 Configuring Your System SlideBook will update this preferences file with specific information about your setup An electronic copy of this manual called SlideBook 5 0 Users Manual pdf isin Adobe Acrobat format and can be opened by an Acrobat reader
8. 2 Follow steps 2 4 in the above section 3D Capture Setting Capture Window Parameters 3 Create any annotations that you would like to record during capture see Creating Notes below 4 Check the Timelapse checkbox in the Capture Type section of the Capture dialog box You now have the option to define a variety of timelapse parameters Enter the desired values in the following fields Timelapse Capture Capture Type of Time Points 10 3D Duration 20 min E Interval 2 i i Timelapse Display Renormalize to t 0 min Stereology e of Timepoints Number of timepoints that will be captured e Duration Total length of time for the experiment Units of time in milliseconds ms seconds s minutes m or hours h can be selected from the dropdown menu Due to the inability to predict time required for hardware movement this option 1s only available when the interval for capture 1s over 30 seconds e Interval Delay between the beginning of one timepoint and the beginning of the next timepoint The interval unit can be selected from the dropdown menu If the capture sequence at one timepoint takes longer than the interval SlideBook will capture the next timepoint immediately following the preceding timepoint NOTE As you type in two of these values the third field will be calculated automatically e Renormalize to First Timepoint When checked the minimum and ma
9. Multiwell Setup Layout Mame Microscope Components Objective 101D vr Mag Changer 10x vr Plate Setup Rows Columns s spacing Y spacing Well Diameter Calibration Aow Calibration Columri Channels 2 Offset o um Auto Calibrate Exposure f Exposure 00 mg Flat Field Capture Parameters o f mages at center of well i i i E Image single center frame E grid Separation o um C mage pattern Chapter 7 Advanced Capture Autofocus Enable Autofocus AF Channel 340 F AF Type AF Frequency 0 AF Exposure Time o me AF Total Search Range o um Feak Delta Threshold 0 8 Bin Factor fj 7 fi af ats gs caed_ se 3 Enter the Layout Name in the edit field This layout will be saved so you may use 1t again for future experiments 4 Select the Microscope Components from the Objective and Mag Changer dropdown menus 5 Enter Plate Setup parameters by either entering numbers or using the up and down arrows e X spacing Y spacing center to center distance in x and y directions in microns can be determined by contacting the plate manufacturer values show in above dialog box are typical for a standard 96 well plate e Well Diameter can also be determined by contacting the plate manufacturer e Calibration Row row used for setting top of upper left and upper right wells see below You can generally specify this to be 1 e g r
10. p Polygon Tool click to draw a series of straight lines double click to close the shape ES Free Hand Tool click and drag to draw freehand release mouse button to close the shape e a ROI Selection Tool use to select an ROI Holding shift and clicking on multiple regions will allow you to select more than one region Clicking and dragging on a selected region will move the selected ROIs If the ROI number is displayed in white this indicates that the ROI is selected 3 Continue to draw regions as desired The following operations can be performed by selecting the ROI with the ROI selection tool and then right clicking to bring up the ROI menu e Set as background uses the selected ROI for background subtraction during graphing e Duplicate creates a copy of the selected ROI e Delete removes the selected ROI s 4 Once you have created the desired regions of interest and begun capture you can display a graph by selecting the desired channel in the Graph Channels section at the bottom left of the Capture Controls and then clicking on the Show button The regions of interest that you have created can also be displayed after capture as discussed in Displaying Annotations on page 181 To recreate the graph post capture please see page 251 7 2 5 Initiating and Monitoring Timelapse Capture In the Capture dialog box click Start to begin capture The Capture Controls will appear and display the current
11. 8 2 3 4 Arranging your Tile View You may wish to change the way your Tile View is displayed For instance the default tile display may choose a 4 X 6 arrangement while you desire a 6 X 4 arrangement You may also wish to change the background color between the tiles To alter these settings 1 With a Tile View displayed select View gt Settings or click on the E Setting icon in the View window The following dialog box will appear View Settings Tile View W Specify Dimensions Manually Tiles Per Aow E Display Styles Background Channel Blend 12 100 50 Set Grey Values Below Background Loop Speed Loop FFS i T Background Color Mew background color Cancel l 2 Select the Specify Dimensions Manually checkbox then enter the Tiles Per Row that you desire in the edit field 3 In the Background Color section click on the colored square next to View background color 4 Select the desired background color from the Color dialog and click OK 5 Click OK to update your Tile View 166 Chapter 9 Preparing an Image for Analysis or Export Y 3 ratiosample Untitled 1 k 0 E fimz 60 2 22 0 22__Ratio1 661 _Fura 2 3801 00 05 16 3 00 06 03 v 8 2 4 Displaying a Multidimensional Channel View The Multidimensional Channel View is most useful for displaying multi channel images The Multidimensional Channel View displays each channel independently plus
12. Chapter 2 Quick Tour 2 3 Using the Info Tool Bar The Main View window contains an Info Tool Bar placed above the image of the data The Info Tool Bar displays a variety of useful information and gives you quick access to tools you will use to explore and manipulate the view The Info Tool Bar is present in all data views namely the Main View Three View Tile View and Channel View X Y Z T Position Adjust Renormalization Mask Menu Axis Menu Set Default Show Graphs View View Settings Pixel Intensity Values Zoom Controls A Quick Tou INI Metap VA g Br Cell 2 a x Red Menu lwz E 013 04 JU Cra Green Menu Plane Selection ala FITC a eee E 205 DAPI Blue Menu 4 A t E di cae Hone Tool Men Background Menu 2 3 1 Channel Menus and Data Values On the far right of the Info Tool Bar are three pop up menus with colored red green and blue text next to them These menus allow you to select the fluorescence channel that is displayed for each color The Main View in this data set has the red menu set to the CY3 channel the green menu set to the FITC channel and the blue menu set to the AMCA channel 25 SlideBook 5 0 User Manual 26 Click on one of the Main View images to select 1t as the active window Click on the CY3 bar red menu and select FITC from the pop up menu Notice that the FITC label in the view now appears yellow from the combination of red and green pixels Click on the red menu and
13. Controlling the Camera and Microscope Hardware Focus Window 3 Click on the Z tab Now you will need to set the z limits for capture 105 SlideBook 5 0 User Manual 106 4 10 Scope Z xy Camera Stream Top Set Top Capture Information Mot Set Number of Planes H Center Total Travel um HA HA Go Step size um 0 27 a 63 Ol Clear All Referente Set Mot Set Bottom Not Set Set Bottom a 3 Starting at the middle of the focus range move the stage or nosepiece up until you are at the top of the volume that you wish to capture This can be accomplished either by repeatedly clicking on one of the up arrow buttons or by manually adjusting the fine focus of the microscope Z Stage Simulated Z Stage 323 5 um gt gt al en Ele El A 10 1 1 gt 5 429 um NOTE When moving the z position by computer control you may elther use one of the fixed increment buttons 10 1 or 1 micron steps or manually enter a step size in the adjacent edit box Click on the Set Top button SlideBook will save the current z position and display it to the left of the Set Top button Focus in the opposite direction until you are at the bottom of the volume of interest Click on the Set Bottom button SlideBook will save the current z position and display it to the left of the Set Bottom button Return to the point of best focus the brightest plane using the slider Go button or micr
14. Selecting Channels and Setting Exposure Times Selecting the Area to be Imaged Entering Image Information and Optical Parameters before Capture Capturing a 2D Image Performing Auto White Balance Importing an Image Getting and Editing Image Information after Capture Advanced topics such as 3D timelapse 4D montage and multipoint imaging are covered in Chapter 7 Advanced Capture 6 1 Selecting Channels and Setting Exposure Times Exposure times are set by generating a series of test images for each channel that will be exposed If you are performing a transmitted light capture with a color camera or color LCD filter you may wish to first perform an auto whitebalance see below 1 Once you have a sample in focus open the Capture dialog by selecting Image gt Capture or by selecting the Capture icon in the toolbar oi Note that you must have a slide file open in order to open the Capture dialog The dialog box that appears is divided into eight parts starting from the upper left corner capture settings image extent and binning factor multiplane capture multipoint capture exposure settings by channel image histogram optical parameters and image info The Capture dialog uses the same Live View as the Focus Window 89 SlideBook 5 0 User Manual Capture Current Parameters Default Extent Offset and Binning pixels Image Bin Factor Width 1392 Height 1040 Update O Autofocus 1x1 a X Of
15. volume in cubic microns or square microns in 2D or timelapse images Note that the image must have the correct objective and optovar selected for these values to be accurate see Defining Objectives on page 62 and Defining Magnification Changers on page 73 Surface area um Perimeter um the area of exposed voxel faces for 3D objects or exposed pixel faces in 2D or time lapse images Note that these values should only be used as rough estimates as area or perimeter measurements are highly affected by the resolution of the image Center of Area Volume the coordinates of the center of the object unweighted by intensity values Major Axis Coordinates the coordinates of the two edge voxels that lie along the major axis Major Axis Length the length of the longest line that runs through the center of volume also called the axis of minimal intertia Minor Axis Length the length of the minor axis in 2D or both minor axes in 3D Longest Chord Length the distance between the two furthest voxels in the object Major Minor Shape Factor The ratio of major axis to first minor axis 1 e width vs height Intensity Center Of Intensity the coordinates of the center of the marked object welghted by intensity calculated for each channel Mean Adj Center Of Intensity the coordinates of the center of the object weighted by intensity values minus the mean intensity of the object Mean In
16. 3 Select the desired capture order If you would like to reduce the number of filter switches that occur during capture select Capture one channel at a time for all z positions 4 Click OK and then proceed to the Capture dialog box to set up for 4D capture as described above 7 4 Multipoint Capture If your stage is equipped with a motorized xy stage SlideBook allows you to select multiple locations to visit throughout the course of capture You may perform timelapse and or 3D capture at each location As with the other types of capture you will set parameters in the focus window and in the capture window 7 4 1 Setting Capture Preferences Depending on the type of capture you plan to perform 2D timelapse 3D or 4D you may wish to select capture preferences such as periodic capture midvolume capture multi channel z options autofocus and spooled capture as described in the sections above and below see pages 113 115 125 127 and 146 127 SlideBook 5 0 User Manual 7 4 2 Setting Focus Window Parameters You may choose to select new points or you may load points that you have saved previously 7 4 2 1 Setting Points 1 Open the Focus Window by either selecting Window gt Focus Window or clicking on the focus window icon in the SlideBook toolbar The Live View will open and begin updating 2 Select your objective magnification changer and filter configuration and bring your sample into view and focus a
17. 81 A AA Sette cle ido a tation leds asa ode alee roo led rt E A OOE l 5 1 3 1 Sel TOD Se Bolton Set Reference Center aesae a aa 82 5 1 3 2 SIET ah ce ee cme Sah tel Shh ddan Sakti a a lees ie 83 5 1 3 3 Capture Mora dl N 83 VI DN SAA SAA AA 53 ADT EME A AS AAA EA PE REED EDT EDS A 84 5 15 1 IPS 00 OF Lies cees tras cere asec sac emt a eee ean uate a ae eee 84 Sal CT A A e eels 85 5 15 3 PB ate aa ta tle wet the atte wae tke ed tee tats wai ada vane tae 85 5 1 5 4 A ak iigdatgee 85 SA a o MtensiicaO ina 85 5 1 5 6 Parani A pon 5 O o O centeatices A 85 al es a SA e a 85 dz USING THB FOCUS WINDOW odas 85 LATP sSNGD DINGO LINGO Cis iether Me eta dd dit abies ie it aos 56 5 2 2 Closing the Fluorescence Shutter Automatically when Closing the Focus WindOW cccccccccccceeeeees 56 32 5 Labelime ihe Ocular Photo Prism POSTION oi ios ete les end eee iene edie a 8 amp 7 324 HOW 10 Perform SW eAMine Capire AS A bee ce ee ee 7 6 IMAGE CAPTURE AND IMPOR YW N AEN O ANE 89 6 1 SELECTING CHANNELS AND SETTING EXPOSURE TIMES sesion dis cadaver vest donmneat 89 6 2 SEEECTING THE AREA TO BE IMAGE Droi dl E A T N 92 621 Osme thelmase FCAT MCI siete dacs in EEEE A guest RE 92 O22 AA A E E E S 93 6 3 ENTERING IMAGE INFORMATION AND OPTICAL PARAMETERS BEFORE CAPTURE sssseeeeeeeeeeeeeeeeeeeeeeees 93 6 4 CAPTURING A SINGLE OR MULTI CHANNEL 2D IMAGE cssssssessssssssssssssssssesssssssssssesssssseessesssssessseesees 93 6
18. An advanced debugging feature that will generate a series of TIFF files in a numbered directory created in SlideBook program directory e Save Logfile An advanced debugging feature that creates an autofocus log file in the SlideBook program directory 6 Press the Autofocus button to initiate a single auto focus scan When finished SlideBook will report the optimal focal z position in the Best Z information box and automatically move the z stage to this location In addition timing statistics for the autofocus routine are given in the Total Time and Image Time information boxes NOTE If SlideBook is unable to determine the optimal focal plane in the sample it will return one of two messages in the Best Z information box The No Gradient message indicates that SlideBook was unable to find a focal gradient within the specified search range In this case the peak delta threshold used for autofocus computation may need to be increased or the search range may need to be increased The At Edge message indicates that the optimal focal plane appears to lie above or below the specified search range Since SlideBook will automatically move the z stage to the best focal plane within the specified search range simply running the auto focus routine again starting from this new z position will often find the optimal focal plane 7 Press the OK button in the Auto Focus dialog box 8 Use the focus window to evaluate the success of the autofocus r
19. Lamp None at fort ab M Objective Turret hone at pf X LCD BF Filter None at off v Mag Changer None v at of y Ad d Lightpath baling al ae Ocular Photo hone at Or X Auxiliary ND None a for y CCD Video None v at Off ue Auxiliary wheel None at off v SA Correction None v at Diagnostic Mode OK 3 Name the system in the System name edit field This should be descriptive and include the laboratory name so that the preferences file can be identified by 31 technical support 4 Select the components that are present from the drop down menus set to None in the dialog box above 5 For each component that is controlled through a COM port or USB port mapped to a COM port select the COM port to which it is connected in the drop down menus to the right of each component If a component is not present or you would not like it to communicate with SlideBook be sure the COM port is set to Off NOTE A typical PC has two serial COM ports port 1 and port 2 located at the rear of the machine Additional COM ports are often added e g through use of an Edgeport adapter or PCI card These additional ports are then typically termed port 3 port 4 etc instead of following the labels on the adapter 57 SlideBook 5 0 User Manual Select OK Restart SlideBook to register the changes and begin hardware communication If you plan to control your hardware via TTL control please c
20. M 0 1 1010 4 SlideBook 5 0 Version 5 0 0 0 for Windows XP User Manual Latest Version 3 23 09 End User License Agreement This software interfaces with LibTiff version 3 6 1 1 Copyright c 1988 1997 Sam Leffler Copyright c 1991 1997 Silicon Graphics Inc Permission to use copy modify distribute and sell this software and 1ts documentation for any purpose 1s hereby granted without fee provided that 1 the above copyright notices and this permission notice appear in all copies of the software and related documentation and 11 the names of Sam Leffler and Silicon Graphics may not be used in any advertising or publicity relating to the software without the specific prior written permission of Sam Leffler and Silicon Graphics THE SOFTWARE IS PROVIDED AS IS AND WITHOUT WARRANTY OF ANY KIND EXPRESS IMPLIED OR OTHERWISE INCLUDING WITHOUT LIMITATION ANY WARRANTY OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL SAM LEFFLER OR SILICON GRAPHICS BE LIABLE FOR ANY SPECIAL INCIDENTAL INDIRECT OR CONSEQUENTIAL DAMAGES OF ANY KIND OR ANY DAMAGES WHATSOEVER RESULTING FROM LOSS OF USE DATA OR PROFITS WHETHER OR NOT ADVISED OF THE POSSIBILITY OF DAMAGE AND ON ANY THEORY OF LIABILITY ARISING OUT OF OR IN CONNECTION WITH THE USE OR PERFORMANCE OF THIS SOFTWARE SlideBook 5 0 User Manual End User License Agreement for 3i Software IMPORTANT READ CAREFULLY This 31 End User License Agreement
21. Select the UV checkbox for UV objectives and select the appropriate medium from the radio button menu Select the appropriate configurations for the desired transmitted light condenser turrets using the drop down menus on the right Once you have defined the condenser positions for a given objective you may define filters that utilize the appropriate condenser positions Filter definitions are discussed in the next section Repeat steps 1 through 6 for any additional objectives that you wish to add Select OK when you are finished Restart SlideBook to register the changes that you have made 4 3 2 Modifying Information for an Existing Objective You can modify existing settings as follows l 4 5 Select Edit gt Define Optics gt Objectives to bring up the Objective Parameters dialog box Select the appropriate lens using the Objective Lens dropdown list Change the appropriate fields as desired Select OK to save the changes Restart SlideBook to register the changes that you have made 4 3 3 Removing an Objective Select Edit gt Define Optics gt Objectives to bring up the Objective Parameters dialog box Select the appropriate lens using the Objective Lens dropdown list Choose Remove then OK to save the changes Restart SlideBook to register the changes that you have made 63 SlideBook 5 0 User Manual 4 4 Defining Filter Configurations You can add modify and remove filter configurations using
22. allowing control of the lighting angle Useful for determining surface characteristics eC 5 QuickTourWIN3 Capture 8 Cropped CI cropped 1 o O e x 180 Y 00 Z 00 degrees Red __ CY3_ Dolly 1 000 Green FITC1 a sy ol Bela Blue DAPI 10um grid Dynamic Lighting 189 SlideBook 5 0 User Manual e Fixed Lighting uses a fixed lighting pattern for shading approximates surface characteristics r 3 QuickTourWIN3 Capture8 Cropped CI cropped 1 Ee iss x 180 Y 00 Z 00 degrees Red CY3 Dolly 1 000 Green FITCI Ey m la Blue DAPI 10um grid Fixed Lighting e Maximum Intensity Projection MIP The brightest pixels through the axis perpendicular to the screen are displayed This algorithm requires the least processing power and is particularly useful for rendering large data sets and does not require advanced graphics card 6 2 QuickTourWIN3 Capture 8 Cropped Cl cropped 1 e E is x 180 Y 00 Z 00 degrees Red CY3 Dolly 1 000 Green EIGI E E Ol tj Blue DAPI 10um grid MIP e X Ray Similar to MIP however the sums of pixels through the axis perpendicular to the screen are displayed This mode is useful when trying to visualize density or concentration differences 8 5 1 1 Working with 3D Volume Views To open a new 8D Volume View 190 Chapter 9 Preparing an Image for Analysis or Export In a Slide View click on
23. i Fill uncaptured time points with data from last captured time point 7 2 1 2 Opening and Closing Brightfield Shutter Between Exposures For fluorescence shuttering please see the above section Setting Capture Preferences to Open and Close Shutter during Capture on page 108 If you are performing 2D timelapse capture the default fluorescence shutter action in SlideBook is to open and close the shutter if your timelapse interval is non zero If you have a Oms timelapse interval the shutter will remain open to allow you to capture as quickly as possible This default behavior is designed to protect the sample at non zero timelapse intervals yet maximize acquisition speed at Oms timelapse intervals Thus the Open and close shutter on single channel or simultaneous 3D capture checkbox is only applicable when you wish to shutter for 2D timelapse images with a timelapse interval of Oms If you would like your brightfield shutter to close between transmitted light images 1 Select Edit gt Hardware Properties and expand the Advanced Capture Settings list 2 Click on ShutterBrightFieldTimelapse The right side of the dialog box will update 113 SlideBook 5 0 User Manual Edit Hardware Properties E Advanced Capture Settings Property Information ShutterBrightFieldT imelapse Hame ShutterBrightFieldT imelapse ShutherAltS ounce ways ShutterAltSourceMinD elay Category Advanced Capture Settings EnableT TLD isabled ntormation TTLTrigg
24. oO Ending timepoirt E Insert image at end of slide Result image 32 timepoint 5 14 6 MB Cancel 3 Enter the starting and ending timepoint of the subseries you would like to extract 4 Check the box for Insert image at end of slide if you would like to save the subseries in the same slide as the original data If you do not check this box the image will save below the active image in the Slide View 8 3 4 Rotating an Image You may wish to rotate a view of the image for publication purposes CAUTION Rotation is an irreversible process You may wish to make a copy of the image before rotating see Copying an Image on page 159 To rotate an image 5 Open a Main View of the desired image 6 Select Image gt Rotate The following dialog box will appear 178 Chapter 9 Preparing an Image for Analysis or Export Image Rotation Image Range f Current Image e e e Rotate Clockwise 90 Rotate Counterclockwise 90 Rotate 180 Flip Horizontal Selected Images Flip Vertical Flip in Z C Arbitrary CCM Interpolation Bilinear A E F A A A Metaphase B Cell Cancel 7 Select the Image Range as described below You may also select and deselect images using the checkboxes in the Selected Images list e Current Image Operation will be performed on the selected image only e Current Capture Type Operation will be performed on all images in the slide with the same capture
25. the user will decide whether the capture will be two dimensional three dimensional two dimensional over time or three dimensional over time Parameters set in the Focus Controls such as multiple points in the xy plane and z distances can be imported into the Capture dialog box and utilized during the capture sequence See Chapters 6 and 7 for further explanation of the Capture dialog box 3 1 3 Capture Controls The Capture Controls window shows the progress of a capture sequence The features in this window are most useful when performing timelapse acquisition and are discussed in Chapter 7 3 2 Data Storage Display and Analysis Chapters 8 9 and 10 SlideBook s image visualization and analysis tools are designed around five principal objects image channel view mask statistics and slide This organization is somewhat different than other image processing programs For instance with this organization operations that change data are kept separate from those that only affect its display or analysis 52 Chapter 3 SlideBook Organization 3 2 1 Images and channels An image is a two or three dimensional set of data collected by a digital microscope or imported from another application It consists of one or more channels where each channel corresponds to a different fluorophore For instance one channel may represent a FITC labeled protein while another represents a CY 3 labeled protein An image and its constit
26. then turn the stage off and on to zero the stage You must perform this procedure each time you plan to save or load a multipoint list 3 Once the stage is homed place your sample on the stage and proceed with setting your point list Once the list is complete select Save You will be prompted to enter a description for your point list Save XY Stage Positrons Description point list 1 Cancel 129 SlideBook 5 0 User Manual 4 Enter a description and then select OK Now that you have saved XY stage positions you can load them for subsequent experiments To load them simply home the stage before loading your sample then select Load to populate the point list 7 4 3 Setting Capture Window Parameters 1 Once you have set the appropriate parameters in the Focus Window open the Capture dialog box by selecting Image gt Capture or by selecting the capture icon in the SlideBook toolbar A 2 Follow steps 2 through 4 in the above section 3D Capture Setting Capture Window Parameters in order to select channels to be captured and set parameters for image extent and binning 3 Click on the Entire List Montage radio button in the Multiple XY Location Capture section Alternately if you would only like to capture the current point you may choose the Current Location radio button If you wish to take a single image at each location proceed to step 8 7 4 3 1 3D Multiple Location Capture 4 If you would like t
27. 1 Stage TTL DAQ 2 Stage User Login Properties Volume Rendering Y Stage 3 MoveFieldRightSign MoveFieldD owns igr Stereclogyhontagel verlap FulllageM ontagel verlap Enable Y Position Query Rotate Computed Montage 90 Degrees Enable Home Command Override Yokogawa CSU Properties Zeis Asiolmager Zeis Awol bserver Zels 4xioplan 2 Axiovert 200M Zels MEU 28 Property Information Name MoyveFteldAightSign Category Y Stage Description Do position values Increase 1 or decrease 1 when panning right field moving to the right Apply Change Default Value q AA Revert To Default 7 Enter the appropriate value 1 or 1 based on the change in x position values when panning right in the edit field and select Apply Change 99 SlideBook 5 0 User Manual 8 Repeat steps 6 and 7 for the y axis by clicking on the MoveFieldDownSign 9 Close the dialog box using the Close button 4 2 3 Configuring an Image Splitter SlideBook supports the use of image splitter devices An image splitter allows you to capture two separate channels for the right and left halves of the camera To configure an image splitter 1 Select Edit gt Hardware Properties and click on the sign to expand the Simultaneous Capture section 2 Click on Split Capture The left side of the dialog box will change Edit Hardware Properties x SAC Property Information Sensilam Mame SplitCapture Sigma Kaki Simultaneous Captur
28. 1 WV Obs O wath o D Volume VIEWS sipaan A cl A E NT 190 8 5 1 2 W Orkin wart 4D Volume VIEWS a an hy Sel Ane a ln cl O de 193 O92 DISPARADO SULACE S Wen ds cei incor td 193 8 5 3 Generating a Physically Proportional Rendering Of 3D Data o oo 195 ne ATOE SCHICS IO VIS A ted ctas 195 8 5 4 1 A ee Ae ee Ree ee ee een RO 195 8 5 4 2 AA T ee E r EE IA AE ET IESE EAE T P ot ao EA 196 8 6 E TORING MIE WS E NA 196 8 6 1 Exporting a Main View Three View Channel View or Tile View as a TIFF 197 8 6 2 Exporting a 3D or 2D Timelapse View as a TIFF or TIFF Series o n 197 Table of Contents 8 6 3 Exporting Default Views of All Images in a Slide ooononnnnnnnnnnnnnnononooaoonooonnn nono nonnnnnnnnnnnnnnnnnnnnnnnnnonons 197 8 6 4 Exporting Volume View and Surface View MOVIES ooooooonnoconnnnnnnnnonnnnnonononononnnnnnnnnnnnnnnonnnnnnnonononininoss 198 9 PREPARING AN IMAGE FOR ANALYSIS OR EXPORT o o occcccnnnncoccccnnnnccccnnnnnccccnnnnnccccncnncccnnncnccccnnnoss 200 9 1 MANIPULATING INDIVIDUAL C HANNELS ias 200 Old Ansertineanimaceas a CNAE OS 200 OLD IeCMOVING d CRANN OL A A A A A A a ee 201 Ode JUSTAS Channel MA wot cath AA A A aaa ss 202 91 4 Creatine a Timelapse Composite Channel acids Quasi A hws 204 92 CROPPING AN IMAGE S ENE 204 9 3 ALIGNING AN IMAGE ice si Os 204 9 4 MANIPULA NNG LIMELAPSE SERTE Sian 206 9 4 1 Removing Timepoints from a Timelapse Series ooonnnnnnnnnnnanananananananananananan nono nono ono n nn nn nana nn nn nn nn r a
29. 1 microns In addition an user defined stage movement increment value can be entered in the edit field below the last set of buttons on the right The default value of the edit field will be set to the theoretical z resolution for the selected objective With few exceptions the up and down buttons correspond to the direction of mechanical movement For instance on an upright microscope with either an integrated or external stepper motor the direction corresponds to stage movement so up means focusing further past the cover slip and towards the slide since the stage is moving up On an inverted microscope up corresponds to moving the nosepiece up and therefore also means focusing further past the cover slip The Auto focus Button is used to define and test parameters for auto focusing during capture Use of this feature is covered in Chapter 7 5 1 1 6 Neutral Density If you have defined neutral density positions in your hardware configuration you may select the position from the Neutral Density drop down menu 5 1 2 Scope The Scope tab lets you observe and control other encoded and automated features of your system Scope Z KY Camera steam Objectives Emission Selection Stage Limits 10 Dry 20 Air C Confocal Lamp en ADs Air iT Wideteld f 100 Eves Condenser 100 Oil ae i agnification perture r y Changer O Cemas B oa Position Pas 5 5 1 2 1 Emission Selection On
30. 32 3 03 053 14 26 07 177 67 10005 54 5 06 22 66 4 04 053 14 26 08 17731 10004 1 2 455 0 30 5 05 054 14 26 09 177 63 10005 08 470 22 44 E 06 055 14 26 10 177 97 10005 66 4 35 22 29 T 07 056 14 26 11 177 32 10005 60 441 24 17 g 08 056 14 26 12 176 64 10006 64 4 52 23 80 3 03 056 14 26 13 176 55 10005 88 4 32 22 00 10 10 056 14 26 14 179 21 10007 29 4 39 24 46 11 11 057 14 26 15 173 36 10008 58 4 24 04 12 12 057 14 26 16 179 29 10007 19 427 20 55 13 13 057 14 26 17 12311 10007 74 4 46 22 40 14 14 057 14 26 18 17351 10007 87 474 21 57 15 15 057 14 26 19 179 18 10007 26 4 64 21 43 16 16 057 14 26 20 178 66 10005 00 4 53 20 57 17 17 NAA 1420291 178 79 1000F 41 R17 AA AA E se Output Grouped by Timepoint 101 x Image test Mask Mask 4 Capture Date 12 03 2003 14 26 04 Clock Time 14 26 04 Time Port jo a OF 30 total time points Elapsed Time 2 tHhm 00 000 atandard D Standard D 10006 33 10002 93 10002 79 10003 38 3 Check all the statistics you want to compute SlideBook offers the following options for statistics for a single mask Morphometry e Object Count number of objects This statistic is only available when you select Entire Mask in the Mask Scope section of the statistics dialog 245 SlideBook 5 0 User Manual 246 Volume voxels Area pixels total number of voxels Note that in a 2D or time lapse image you will always get an area measurement Volume um Area um
31. Default Settings dialog by selecting Annotation Settings l E z L l from the View icon or by going to Annotations gt Settings The following dialog will appear Annotation Default Settings Notes Object IDs Regions Timestamps Scale Bars Lookup Tables Placement Mi se Text Color Font pt Format a 2 Select the annotation that you would like to format The dialog boxes for annotations are shown below Scale Bars You may define the length of the scale bar choose color placement and font and include text or not Annotation Default Settings Notes Object IDs Regions Timestamps Scale Bars Lookup Tables Length a Bar Color Placement Lower left comer Include Text net Font 26pt 182 Chapter 9 Preparing an Image for Analysis or Export Lookup Tables You may choose the size of the lookup table as a percentage of the image width and height as well as placement text color and font Annotation Default Settings Timestamps Lookup Tables Placement Upper right comer id Text Color Font 18pt Annotation Default Settings Timestamps Scale Bars Lookup Tables Notes ObjestIDs Regions Placement Upper left camer Text Color Font Ariel 1 Ant Object IDs You may select text color and font Note that object IDs only appear if you have a mask displayed that has been divided into objects Annotation
32. E E A Sacet 137 POIS Dele cine ens OF CAM o aso 137 100 IMtatin and MONTORO CA o cis 137 TO APON ROS ai E E N E NE Momeni seeeaemenudae 138 TA SIMUL TANEOUS CAPTUR sti dci 138 7 8 CONFIGURING YOUR HARDWARE FOR SPEED ooccccccccncoooncncnnnnnnnnnnncnnnnnnnnnnnnnncnnnnnnnnnnnnncnnnnnnnnnnnnnccnnnnnnnnnnnss 138 Ll Photomeri cS COMAS A ias 138 Lo DUNI TAG A E N 139 LO A OS porns nite T ri A EN 139 7 8 3 1 ASUMA CURE e e ds 140 7 8 3 2 Physik Instrumente PIFOC Piezoelectric Focusing Collar oococccccnnnnnnncnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnninnns 141 7 9 PRU VOB OCU S aii 144 FiO Determining AUTO Focus LATA de o 144 7 9 2 Adding Auto Focus to an Image Capture SequenCe ooooooononnnnnnnnnnononnnnnnononnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnss 146 7 10 VARYING CAPTURE RATES DURING TIMELAPSE CAPTURE SEQUENCES ccccccseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 149 7 10 1 SANE Capture P TSOR CS dd altel cas 149 7 10 2 Setting Capture Dialog BOX Paramete S coe A einen iste 151 7 10 3 Examples Jor Variable CAPTURE A A A A anne eis 152 FIOL A Tarin a AEE ALA A N A ae 152 TAOS ol AAA A O ee O A a 153 7 11 SAVING IMAGES TO DISK SPOOLED CAPTURE cooooocconcnooonooncnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nn nn rn nn nn 153 TZ SAVING CAPTURE PARAMBIERS latina ici a a pte 154 8 IMAGE DISPLAY AND MANIPULATION VIEWS cccccccssssssssssssssscssssssssccccccssssssscsscssssssssssssssssees 156 8 1 INTRODUCTION TO SLIDE AND IMAGE DISPL
33. Excel 7 Click Close to close the dialog box 10 7 2 2 Method 2 Point and Click From a main view select the point selection tool from the tool menu Press Ctrl and left click on any object in the image An intensity time profile will appear as shown below 250 Chapter 10 Using Masks for Image Analysis Ratio Timelapse Data 000 200 400 600 3 00 10 00 1200 1400 16 00 13200 20 00 2200 2400 2600 2600 30 00 Range 94 95 to 99 100 Mean 0 425 50 0 970 Channel E y Radius 3 piselz Export The intensity displayed 1s the average intensity of an area around the point where you clicked of the specified radius You can alter the radius edit field and choose to export the statistics as a tab delimited text file txt 10 7 2 3 Method 3 Using ROIs If you have drawn regions of interest to monitor timelapse capture you can create graphs of these regions post capture 1 Open a view of the desired image 2 Select any of the region tools or go to Annotations gt Regions to display the ROIs 3 Select View gt Graph The following dialog will appear New Graph View Intensity Intensity Intensity Min Intensity Max Ratio Cancel 4 Select the type of graph you wish to display and the channels that you wish to display 251 SlideBook 5 0 User Manual 5 Select which lines you would like to display only applies when graphing intensities not ratios 6 Click OK
34. Focus Window Parameters 1 Open the Focus Window by either selecting Window gt Focus Window or clicking on the focus window icon in the SlideBook toolbar El The Live View will begin updating 2 Select your objective magnification changer and filter configuration and bring your sample into view and focus as discussed in Chapter 5 Controlling the Camera and Microscope Hardware Focus Window on page 77 3 The XY tab of the focus window has a panel of buttons that assist in selecting the range of the montage Montage Extent F A K El u 4 These buttons are used to define the boundaries of the entire region to be included in the montage image SlideBook needs enough coordinate information to generate an imaginary box around the region to be imaged Thus the extent of the montage image can be defined by selecting one of the following sets of coordinates upper left lower right upper right lower left top bottom left right In the following example the upper left and lower right buttons are used to define the montage extent 5 Navigate to the upper right corner of the region that you want to image by either moving the stage controller s joystick or using the up down left and right arrows that appear in the Focus Controls 6 Click on the Al button The x y and z coordinates of that position will appear in the XY tab s list of points with a UR designation next to it 7 Now search for the lower le
35. Hand LO iio a tos at e a Ecal 29 LAA E E A A a 30 Hd MO E E a tales 31 ZO O a E 32 2 4 GENERATING A THREE VIE Wi sto 33 25 SPAWNING A DIEE VIEW sado 35 2 6 PERFORMING VOLUME RENDERING cut dida 36 2 CREATING MASKS AND GENERATING STATISTICS cnenocnrosnnnnnaara ninia donaci n 39 2 7 1 Creating a Mask using Threshold Techniques oococnnnnnuuuononononononanananononanannn nono non nono nn nono nn non anno nora aaa aaa aaa 39 2 7 2 Creating Masks Manually Background Subtraction Example ooonnnnnininininininnnnannanananannnannnnrn non n ono nn nono 41 vi 3 Table of Contents LID DMEM LSO ES ts isiican 45 SLIDEBOOK ORGANIZATION ci ici 52 3 1 HARDWARE CONTROL AND IMAGE CAPTURE CHAPTERS 5 6 AND 7 cccccceeeeseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 52 A OCASO MOS a ie oleae bak D2 Jl NINA ia eS A Vall Sl al GA laa te 32 IAr IR A A A eee 32 3 2 DATA STORAGE DISPLAY AND ANALYSIS CHAPTERS 8 9 AND 10 ccccccesseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 52 SAT A ala etadlateeheet cia cieatieceltad Deda dlatabty tats tnt cant 53 OZ VA CWS seta tec aot dai as Gia aati sh tetas niacin isin ba ie tsetse 53 Ir TAS SA In A a ite tie eleadat 53 Se SSH CN sidiaie inde a ere cia tlie alessio ak lca paty ahd lst a actaabad ah s ad deotoah bak aint ah delta hts Ae dd cele at 53 CONFIGURING YOUR SYSTEM eissoes eeno eo onnee a o eaaa e a oaao ane a are Eako ron a oE Raa sas 54 4 1 DESIGNAN SERIOS one ade blo dd io 54 4 2 CONFIGURING HARD
36. Mask gt Mark Selection to fill in your selection To remove the box choose Mask gt Unmark Selection o P single pixel pencil click and hold the mouse button to generate a single pixel width freehand line NOTE the resulting line 1s guaranteed to be continuous no gaps as long as you have the mouse button down e Small pencil click and hold the mouse button to generate a thin freehand line o de Large pencil click and hold the mouse button to generate a thick freehand line e P Small eraser click and hold the mouse button to remove small amounts e Y Large eraser click and hold the mouse button to remove large amounts Paint bucket fill a shape by single clicking within any closed shape drawn with the small or large pencil Additionally you may generate a 2D or 3D rectangular volume by first defining a volume as discussed in Making a 2D or 3D x y z or x y t Selection on page 186 then choosing Mask gt Mark Selection Use the tool to add or remove pixels The selected pixels will be shown in blue If you are working with a 3D or timelapse image and would like to copy your selections to other planes in the image select Mask gt Copy This Plane The following dialog box will appear 227 SlideBook 5 0 User Manual Copy mask lt planes lf Copy mask in current z plane to f all planes all subsequent planes f the next i planeja C planes E through 43 Timepolnts ES timeponts s
37. Mask Menu Axis Menu Set Default Show Graphs View View Settings Pixel Intensity Values Zoom Controls A QuickTou wi 1 Metap su 3 Cell 2 a Red Menu 200 6 113 0 0 380 Green Menu Plane Selection 219 FIT 205 DAF Blue Menu NONE Tool Men X Y Z T Position Zoom Controls Channel Menus Pixel Intensity Values El Up and down buttons Tool Menu Mask Menu 170 Background Menu Shows and continuously updates x y z 3D images or x y t timelapse images position of the mouse pointer The location follows x y z or f order regardless of the invisible axis that is displayed Displays and manipulates the magnification or zoom factor of the image Allow the user to select the channels that correspond to R G and B or monochrome displays default display corresponds with filter configuration definitions Defining Filter Configurations on page 64 Shows and continuously updates pixel intensity values corresponding to the mouse pointer location for a given channel Main View used to navigate through the invisible axis Three and Channel View used to navigate the z or f axis 4D Tile View used to navigate z axis Clicking once will move the displayed plane by one holding continuously will scroll through the planes Not used for 2D images Allows the user to choose the function that the mouse and its cursor perform as described in the section Using the Tool Menu This menu has
38. Note that the mask persists throughout the entire image Close the Segment Image dialog box by pressing the OK button Change the red channel from Cy3 to None Observe the mask that is displayed Click on the mask menu button Kal in the tool bar and select None The mask has been removed from the display Select Mask 1 from the mask menu The mask is now displayed To delete the mask select Mask gt Delete The following dialog box will appear Chapter 2 Quick Tour f Delete from current image C Delete from all images in current slide Delete mask hd ask C Delete all masks Cancel 14 Click OK to delete the mask from the image 2 2 Creating Masks Manually Background Subtraction Example SlideBook also offers a variety of tools that can be used to manually select regions of interest Note that this example illustrates multiple features of SlideBook dE 2 Click on the Main View of the Fibroblast image Go to the middle of the focus range by selecting View gt Go to Plane entering 7 in the edit box and selecting OK Next select Create Empty Mask from the mask menu or go to Mask gt Create The following dialog box will appear Create New Mask f Inthe curent image Cancel Click OK to create a new mask for the current image Often when you perform analysis you would like to specify a region to be used for background subtraction This can easily be done using the
39. Offset and Binning pixels Image Bin Factor Width i392 Height 1040 y 1x1 256 m Xx Offset 51 Y Offset lo Full Chip You may select to add yellow guides in the test view that correspond to 256 x 256 512 x 512 and 1024 x 1024 pixels By default SlideBook reads from an area centered on the chip with the provided dimensions To add the guides you must select the Guides checkbox in the Focus Controls KE Camera 1 Test Camera 0 29 4 92 Chapter 6 Image Capture and Import 2 If you want to move the region that is read to some other location on the chip enter offset values for both the x and the y axes in the X Offset and Y Offset fields 3 Select Test to update the Live View so that 1t has the desired size 6 2 2 Using the Test View Alternatively you may choose a region of the chip to be imaged by directly selecting 1t from the Image Window 1 Be sure that the E button at the top of the window is depressed 2 Click and drag the mouse over the desired portion of the test view 3 Select Update to register the new image size 4 Select Test to update the Live View so show the new image size Clicking on the Full Chip button will automatically select the maximum extent of the camera NOTE After you have selected the area to be imaged you may wish to adjust the exposure times using the Test and Once buttons to take advantage of the full dynamic range of the camera 6 3 Entering Image I
40. Reference position posman position Available 1 61 GB Required 0 00 KB Available 1 61 GB Required 0 00 KB Available 1 61 GB Required 0 00 KB 30 Capture 3D Capture 30 Capture Lise current Range um s e maths Range um ls e ee Range um ls osition position position Planes s Planes 5 lca ref Planes 5 Use reference i Use reference l ie reference position Step Size um 1 position Step Size um 1 position Step Size um 1 Use top and as Use top and se yr Use top an O P 2 bottom positions Offset um J 2 5 bottom positions Offset um Jo bottom positions Offset um I V Range around reference Return to reference position after capture Range around reference TF Range around reference Return to reference position after capture Return to reference position after capture Fig 7 3 This figure shows how Method 2 is performed when a reference position is selected in the Focus Window Note that the offset is different for the two situations on the right 7 2 Timelapse Capture In order to perform timelapse capture first bring a sample into view and focus as described in the Using the Focus Window section of Chapter 5 on page 85 Then proceed as follows 7 2 1 Setting Capture Preferences Before beginning a timelapse capture you may alter your capture preferences to do the following e Change capture frequency for a single channel in a multi channel timela
41. Scale change the size of the movie with respect to the original image dimensions o Width Height manually enter the dimension you wish to export e Movie frames per second adjust the speed of your movie Once you have selected the appropriate parameters select Export A standard Save As dialog box will appear Type in a filename and then click Save and you are finished 199 SlideBook 5 0 User Manual 9 Preparing an Image for Analysis or Export Before performing statistical analysis on your images you may wish to perform a varlety of pre processing operations In this chapter you will learn how to Manipulate Individual Channels Crop an Image Align an Image Manipulate Timelapse Data Perform Flat Field Correction Perform Photobleach Correction Perform Background Subtraction Create a Projection Image Create an Interpolated Image Apply Filters Perform Deconvolution 9 1 Manipulating Individual Channels A channel denotes the image data collected for a specific fluorophore SlideBook allows you to manipulate channels prior to performing quantitative analysis on your images You may insert a channel from one image into another image remove a channel or create new channels using channel math For timelapse images you may also create a timelapse composite image The timelapse composite channel adds the previous timepoints to each timepoint Thus timepoint 3 in a timelapse composite channel is the sum of ti
42. Slider PolyTech Pl M ShortioveSerialD elay EnableLongD elay Shothovel TLD elay LongMovel TLDelay Prior PYOAM Olmaging Properties SAC Sensilam Sigma Koki Simultaneous Capture SlideBook Info Statistics Properties SuperPro Network Settings Sutter 10 2 Settings Sutter 10 3 Settings Chapter 7 Advanced Capture Property Information Mame ShotMoveS erallelay Category PolyT ech Pl Description The number of milliseconds to Walt after a seral stage move 25 me default Appl Change 7 vrs Default alue p Revert To Default Close 7 Click on EnableLongDelay the right side of the dialog will update Edit Hardware Properties Leica CTR Leica DM 000 Lifetime Ludi Info Marzhauser Corvus Memory Cache MAL Fiber Switcher Nikon TE2000 Ocular Lightpath Olympus OP Camera Olympus DSU Settings Olympus MAL Olympus TTL TIRE Slider Fal Tech Pl ShorthMoveS erialD elay EnableLongDelay Shotklovel TLD elay LongMovel TLDelay Prior PYCAM Qlmaging Properties SAC Sensilam Sigma Koki Simultaneous Capture SlideBook Info Statistics Properties SuperPro Network Settings Sutter 10 2 Settings Sutter 10 3 Settings E E E 6 E E 6 E E E E 8 Select Yes and then Apply Change Property Information Mame EnableLongDelay Category PolyT ech Pl Description Should a serial move of larger than 2 microns increase the stage delay This i
43. The graphs can be explored as described in Creating Graphs to Monitor Regions of Interest on page 120 NOTE You can add ROIs by using any of the ROI tools to draw regions Please see Creating ROIs and Graphs to Monitor Regions of Interest on page 118 for more information on creating and deleting ROIs 252 Appendix A Smooth Curve Analysis Appendix A Smooth Curve Analysis Kymograph Smooth curve analysis allows you to follow time dependent lateral intensity changes along a static object For example an action potential moving along a neural spine could be displayed as an x vs t image using smooth curve analysis You could then extract velocity and displacement information from this image SlideBook s Smooth Curve Analysis is a mathematically rigorous and flexible operation with several adjustable parameters These parameters are defined using the following figure 84 86 88 90 92 94 Figure Al Representation of Smooth Curve Analysis Kymograph The above figure 1s a schematic of a pixel map of a 2D image When performing kymograph analysis you first define a curve along a static object that will serve as a backbone for your analysis Once this curve is defined you may perform a smooth curve analysis Smooth curve analysis uses the following parameters e Spline parameters Knot Interval discrete pixels the default is set to five A spline is fit between two knots Multiple splines are used to
44. a montage image e Load allows you to load a saved x y z point list e Save allows you to save an x y z point list e Home allows you to calibrate your x y stage before loading a point list Using these features in the context of preparing to capture an image is also discussed in Chapter 7 Advanced Capture NOTE If your system has a motorized xy stage but not motorized z focus then the XY tab works the same way except with xy rather than xyz locations 5 1 5 Camera The Camera Tab lets you control various settings on the CCD camera as well as view a histogram of the pixel intensities for the image in the focus window scope z xv Camera stream LCD Temp 112 Info NA Scale Image Displa g p ap Speed lo e Low E Gain o l High 65535 Intensification 0 O Mas 9044 65538 M J W Show Full Dynamic Range E Optional Param lo 5 1 5 1 Histogram The histogram shows a plot of pixel intensities values for the focus window image Pixel intensity values are plotted on the x axis while numbers of pixels at a given intensity are plotted on the y axis In addition the minimum maximum and mean pixel intensity values are reported beneath the histogram This data 1s continuously updated when the camera is actively acquiring images The histogram can be operated in two modes as shown in the Scale Image Display region of the window Manual and Auto In manual mode you may drag the red and green bars to
45. be used If your microscope has an automated brightfield lamp the lamp will be switched on and off or shuttered for each brightfield image that is acquired Also if you have a motorized condenser turret you may select whether a transmitted light channel is brightfield darkfield DIC Nomarski or phase If you check the Prompt if mode is not available for current objective checkbox SlideBook will automatically warn you if objective and filter definitions are inconsistent For fluorescent channels input the emission wavelength in microns in the edit field You should also indicate whether the fluor requires a UV objective SlideBook will automatically check for consistency between objective type and fluorescent channel type and prompt you if there is a problem NOTE You MUST have condenser turret positions defined for objectives if you would like the condenser turret position to correspond to a specific transmitted light channel see Adding a New Objective 4 4 2 2 Filter Set SlideBook allows you to assign a particular filter to one of six groupings or sets The default sets are Widefield Confocal Transmitted TIRF Life Time and FRAP You may also edit these set names as shown on page 64 These sets can be chosen in the Focus 65 SlideBook 5 0 User Manual Controls and the Capture Controls Sets allow you to work with many different filter definitions in an orderly fashion 4 4 2 3 Filter Positions SlideBook permits filte
46. before the image 1s captured NOTE If you see these prompts in error you need to make sure that you do not have positions defined for hardware components that are not motorized For example 1f you have a filter cube reflector turret that 1s not motorized make sure that your filter definitions are designated Unmounted in the filter turret position 2 Move the hardware into position and click OK The Live View will show the image in color as 1t is being acquired If you have more than one channel selected for capture it will show a combined image Thus if you have designated your DAPI and FITC filters to display as blue and green respectively the Image Window will first display the DAPI image in blue and then overlay the FITC image in green In addition to the Image Window updates the Capture Controls will become active and report the progress of your capture Capture Controls Status FRAP Notes Live Stages Mean 1006 Scale Display Manual Auto l Show Full Dynamic Range Next Capture 00 00 02 Time Remaining 00 00 07 Elapsed Time 00 00 05 Capturing channel DAPI timepoint 2 of 12 Graph Channels Regions Select All Set Background Cancel Pause The default Status tab of the Capture Controls displays the current channel in monochrome The features of the Capture Controls are best utilized when performing timelapse imaging and are discussed in Timelapse Capture on page 112 Chapter 6 Image Captu
47. button Alternatively you may click and release once at the start point and click and release at the end point The length of the line that you have just drawn will be displayed in microns in the info tool bar Note that these distances cannot be saved to disk from within SlideBook In order to perform measurements that are to be saved see Chapter 10 187 SlideBook 5 0 User Manual 4 To remove the line from the view click once anywhere on the view 8 4 3 2 Making a Measurement on a 3D or 2D Timelapse Image l 4 Open a Three View of the desired image by clicking once on the thumbnail image and then selecting View gt Three View Click the mouse button on the image at the point where you would like to begin measurement This point may be in any of the three panes Click the mouse button on the image at the point where you would like to end measurement Again this point may be in any of the three panes The length or velocity between the two points will be displayed in microns in the info tool bar area Note that these distances can not be saved to disk from within SlideBook In order to perform measurements that are to be saved see Chapter 10 To remove the line from the view click once anywhere on the view 8 4 3 3 Making a Measurement on an Imported Image 1 2 Import the image as discussed in Importing an Image on page 96 Make sure that the objective definitions exist by selecting Edit gt Defin
48. f 1x1 2x2 U dxd f 8x8 Enter the exposure time and bin factor for the uncaptured auto focus channel These parameters will be used to determine image capture for the purposes of autofocusing Click the OK button and proceed with setting up the image capture Now the channel that is being used for autofocusing does not need to be exposed during the capture series Selecting Expose uncaptured auto focus channel also provides the ability to specify a region of interest that will be used for auto focusing This is useful for auto focusing on samples that have features with heterogeneous optimal focal planes In this case a full chip image extent can be captured at each timepoint while using only a small subregion to determine optimal focus Specifying an auto focus region of interest 1s done in the Capture dialog box Extent Offset and Binning pixels Image Bin Factor Width 256 Height 256 f Autofocus ixi oF x Offset Y Offset d Chin White Balance E E oe When the Expose uncaptured AF channel checkbox is selected in the Capture Preferences dialog box the Autofocus radio button is active in the Extent Offset and Binning pixels section of the Capture dialog box This menu is used to alternate between designating the image extent for captured channels and the image extent for auto focus To define a subregion for autofocus simply select the Autofocus radio button and specify a Width Height X Offset and Y O
49. fit a given curve The knots are shown in pink in the figure above e Measurement dimensions 253 SlideBook 5 0 User Manual o Radius Perpendicular to Curve pixel units shown as dimension r2 in the figure above o Radius Parallel to Curve pixel units shown as dimension rl in the figure above o Increment Along Curve pixel units center to center distance between rectangular areas used to calculate smooth curve In the above figure the boxes are drawn every two pixels units along the curve The resultant smooth curve analysis with have half the resolution of the original image Once these parameters are defined the smooth curve algorithm averages the areas at each timepoint and creates a new image in two dimensions distance in x vs time in y 254 255
50. following file types are supported by SlideBook e SlideBook Spool spl e TIFF e TIFF Sequence SlideBook Spool is the file type used when capturing images to disk Please see Saving Images to Disk Spooled Capture on page 153 96 Chapter 6 Image Capture and Import 6 6 1 Importing SlideBook Spool Files To import a SlideBook Spool file 1 Select the destination for import by either opening a new slide or opening an existing slide file You may do this by selecting File gt New Slide or File gt Open Slide If the slide is already open make 1t the active window by clicking on it 2 To import an image select Image gt Import gt SlideBook Spool The following dialog will appear Look in mM SlideBook 5 0 do E Name Date modified Type de Exceptions 3 21 2009 4 59 PM File Folder Je Global Preferences 3 21 2009 4 59PM File Folder are 4 59 PM _ File Folder 1 File name Capture 220090321_181635 0 Files of type lideBook Spool Files spl 3 Navigate to the desired spool file and select Open The Load Spool File dialog will appear 97 SlideBook 5 0 User Manual T 8 Load Spool File Load Load time points m to 4 Mar 4 Memory Size 13 MB Cancel aD Pre process Sub sample E f del 2x2 ded BE Tr Dual View Image Mame Capture 2 Comments Capture Info Date Unknown Capture type Channel 1 Channel 5 Channel 2 Channel 6 Channel 3 Channel
51. gt Ocular Photo switch Fluorescence lightpath hardware gt Filter wheels gt Filter turret gt Shutter Automated xy and z stages Image Splitters Transmitted brightfield ightpath hardware gt Shutter gt Lamp gt LCD filter TTL controllable hardware Chapter 4 Configuring Your System When you start SlideBook for the first time it will notice that a preferences file which is stored along with the application does not exist SlideBook will create a default preferences file As you configure your hardware and define optics SlideBook will update this preferences file with specific information about your setup 4 2 1 General Hardware Configuration To configure your hardware 1 Start SlideBook by selecting the program from the Start menu 2 Select Edit gt Hardware Configuration The following dialog box will appear Hardware Configuration Cameras System name Last modified 02 24 2009 11 41 Camera 1 No Camera M Fluorescence Lightpath Camera 2 No Camera Excitation wheel hone at or v Camera 3 No Camera m Filter turret hone at off v Camera 4 No Camera h Emission wheel None at of X Shutter None v at Off Stage ND Selection None at of X xY Stage None x at of y Altemative Source None z at lor y Primary Z Stage None at log m Transmitted Lightpath Auxiliary Z Stage one at lor Shutter None v at lof gt Microscope Automation
52. image by selecting Mask gt Copy This Plane The following dialog box will appear 241 SlideBook 5 0 User Manual Copy mask lt planes aa planes jo through aooo Timepoirts i Copy mask in current timepaint fall timepoints f all subsequent timepoints C the next i timepaints s C Emepoint p through 23 5 Use the radio buttons to select the desired action and click OK 6 Select Mask gt Advanced Operations gt Smooth Curve Analysis The following dialog box will appear Smooth Curve Analysis Generate Image Measurement dimensions Radius perpendicular to curve peel units i Radius parallel to curve piel unita 1 Increment along curve pixel units Spline parameters Enot interval discrete pixela 5 Cancel 7 Adjust the dimensions as necessary please see Appendix A for a detailed description of Smooth Curve parameters and select OK A new image will appear in your slide 8 Open a main view of the Smooth Curve Image and select the y axis from the axis menu B on the info tool bar You are now able to display a 2D view of your 3D 242 Chapter 10 Using Masks for Image Analysis selection You may perform velocity measurements of timelapse data with the ruler tool see Making Distance or Velocity Measurements on page 187 10 6 Generating and Exporting Mask and Object Statistics You can answer many quantitative questions about a mask through mask statistics SlideBook
53. in Exporting Views on page 196 The scale bar will be present in the exported image This image can then be further modified using a drawing program 8 5 Using Display Views Creating Renderings and Movies Besides data views SlideBook can generate two types of movie displays volume renderings and series movies Both must be created from an open data view Main Three Tile or Channel View and will use the same color assignments and renormalization values as that data view The volume rendering will be generated and displayed from within SlideBook while the series movie must first be saved and then later viewed in QuickTime The volume rendering can also be exported and viewed using QuickTime 8 5 1 Displaying a 3D or 4D Volume View Volume Rendering SlideBook can perform interactive volume renderings using the 3D Volume View The following rendering types are available When you go to View gt 3D Volume View two options will be available High Speed and High Quality High Speed will render the image at the highest speed possible High Quality will render the image with a more rigorous algorithm that may improve the quality of the image with a small decrease in the speed performance These setting apply to all modalities of volume rendering but are most noticeable when using Dynamic Lighting mode Examples of the various modes of volume rendering are shown below e Dynamic Lighting allows you to illuminate your object as you wish by
54. in the invisible plane as well To illustrate this point move to different z positions in the image using one of the methods outlined above Notice that the marquee selection only appears in the plane in which it was originally drawn With the marquee region still selected go to View gt Select Rect in All Planes Again scroll through the different z planes in the image Notice that now the marquee selection exists in all planes of the image Click once on the image to remove the marquee from the view NOTE There are other ways to select 3D regions that will be demonstrated later in the tour 2 3 3 2 Zoom Tool The zoom tool is used to expand or shrink the size of the window and the corresponding zoom factor of the displayed image 1 2 28 Click on the tool menu and select the zoom tool Click on the Main View image two times zooming in on the image Note that the percent magnification is displayed in the upper left hand corner To zoom out on the image simply hold the shift button down while the zoom tool is selected and click on the Main View image Chapter 2 Quick Tour 4 Alternately you may click on the and buttons or utilize the drop down menu in the upper left hand corner of the view window as illustrated below 206 Era 184 FTE b3 DAPI None 4 QuickTourWIN1 Metaphase B Cell 2 ef200 6 06 30 1600 200 400 2 3 3 3 Hand Tool The hand tool lets you move
55. in other programs 8 6 2 Exporting a 3D or 2D Timelapse View as a TIFF or TIFF Series Three dimensional Main Views may be exported as a single TIFF file containing multiple planes or as a series of TIFF images l 2 Open a Main View of the image and adjust the look of the data as desired Select View gt Export gt TIFF to export the view as a single TIFF tif file or View gt Export gt TIFF Series to export the view as a series of TIFF images one image for each z or t plane Enter the file name and select Save The TIFF image s can now be used in other programs 8 6 3 Exporting Default Views of All Images in a Slide SlideBook allows you to use a single command to export a series of tiff files one for each image in the slide Each TIFF file will be a single plane that has the same look as the thumbnail image default view To use this feature i Alter each image to the desired look as described in this chapter This may include altering the color display and renormalization parameters or cropping the image Select View gt Export gt Default Views of All Images as TIFFs Choose a directory for the files and select OK The TIFF images will be saved under the individual image names in the chosen directory and can be used in other programs 197 SlideBook 5 0 User Manual 8 6 4 Exporting Volume View and Surface View Movies You may wish to export your volume or surface views so that they may be displ
56. in the Microscope Step Size field When you import files from other applications SlideBook will assume a default interplane spacing in microns that is defined in the Default Import Spacing field 76 Chapter 5 Controlling the Camera and Microscope Hardware 5 Controlling the Camera and Microscope Hardware Focus Window SlideBook provides a Focus Window for automated hardware control and semi live camera readout The Focus Window will allow you to choose the appropriate optics and bring your sample into focus It will also allow you to set parameters for advanced capture including top and bottom z limits for 3D imaging xy coordinates for multipoint acquisition and boundaries for a generating a montage This chapter covers the following topics e Focus Window Features e Using the Focus Window The use of the Focus Window for advanced capture is discussed in detail in Chapter 7 5 1 Focus Window Features The Focus Window can be opened by either selecting Window gt Focus Window or clicking on the focus window button in the toolbar fal The Focus Window consists of a Focus Controls dialog box and the Live Window which operates in a live mode when the Focus Window is selected The Live Window is also used for capture as discussed in Chapters 6 and 7 Focus Controls Scope 7 XY Camera Stream Objectives Emission Selection Stage Limits 2 10 Dry 0 Air l C Confocal Lamp owns ADs Air ji E Wide
57. lets you compute three types of mask statistics statistics for the entire mask statistics for each timepoint in a time lapse image and statistics for objects defined above on page 223 SlideBook allows for a wide range of statistics to be generated from simple to sophisticated 10 6 1 Counting Objects in a Mask You may wish to count the individual objects in your mask If you do not wish to obtain other statistics you may count objects by generating objects see page 229 You may also get an object count by generating the Object Count statistic see next section This 1s especially useful if you wish to count objects for multiple images in a slide 10 6 2 Generating Statistics for Masks and Objects SlideBook allows you to generate a wide varlety of statistics for regions of interest in 21 and 3D data To generate statistics from an open view of an image 1 Display the mask of interest by selecting it from the mask menu 2 Select Statistics gt Mask Statistics The following dialog box will appear Mask Statistics Image 5cope Features Output Grouped By O Compute Category E 0 Date OF Capture none C E 0 Morphometry none H E Intensity none Description f Time Point H 0 Cross Channel none f Entire Mask f Object Export All Images in Slide with the Same Mask Name Export Only Primary Mask M ask 1 Cancel Objects will be automatically defined Remove objects small
58. lower bounds on the size of the regions that should be made into objects 3 If you would like to set limits on the size of the objects select Gate Objects by Size and choose a minimum and maximum size The size bounds can be specified in either voxels or cubic microns square microns for a 2D image 4 Select the Generate for all similarly named masks in current slide checkbox to perform the object definition operation on the entire slide NOTE This process does not create multiple masks but rather assigns a number to each isolated contiguous subregion of the existing mask 5 select OK A dialog box will appear that gives the status of the object generation When object generation is complete a dialog box will appear noting the number of objects generated When objects are defined for a 2D timelapse image SlideBook will display the maximum number of objects generated at any given timepoint SlideBook i The maximum number of objects created in each image Image 0 48 6 Click on OK If you now position the cursor over one of the objects in the Main View the object number appears to the right of the cursor location coordinates in the info tool bar 230 Chapter 10 Using Masks for Image Analysis 7 If you would like to remove objects select Masks gt Update Object Definitions The Define Objects dialog will appear as it did when selecting Masks gt Define Objects in Mask 8 Select Gate Objects by Size and
59. marquee tool Click and drag over a region that would be suitable for calculating the background intensity Select Mask gt Mark Selection and notice that the region is highlighted in blue Al SlideBook 5 0 User Manual 5 QuickTourWIN2 Fibroblast 1 o O sa ES 100 Y 113 18 7 488 Cy3 285 FITC gt 208___DAPI_ e t a i ead fice None 7 This mask only exists in the current plane To copy this region to multiple planes go to Mask gt Copy This Plane The following dialog box will appear Mask Copy Plane Copy mask in current plane to f all planes f all subsequent planes the next ia plane s f planes E through 14 Cancel 8 Leave the all planes radio button selected and click OK 9 Scroll through the image and note that now the same masked region is selected in all planes 10 You can use SlideBook to get statistics from masked regions Go to Statistics gt Mask Statistics The following dialog box will appear 42 Chapter 2 Quick Tour Mask Statistics Image Scope Features Current 2D 3D Image Compute Category C Date Of Capture none C Morphometry none C Intensity none Description C Cross Channel none Mask Scope Display C Entire Mask Object Export Primary Mask Mask 1 Cancel Objects will be automatically defined Remove objects smaller than i 0 O O C All Images in Slide with the S
60. me Independent Channels DAFI at 0 mes Independent 7 Enter image information as outlined in the following section 8 Click OK after you have entered the information The imported image will appear as a Main View You may always go back and edit the image information by selecting Image gt Get Info 6 7 Getting and Editing Image Information After Capture You can get information about an image after capture or import The Image Info dialog box gives you the name of the image comments associated with the image and the time and date of capture if the image was collected using a digital microscopy workstation as opposed to imported It also gives you the optical settings as well as which filter configurations were used to collect each channel along with the exposure time If you would like to add optical information to an imported image you must first define the objective magnification changer and filter configurations as described in Chapter 4 Configuring Your System You may view and edit information as described below 6 7 1 Images that were captured in SlideBook 1 Open a Main View of the desired image by either double clicking on the thumbnail in the slide file or by clicking once on the thumbnail and then selecting View gt New Main View 2 Select Image gt Get Info The following dialog box will appear View and edit the Image Name and Comments fields as desired 101 SlideBook 5 0 User Manual Image N
61. oO through st 8 Use the radio buttons to select the desired action depending on your capture type and click OK 9 To further refine your mask you may select a variety of operations by selecting Mask gt Mask Filter The menu items are described below You may use these operations in any order or as many times as desired e Erode Applies a kernel to the entire mask that removes a user defined number of voxels from the perimeter of the mask This function can be applied to in only the x y dimension 2D or in the z dimension 8D as well e Dilate Applies a dilation kernel to the entire mask and thickens features The user defines a radius of the number of pixels the current mask will be expanded by and whether the mask should be dilated in 2D or 3D e Skeletonize Reduces the mask to a set of connected junctions This function can only be applied to single plane 2D images NOTE These operations do not create new masks but simply edit the current mask 10 2 2 3 Moving the Mask You can shift the mask in any direction by selecting Ctrl arrow keys 228 Chapter 10 Using Masks for Image Analysis 10 2 2 4 Copying a Manually Created Mask to Other Planes or Other Images Once you have created a mask you may choose to copy the mask to other planes in the image if you have a 2D timelapse or 3D image To do this 1 Select Mask gt Copy This Plane The following dialog box will appear Copy mask Z planes i Copy
62. on Hamamatsu cameras the parameters menu allows you to select the camera offset 5 1 5 7 Test Dual View The Test Dual View check box is used with systems capable of performing dual camera simultaneous capture It is also available when SlideBook is configured with an image splitter such as the Optical Insights Dual View see Defining Filters when using an Image Splitter on page 72 and Configuring an Image Splitter on page 60 This option is only available when multiple cameras or an image splitter have been configured in SlideBook When this box is checked the focus window display will rapidly alternate between images from camera 1 and camera 2 5 2 Using the Focus Window The following general procedure describes how to use the focus window to focus on a sample If the microscope that you are using is not automated you will need to move the optics manually Procedures for using more advanced features of the focus window are detailed in Chapter 7 Advanced Capture 1 Open the Focus Window by either selecting Window gt Focus Window or clicking on the focus window button in the toolbar The Focus Capture Window will begin updating as soon as the Focus Window is opened 2 Choose the desired objective and magnification changer from the Scope tab 85 SlideBook 5 0 User Manual Choose the desired filter configuration from the Focus Controls below the Scope tab Place the sample on the stage Click on Open Fluor or Open Bri
63. on any of your cameras in the Camera tab and the prisms will move to direct light to that camera Additionally if you have defined filters for specific cameras the prism positions will move to the appropriate camera when you select a filter in the Focus Controls 61 SlideBook 5 0 User Manual 4 3 Defining Objectives Once an objective is defined it can be controlled if the objective turret is motorized from the focus window If the objective turret is not motorized it may be selected in the capture window and the objective definition will automatically be stored with the image information If the objective was used to capture an image that has been imported the objective information can be added to the image information 4 3 1 Adding a New Objective In order to define a new objective for your system do the following 1 Select Edit gt Define Optics gt Objectives The following dialog box will appear Objective Parameters Objective Lens HA Parameters Name 10s Dry yh Medium Magnification ho le Air C pil Numerical aperture N 4 0 3 Add elsa Remove a Cancel C water Transmitted Light Configuration Brighthield condenser Unmounted turret position Working distance in um dk 5200 um pisel at 18 mag changer h 295 Position I Parfocality and Parcentricity Parfocal offset cam eyel 0 Darkfield condenser Unmounted gt turret position DIC condenser turre
64. out on the image display Zooming in on the display can be particularly useful for focusing on small objects Selecting the Guides check box will bring up yellow guides that mark a 256 x 256 512 x 512 and 1024 x 1024 pixel region in the image The Color check box is used to display a color brightfield image in the focus window Use of this feature requires specific hardware for capturing color images such as an LCD color filter changer or color camera 5 1 1 2 Open Fluor Open Bright Open Alt The Open Fluor button is used to toggle the fluorescence light source if there is a computer controlled shutter or dark slider in the reflected light path The Open Bright button is used to toggle the brightfield light source if there is either a computer controlled shutter in the transmitted light path or the microscope has computer control over the transmitted light source voltage The Open Alt button is used to toggle an alternate source if it is present For example if you have a NEOS AOTE laser system or a shuttered TIRF laser Open Alt will toggle the laser light on and off 5 1 1 3 Filter Controls You may select a filter set using the filter set drop down menu Each button corresponds to a filter configuration that you have defined for the system For more information on creating new filter configurations please refer to the section Defining Filter Configurations on page 64 If filter configurations have been assigned to a particular filter
65. select None Note that there are no data displayed in red Next set all color menus to the same channel FITC and observe that the image is now displayed in black and white A QuickTourWIN1 Metaphase B Cell 1 o E oHm 138 192 0 p H 155 ejt m aa 10 35 15 Try changing the settings for each menu noting how the display changes Reset the red menu to CY3 the green menu to FITC and the blue menu to AMCA To the left of the channel menus are colored numbers These numbers correspond to the intensity of the voxel that the cursor is positioned over for each channel Move the cursor over the image displayed in the Main View Note how the intensity values increase as you move over brighter regions and decrease as you move to darker regions Visualize just the DNA by setting the red green and blue menus to DAPI As you move the cursor over the image note that the red green and blue values are equal Chapter 2 Quick Tour 8 Close this Main View window by clicking on the X in upper right corner One Slide View and one Main View window should now be open 2 3 2 Up Down Arrows Scrolling through a 3D image The Dividing B Cell image is actually a 3D data set containing 50 planes captured at 0 2 micron spacing You may navigate through the stack in a variety of ways 1 Click on the remaining Main View Scroll through the z axis using the small arrow buttons lon the far left of the in
66. set the min and max values that are displayed in the live image You may also enter numbers in the Low and High edit fields In auto mode the live image will use the minimum and maximum intensity values of the live image to set 84 Chapter 5 Controlling the Camera and Microscope Hardware the contrast in the image Thus if you increase the exposure time the live image will update and automatically reset the contrast of the image to match the range of intensities in the live image You also have the option to display the full dynamic range of the camera using the Show Full Dynamic Range checkbox When this box is checked the histogram will not show the minimum and maximum intensities of the image but rather the minimum and maximum possible intensities based on your camera s readout 5 1 5 2 CCD Temp On systems with a camera that has electronic temperature readout this field reports the current sensor temperature in degrees Celsius 5 1 5 3 Speed On cameras that support this adjustment the CCD read out speed can be adjusted using this dropdown menu 5 1 5 4 Gain On cameras that support this adjustment the gain can be set using this dropdown menu 5 1 5 5 Intensification On cameras that support this adjustment the intensification factor can be adjusted using the slider and edit field located below the speed menu 5 1 5 6 Parameters This dropdown menu allows for support of additional features on certain cameras For instance
67. systems with a motorized emission light path these radio buttons let you choose between letting the light go 100 to the camera 100 to the oculars eyepleces or to 50 each if available 5 1 2 2 Magnification Changer On systems with an encoded magnification changer this field reports its current position 80 Chapter 5 Controlling the Camera and Microscope Hardware 5 1 2 3 Objectives On systems with a motorized objective turret you can move to a different objective by selecting the corresponding button For information on adding objectives to those defined for your system please refer to the section Defining Objectives on page 62 If you try to move between objectives that use differing immersion media such as an oil objective to an air objective SlideBook will bring up a dialog box warning about the pending move CAUTION On most systems with motorized objective turrets you can safely move between objectives of similar immersion media e g air oil However there are situations where you will definitely not want to move between objectives automatically For instance if you are using a water immersion objective on an upright microscope where the objective is dipped into the chamber trying to move the objective could break the chamber or damage the motorized turret 5 1 2 4 Stage Limits On certain microscopes with motorized z focus drives stage limits are used to control the maximum and minimum position of the st
68. the Filter Configuration Parameters dialog box The components of this dialog box and instructions for adding modifying and removing filter configurations are explained below 4 4 1 Defining Filter Sets SlideBook allows you to assign a particular filter to one of six groupings or filter sets These sets can be chosen in the focus window and capture window The default sets are Fixed Live User 1 User 2 User 3 and User 4 Sets allow you to work with many different filter definitions in an orderly fashion To change these default group names 64 Select Edit gt Hardware Properties The Filter Configuration Parameters dialog box will appear Scroll down to find the property Filter Set Information and click on the sign to expand as shown below Edit Hardware Properties 8 E E E H H H H H H E E H H H H H Diagnostic Instruments Camera Properties Dongle Info Dummy Camera Dummy Camera 2 Dummy Camera 3 Dummy Hardware Filter Set Information S Filter Set 1 Label i Filter Set 2 Label Filter Set 3 Label Filter Set 4 Label Filter Set 5 Label Filter Set 6 Label Focus Window FRAP Parameters Hamamatsu Properties Hardware Properties Version Info Laser Launch Test Leica CTR Leica DMs000 Lifetime Ludl Info Marzhauser Corvus MATLAB Verification Memon Cache MAL Fiber Switcher Mikon TEZO00 Ocular Lightpath Olympus OP Camera Olympus OSU Settings Property Information Mame
69. the following voxel dimensions Current Capture Type Pixel Size square microns 0 11 f All images with Same Channels Z Step 2 00 Recommended expansion sizes Selected Images Capture 1 Timepoint 1 Sy haie 1 2 Ratio 19 Expansion you want to pertorm EY Ratio i Z Ratio E Cancel 215 SlideBook 5 0 User Manual The dialog box recommends expansion parameters based on the calibrated pixel size and z step size used for capture These recommended parameters will generate an image with the approximate dimensions of the actual sample You can use the recommended expansion parameters or enter custom values in the edit boxes 3 Select the Image Range as described below You may also select and deselect images using the checkboxes in the Selected Images list e Current Image Operation will be performed on the selected image only e Current Capture Type Operation will be performed on all images in the slide with the same capture type 2D 3D 4D etc as the selected image e All Images with Same Channels Operation will be performed on all images in the slide with the same channels as the selected image 4 Select OK and a new image s will be generated NOTE The um pixel value for the objective that was used must be accurate for this feature to work properly Before generating the isotropic image check the image information to make sure the um pixel value is set properly see Getting and Edit
70. the image s masks A new data view can be spawned either from the slide or in one case from another view Display views on the other hand are always spawned from a data view and are set to the channel mask and renormalization selections of that view Data views focus on interactive analysis while display views focus on visualization This chapter covers the following topics Introduction to Slide and Image Display in SlideBook Using Data Views to Display Images Manipulating Data Views Using the Info Tool Bar Using the Tool Menu Using Display Views Creating Renderings and Movies Exporting Views 8 1 Introduction to Slide and Image Display in SlideBook When you start SlideBook a new slide called Slidel is created and unless you open another slide 1t 1s into this slide that all new and imported images are inserted Typically a slide corresponds to all of the images taken from a particular sample A QuickTourWIN2 lo El kE Capture Type El Fibroblast DAPEDMA FITC Tubulin C 3 Phalloidin 7 31 2003 19 44 39 3D Capture 3 678x431x15 25 1MB 0 B HeLa Cells mitotracker red sytox green 10 20 2001 17 52 44 2D Capture 2 488 x 422 604 4 KB 0 Each image in a slide 1s represented in a slide window as a thumbnail and some associated data The image may be either a single image or a set of images depending on the type of capture that was performed e g 2D 3D or timelapse The thumbnail is either a shrunken renderin
71. the image and allows all of the tiles to be readjusted simultaneously 90 SlideBook 5 0 User Manual Close the Tile View Open a Tile View from the Main View by first clicking on the Main View to remove the marquee selection and then selecting View gt Tile View Notice that the entire image space is now displayed NOTE In order to display a Tile View of a portion of the image from the Main View you must first define a 3D selection Close the Tile View You may close the slide by either clicking on the X in the upper right hand corner or by selecting File gt Close Slide 2 6 Performing Volume Rendering SlideBook can perform interactive volume renderings using the 3D Volume View The following rendering types are available When you go to View gt 3D Volume View two options will be available High Speed and High Quality High Speed will render the image at the highest speed possible High Quality will render the image with a more rigorous algorithm that may improve the quality of the image with a small decrease in the speed performance These setting apply to all modalities of volume rendering but are most noticeable when using Dynamic Lighting mode 36 Dynamic Lighting allows you to illuminate your object as you wish by allowing control of the lighting angle Useful for determining surface characteristics Fixed Lighting uses a fixed lighting pattern for shading approximates surface characteristics MI
72. type 2D 3D 4D etc as the selected image e All Images with Same Channels Operation will be performed on all images in the slide with the same channels as the selected image 8 Select the desired rotation angle and select OK The image will be rotated as specified 8 3 5 Changing the Invisible Axis SlideBook allows for 3D and timelapse data to be viewed in all possible dimensions By default a Main View will display the x y plane invisible z axis for 3D and t axis for timelapse However you can change the invisible axis to the x or y axis if you wish To view your image with a different invisible axis 1 Open a Main View of the desired image as discussed above axis from the dropdown menu The up and down arrows in the info tool bar will be tied to the new invisible axis 179 SlideBook 5 0 User Manual NOTE The axis menu is not available in the Tile View or Channel View If you would like to view a different invisible axis in the Tile View you should first open a Main View change the invisible axis make a 3D selection and then spawn a Tile View as described above Displaying a Timelapse Tile on page 164 8 3 6 Scrolling through the Invisible Axis in a 3D or Timelapse Image There are several ways to scroll through the invisible axis in Main Views Channel Views and 4D Tile Views e e Using the up and down arrows in the info tool bar one click on either arrow will move one plane up or down holding conti
73. you select Apply only the current image will be segmented e Current Image Segments the current image only e Current Capture Type on OK Segments the current capture type only For instance if you have a 2D timelapse image open in a slide with at least one other timelapse image as well as other capture types only the 2D timelapse images would be segmented e All Images with Same Channels on OK Segments images that have the exact same number and type of channels For instance if you wish to segment a FITC channel and one image contains a FITC channel while another contains both DAPI and FITC both images will NOT be segmented Only the active image will be segmented NOTE All images in the slide must be of the same size for this operation 6 Set the thresholds for segmentation You may do this in one of two ways a Move the red and green bars to the right and left by clicking and dragging them Note that when you release the mouse button the image will be segmented and the mask will be displayed OR b Enter intensity values in the Low and High edit fields then click Apply to register the change and move the red and green bars SlideBook has selected or masked any pixel that has intensity values that he between the red and green bars in all of the three channels In other words segmentation is performing an AND operation across the bounds defined in all channels 7 If working with a 3D image scroll through th
74. your graph will remain open for viewing 4 To export your graph simply select the icon The regions of interest that you have created can be displayed as discussed in Displaying Annotations on page 181 To recreate the graph post capture please see page 251 7 2 5 2 Focusing During Capture You may adjust the focus of your image during capture To do so go to the Live tab The Live View works similarly with the Capture Controls as it did with the Focus Controls NOTE Before using the Live tab you must first select Pause Once you are finished focusing you must select Continue to resume capture The window has the following features 121 SlideBook 5 0 User Manual Capture Controls status FRAP Notes Live stages Z Stage z Stage XYZ Position Adjust the Z position using software or hardware When you ei the new position Update xy E m Filter Filter TxRed Go Captured Images Show oe Shutter Buttons 7 a Next Capture 00 00 08 Time Remaining 00 16 38 Elapsed Time 00 00 02 Capturing channel TxRed timepoint 1 of 100 Graph Channels Regions e _ Show Red y DO Select All _Set Background e Start Stop begins or ends the semi live camera readout e Snap snaps a single image e Shutter Buttons toggle fluorescence brightfield or alternate source shutters open and closed e Filter moves motorized filters into position when you select Go e Z Stage allows you to refo
75. 0 planes of the 3D image acquire the midplane channel and then acquire the remaining 5 planes of the 3D image To have SlideBook capture the midvolume image at the end of the 3D capture select the Capture midvolume plane at end of 3D acquisition checkbox When this option is selected SlideBook will capture the entire 3D volume return to the specified midvolume plane and then capture the selected channels 5 Select the midvolume channels that will be captured at each timepoint during the 4D capture In the edit box to the right of each channel specify the exposure time that will be used to capture the channel Importantly channels that will be 126 Chapter 7 Advanced Capture captured at midvolume planes should not be selected for exposure in the Capture dialog box when setting up the 4D capture parameters 6 Click OK and then proceed to the Capture dialog box to set up for 4D capture as described above 7 3 3 Multi Channel Z Series In order to speed up capture you may wish to capture multi channel z stacks one channel at a time 1 Goto Capture Preferences dialog box by pressing the Advanced button in the Capture dialog box 2 Select the 4D tab from the capture preferences dialog box The lower half of the dialog box appears as follows Multi channel Z Series f Capture all channels at each z position AB move z AB move z AB C Capture one channel at a time for all z positions A for all z then B for all z m m
76. 04 Color C Opacity 194 Chapter 9 Preparing an Image for Analysis or Export To display or remove surfaces simply check or uncheck the appropriate channels in the Surfaces list You may also change the color of the surface by selecting the color box for the corresponding channel You may also change the width of the outline using the Outline Width slider 8 5 3 Generating a Physically Proportional Rendering of 3D Data You may notice that your object may appear disproportionately small or squashed in the z direction When performing 3D imaging each plane that is captured is represented by a set of pixels in the image set Each pixel is a perfect cube You will notice that the z dimension in the 3D rendering appears to be different than what was actually captured This is because the z step size that was selected during capture is not actually the same as the length of the z dimension of the pixel In the image set planes are simply stacked on top of one another with no regard for the actual z distance traveled this is done in order to preserve data fidelity To generate a proportionate rendering 1 Open a data view Main Three Tile or Channel View of the image as discussed above 2 Alter the renormalization parameters and color display as desired see above 3 Interpolate between planes and expand your z dimension to represent the actual z distance traveled by the microscope by generating an isotropi
77. 31 e Rental You may not rent lease or lend the SOFTWARE PRODUCT e Support Services 31 may provide you with support services related to the SOFTWARE PRODUCT Support Services Use of Support Services is governed by the 31 policies and programs described in the user manual in online documentation and or in other 3I provided materials Any supplemental software code provided to you as part of the Support Services shall be considered part of the SOFTWARE PRODUCT and subject to the terms and conditions of this EULA With respect to technical information you provide to 31 as part of the Support Services 31 may use such information for its business purposes including for product support and development 31 will not utilize such technical information in a form that personally identifies you e Software Transfer The initial licensee of the SOFTWARE PRODUCT may make a one time permanent transfer of this EULA and SOFTWARE PRODUCT only directly to an end user This transfer must include all of the SOFTWARE PRODUCT including all component parts the media and printed materials any upgrades this EULA Such transfer may not be by way of consignment or any other indirect transfer The transferee of such one time transfer must agree to comply with the terms of this EULA including the obligation not to further transfer this EULA and SOFTWARE PRODUCT e Termination Without prejudice to any other rights 31 may terminate this EULA if you fail to com
78. 4 Channel FITC LP y e Manual V Low 0 High 1874 Automatic Pidler Calyard Current image Mask Name Mask 1 Apply Close 3 Drag the red bar to the right until the low threshold value is approximately 550 As you drag the bar note the masked areas that appear on the image Also note that the data that is being selected is shown in white on the histogram Pixels with intensity levels that lie in the gray area are not included in the mask Alternatively type 550 in the Low data entry field and select Apply to set the low threshold value Your dialog box will appear as follows 45 SlideBook 5 0 User Manual Segment Image Dj Channel FITC LP f Manual fw Low 550 w High 1874 f Automatic Ridler Calyard f Current image Mask Mane Maski 4 Select OK to create the mask 4 QuickTourWIN2 HeLa Cells 1 o E E S 366 11 0 200 Cra 178 FITELP None More 5 Next select Masks gt Define Objects in Mask The following dialog box will appear 46 Chapter 2 Quick Tour Define Objects Size Filter Generate for all similarly named masks in curent slide Cancel Here you may choose to exclude objects based on minimum and maximum size gates Leave the box unchecked for now 6 Select OK A dialog box will appear that gives the status of the object generation When object generation is co
79. 5 PERFORMING AUTO WHITE BALANCE FOR COLOR CAPTURE ccccccccccccssssssssssssecacccceceeeeesessessstssseenaaaaees 95 6 6 IMPORTING AN IMAGE ds 96 6 0 Importing SlideBook Spool Files ssi is 97 O22 A A aia Cat ales 98 6 7 GETTING AND EDITING IMAGE INFORMATION AFTER CAPTURE 00s0ssesseesssseeseseesseseeeeeeeseseeeeeeeeseeeeees 101 6 7 1 Images that Were captured in deb O eels es ead Meese tle cd veldlles Uae eee tt 101 OZ AI OFL CE TI OOS chs ci oe ea lel e tao Atel ls do Pal alata 104 7 ADYANCED CAPTURE a A A e 105 7 1 I IR OT Ordo ER 105 Td iio AAA A Pe o td GAN SEATTLE AS REAM TANT 105 7 1 1 1 SEL OCASO a a e eee ae 105 HloL Z SELLO Capiure W imdOw Parameters A he ete arencans 107 7 1 1 3 Setting Capture Preferences to Open and Close Shutter during Capture oooccccccccnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnos 108 E E A E RON 109 7 2 TIMELAPSE CAPTURE AAA A as 112 DA SUNE APE PFS CICS Es 112 7 2 1 1 Pertormino Periodic Capa diet 113 Tala Opening and Closing Brightfield Shutter Between EXpOSULES ooooooncccononoooooooononon ono nnnnnnnnnnnnnnnnnnnnn ono nnnnnnnnnos 113 Pale Chaneine the Status Update Frequency ads 114 7214 AIO LOCUS During ee eat 115 Toshi Chaneoime Capiire Frequency ole 115 7 2 1 6 Savine Imanes dos e titi 115 ie Seine Capture Wind Ow IOMA osa 115 TDS CP CAI TIS NO ES AS AE E TOE aed oo ena 116 viii Table of Contents 7 2 4 Creating ROIs and Graphs to Monitor Regions Of Interest 118 7 2 5 Init
80. 7 Channel 4 Channel E Select the timepoints that you wish to load For large data sets it can sometimes be easier to load a subset of data Select if you would like to subsample the data This may speed up processing for large data sets but will decrease the image resolution If applicable select pre processing and Dual View parameters Name the image and add comments Click Load The image will be added to the active slide 6 6 2 Importing TIFF Files To import individual TIFF files or TIFF sequences 98 L Select the destination for import by either opening a new slide or opening an existing slide file You may do this by selecting File gt New Slide or File gt Open Slide If the slide is already open make it the active window by clicking on it Chapter 6 Image Capture and Import 2 To import an image select Image gt Import and select the file type that you wish to import If there are already images in your slide file and one is selected the following dialog box will appear for TIFF files but not TIFF Sequence Image Import Import this file as f A new image planes o A new channel in the current image Cancel Select whether you would like to import the file as a new image or as a new channel in the current image and click OK 3 The following dialog box will appear r 44 Open Look in F Images t ex Ey E Name Datetaken Tags Size Rating ra Recent Places MB i
81. AY IN SLIDEBOOK coooooocccccccnnonononcnnnccnnnnonnnnnnncnnnnnnnnncnnncnnnnnnnss 156 Sdk WOKO WUDLU e cates ise Sn cist ce A es 157 8 1 1 1 Savio des do 157 8 1 1 2 OpeninoSldes tt tl A 157 8 1 1 3 lOs MS US IS A a assi 1 57 8 1 1 4 Ds E R AE E A EEN 157 8 1 1 5 Chanomo the Slide View Wisplay unid dd a a e a doo 158 liza VOPR NIN E A E E E SN 158 8 1 2 1 DE LECH ING an IMA a 158 8 1 2 2 Removing an Image from One Slide and Placing it in Another ooccccccnnnnnnnnnnonnnnnnnnnnnnnnnnnininininininininanns 158 SlideBook 5 0 User Manual 8 1 2 3 Op TINS An SNA OS arora eet gar ces oes en ines A o OO 159 8 1 2 4 Deletino an NAG cdt 159 else sh IACI ITS O vo PENA RE OE O O OOO cara E E ata hentai tet 159 8 2 USING DATA VIEWS TO DISPLAY IMAGE Stella aa A 160 62 1 Displaying Main VTA needa A E 160 022 DiIsPlayinea Three VIGW alii 162 6 23 Spawnino a THE Wi 163 8 2 3 1 Displayinoa D Dile Vid tl ty Se an 2 ld e e bs al e e dd 163 8 2 3 2 Displayine a Timelapse Rat n 164 8 2 3 3 Displaying a 3D or Timelapse Tile View of a Portion of the Image ccccccccccnnnnnnnnnnininnnnnnnnnnnananannnnnnnanannns 165 8 2 3 4 Arraneino your HS VISWE a a 166 8 2 4 Displaying a Multidimensional Channel VieW ooooooonnnnococonononononnnonnnnnnnnnnnonnnnnnnnnnnnonnnnnnnnnnnnnnnnnnnnnnonoss 167 8 2 4 1 Multidimensional Channel View SEUA as 167 Sd DPIN CAMOM A A T E E 169 8 3 MANIPULATING DATA VIEWS USING THE INFO TOOL BAR ccooooccccccconnoconoconanencncno
82. Background 4 Select the Show button next to Captured Images and the following window will appear 123 SlideBook 5 0 User Manual Captured Images Single Frame Single Channel TxRed t Location 1 Time Point 2 _ Po ool Plane 0 E Pol 5 Use the slider to scroll through previously captured images or enter a specific timepoint into the edit field and select Go You may also use the and buttons to move one timepoint at a time 6 If you would like to view a single channel select the Single Channel radio button and the desired channel from the dropdown list 7 The default mode is Single Frame If you are capturing a 3D stack over time you may also choose MIP to view a maximum intensity projection of each z stack at any timepoint 8 Once you are finished viewing images close the dialog box by clicking on the X in the upper right hand corner of the dialog box or selecting Hide from the Live tab 7 3 4D Capture SlideBook allows you to capture 3D images over time To set up for 4D capture simply set up for 3D capture and timelapse capture as described in the above sections then make sure that both the 3D and Timelapse checkboxes are selected in the Capture dialog box and the appropriate parameters are entered in the edit fields before selecting OK The resulting slide file will contain a single 4D image that includes all of the z sections and timepoints NOTE If
83. Capture Preferences Spool Sequence Notes General TTL 4D Focus Periodic FRAP 0 30 Capture lf Open and close shutter on single channel or simultaneous 3D capture Altemate two channel mode ABBAABBA instead of ABABABAB Live Capture Captured Image Updates C Suppressed When Possible Always fastest TUTTI TTT TT TTL TTT TT ewes if Update histogram every B frames i Update displayed frame every BS frames if Update current plane every E frames Suppress TTL optimization of hardware devices a Automatically restore focus and image capture dialogs at conclusion of capture You may suppress all updates for optimal speed performance To do so select the Suppressed radio button If When Possible is selected updates will be provided when the computer processor allows Some updates will be dropped in the interest of speed If Always is selected each frame requested will be displayed at the expense of speed 7 2 1 4 Autofocus During Capture Please see the section Autofocus on page 144 7 2 1 5 Changing Capture Frequency Please see the section Varying Capture Rates During Timelapse Capture on page 149 7 2 1 6 Saving Images to Disk Please see the Saving Images to Disk Spooled Capture section on page 153 7 2 2 Setting Capture Window Parameters 1 Open the Capture dialog box by selecting Image gt Capture or by selecting the capture icon in the SlideBook toolbar dal 115 SlideBook 5 0 User Manual
84. Create Segment Mask Alternately you can reach this dialog by selecting Mask gt Segment The following dialog box will appear Segment Image 152 Channel DAFI Manual W Lowe 182 e High 1545 Automatic Pidler Calward f Curent image Mask Mame Mask 1 Apply Close 4 Select Apply The Generating Mask dialog box will appear and report the segmentation progress by plane Once segmentation is complete your entire image will have a blue overlay SlideBook has selected or masked any pixel that has intensity values that lie between the red and green bars in each of the three channels 39 SlideBook 5 0 User Manual 40 10 11 12 13 Select Cy3 from the drop down menu Move the red bar to the right by clicking and dragging it Note that when you release the mouse button the image will be segmented again with the new parameters and the mask will be updated Set the low threshold edit box to 550 and click on Apply 5 QuickTourWIN2 Fibroblast 1 o E 100 675 430 7 503 Cy3 298 ___ FITC 202 ___DAPI None Channel Cy3 Manual Y Low 550 V High 2715 C Automatic Ri ller Calvard m Current image hd Wa Mask Name Apply Close OK Now SlideBook 1s masking any pixel with a Cy3 channel intensity value between 550 and 2715 Scroll through the image using the scroll wheel or the up and down arrows in the info tool bar
85. Default Settings Timestamps Scale Bars Lookup Tables Notes Object IDs Regions Text Color Font Ariel 14pt 183 SlideBook 5 0 User Manual 8 3 8 3 Setting Default Annotation Settings Once you have adjusted the Annotations to the desired display you can save the settings as defaults by going to Annotations gt Set Default Settings 8 4 Using the Tool Menu The tool menu of the info tool bar allows the user to choose the function that the mouse and 1ts cursor perform Tool Menu Marquee Tool E 4 Ca kc E ROI Selection Tool Hand Tool ROI Tools S Zoom Tool Ruler Tool a Sa N CE Angle Tool G The tool menu may also be accessed by selecting View gt Tool The various features are described in the table below ay Ov OO vi h Pointer Selection Tool Lets you select a 2D or 3D region of the image and is used when spawning a Tile View or a volume rendering Making a 2D or 3D x y z or Marquee Tool x y t Selection on page 186 This tool can also be used to make masks see Chapter 9 Lets you select an ROI in order to perform further operations Holding shift allows you to select multiple ROIs See Creating ROIs and Graphs to Monitor Regions of Interest on page 118 for more information on working with ROls This tool is also available from the SlideBook toolbar Allows you to draw rectangular ROls on an open view by clicking and dragging This and all other ROI drawing tools are ava
86. Feel free to distribute the manual to all users of SlideBook 1 7 Installing Updates You may download software updates at www intelligent imaging com as described above in Copying Software from your Download Site Before installing an update be sure to back up your configuration files To backup your files go to Edit gt Setup Guides gt Backup Preferences You will be prompted for a location If you use the defaults settings a folder called Global Preferences will be created in the SlideBook 5 0 directory After downloading is complete double click on the file that you have just downloaded and follow the installation directions Install the update in Program Files Intelligent Imaging Innovations Inc SlideBook If SlideBook is not located in the above path refer to section 4 6 Working with SlideBook Preferences and Hardware Properties on page 74 22 Chapter 2 Quick Tour 2 Quick Tour This Quick Tour will introduce you to some basic elements of SlideBook By the end of this tour you will know how to do the following Open slides and images Display a Main View Three View and Tile View Use basic tools in the Info Tool Bar to Maneuver within a view Perform volume rendering Create a mask and obtain statistics NOTE Menu operations such as choosing Open Slide from the File menu are written as File gt Open Slide In order to run the Quick Tour first make sure that the SlideBook program and Qu
87. Filter Set 1 Label Category Filter Set Information Description Label for filter set 1 Value Widetield Apply Change Default Yalue Revert To Default Close Click on the Filter Set Label that you wish to change Enter the text for your filter set Examples may be a specific user name or a type of experiment Click Apply Change and then Close to register your changes Chapter 4 Configuring Your System 4 4 2 Filter Configuration Parameters The Filter Configuration Parameters dialog box is found by selecting Edit gt Define Optics gt Filter Configurations Filter Configuration Parameters Filter Configuration Parameters ane co Associated Channel Type Ratio 1 Numerator Light Source Ratio 1 Denominator Ratio 2 Numerator Ratio 2 Denominator Emission 0 457 RGE Color wavelength um FRET Donor Transmitted f Fluorescence Default Color Display Mode Unknarn f None Red Green m f Alternate Source f Blue Position Unmount f Pseudocolor Color f Pseudocolor Intensity Filter set Widefield ee E Excitation wheel position Unmounted Monochrome User defined E Internal turret position I Duk Emission wheel position Unmounted LCC BF filter position Unmaunted Ausiliary filter position Unmounted Camera Any W Requires UY objective 4 4 2 1 Light Source Here you may choose which light source will
88. G IMAGES ci n 221 10 USING MASKS FOR IMAGE ANALYSIS eeessseccccccsssscccecocscscccecccccsssececcocssssceccecsssscececossssceeceossssecceeeossse 223 10 1 DISPLAYING IMAGE HISTOGRAM AND MEAN INTENSITY ccccccccceceeeeessssessessecececccccceeanseassssssssseeeeeees 223 1072 CREATING MASKS AND OBIEC IS id aa 223 10 2 1 Creating a Mask using Threshold Techniques unnnnnnnnnnnnicnnonooooooooaonanoo nono nn nn nono non nn non ono nn nono nnnnnnnnnns 224 10 2 2 Creatine Mask Manta aa 226 10 221 Ereatinesan Empty Maso ia E 226 10 202 ait the Masie Manta A EAA 227 OA Movino He Was AAA O A 228 10 2 2 4 Copying a Manually Created Mask to Other Planes or Other IMageS ooooononnncnonononononooooononooonnn nono nonnnnnononnos 229 10 2 3 DESTA O ciao 229 10 2 4 SPUNE OD Se Oe 231 10 241 Manually Splitting OD cl in lisis 231 10242 Automatically Spl tine Objects ati dias 231 10 2 5 Removing Objects from Edge Of Image oononnnnnnnnnninannnnnnnnnanananannnananannnnananan ono n oran anar nn n nn nn nn nn nnnnnnnnns 232 10 2 6 TRACKING Objects Particle Tracking a ties 232 10 2 6 1 Automated Particle Tracking Tutorial ooonnnnnnnninininonnonoonoooonononononoonnno nono nono nono nono nn nro non nono nro nr iua ieii 232 10 2 6 2 Pertormine Manual Particle Tracker id 238 10 3 DISPLAVING OR DECETINGA MAS esca lua sita iio iaa 239 104 BOOLEAN MASK OPERA TON Sion 240 10 5 gt USING MASKS FOR SMOOTH CURVE ANALYSIS KYMOGRAPH
89. Image Before performing any operations on a given image 1t must first be selected or made the active image To make an image the active image click on an image thumbnail in an active slide window or anywhere in the box surrounding an image s information The icon and attributes of the selected image will turn blue It is on the selected image that all data manipulation commands such as deconvolution mask creation commands and image information commands will operate 8 1 2 2 Removing an Image from One Slide and Placing it in Another To move an image from one slide to another do the following 1 Click on the slide that contains the image you would like to move this makes it the active slide 2 Click on the image you would like to move this makes it the selected image 3 Select Edit gt Cut Image or click on the Cut Image icon X This will remove the image from the slide 158 Chapter 9 Preparing an Image for Analysis or Export 4 Click on the slide that will hold the pasted image 5 Select Edit gt Paste Image or click on the paste image icon B E NOTE Since the image 1s stored internally in a proprietary format these operations do not use the Windows clipboard In other words 1f you quit SlideBook without pasting a cut image you will not be able to get to that image again 8 1 2 3 Copying an Image You may duplicate an image and place it in another slide or in the same slide It 1s often useful to cop
90. Images list 5 Click OK to remove the channel s NOTE Removing a channel is an irreversible process You may wish to keep a copy of the original image or entire slide 9 1 3 Using Channel Math SlideBook allows basic mathematical operations to be performed on specified channels in an image This 1s appropriate when doing background subtraction or when calculating bleedthrough To perform channel math do the following 202 Chapter 9 Preparing an Image for Analysis or Export 1 Make sure that the channels that are to be used in the mathematical operation are present in a single image You may wish to insert a channel as discussed above 2 Select the image by clicking on 1t You may also start from an open view 3 Select Image gt Channel Operations gt Channel Math The following dialog box will appear Channel Math Scope f Current Image First Channel Coefficient Channel Offset i af sdGFP z o Note Batch Operations only include images that match the channels of the selected image e 4 es a E do fii no op Second Channel Result noo Waer dC Channel Name New Channel Herel cr 4 Setting the scope section of the Channel Math window allows channel math operations to be applied to multiple images in the slide Select one of the following options e Current Image Operation will be performed on the selected image only e Current Capture Type Operation will be per
91. M uuunnnnnnnnnnnnninnnnnonacananaann ono corno nro nono non nnnnnnnnos 73 AAF REMOVAL dt el lis 73 4 5 DEFINING VIAGNIFICATION CHANGES dde dd aas 73 a0 Addinsa New Macnijication CAMAS ii 73 4 5 2 Removing or Modifying Magnification Changer Definitions uunnnnnnnninininnnnnnnnnnnnnccacaaaaar nor nn nr nn nn nro nn nnnnos 74 4 6 WORKING WITH SLIDEBOOK PREFERENCES AND HARDWARE PROPERTIES cooooooccccccncnnononcnnnccnnnnnnnnnnnnccnnnnnnos 74 4 0 1 Backing Up SHACROOK COn oural on sccrwiec ccc ieuei E led elctda tate agile and EN 74 40 Retorne SHACBOOK OR SU a 75 NOS ENC PONS EE E E ROOT E Sec ete esld Galan Veet in Iain tea RL Adak tee act atae ome eae 75 4 7 DELNING SYSTEM PARAMETER Sie 0 onthe le BO ae heal ie ia A cee pl tu te SOLOS 76 CONTROLLING THE CAMERA AND MICROSCOPE HARDWARE FOCUS WINDOW ooooooooo 77 5 1 FOCUS WINDOW PE AMOR S dd del Dodd PIM CONTOS dd ios 78 51121 Camera and Display Se un GS sa c6ce acess scadt ica 78 sA Open Fluor OPen Brent Oper Altea uea tec epale lea della ae let ato aso lll la de 79 5 1 1 3 MERC a Ad 79 5 1 1 4 O A E ee etl eat arse he ect A A 79 ILES A OTI TEE TIEA EEEE I E EEIT EE EIE EE E EEE I EE T 80 5 1 1 6 Neutral Densi tc icalctlrcosin 80 vil SlideBook 5 0 User Manual IA A A O en Denn a are eO Ne Tem SU S21 27 Emission CC LUC saa A A A 80 5122 2 Masntiica Ln MAO CM patties eii 80 312 3 A E EA EAE 81 5 1 2 4 A T E EAEE AA ATA 81 5212 5 A A teed etal eat 81 5 1 2 6 o II ee tee enen coe
92. NOTE Views are only used to display and alter the look of an image The underlying data are not affected Now we will open a Main View 1 Double click anywhere on the thumbnail or within the shaded region that surrounds the thumbnail A new Main View window will open that displays the image 2 Create another Main View of the Dividing B Cell image by first selecting the image with a single click then selecting View gt New Main View At this point you should have three open windows the Slide Window and two Main View windows Both Main Views show the dividing B cell in three colors where each color corresponds to a different channel or fluorophore DNA appears in blue AMCA channel surface immunoglobulin appears in green FITC channel and tubulin appears in red Cy3 channel SlideBook QuickTourWIN1 File Edit Image View Annotations Mask Statistics Macro Window Help Osa x e el al ele lo ajololsiolsj de QuickTourWIN1 1 Image Comments Capture Date Capture Type Channels Dimensions g Metaphase B Cell DAPI DNA F 3 16 2000 15 13 34 amp QuickTourWIN1 Metaphase B Cell 2 100 0 0 Red if fa al a E Bkgnd None 3D Capture 3 189 x 194 x 50 5 QuickTourWIN1 Metaphase B Cell 3 fix 6 240 Hilela o at ice 85 Free Memory 44 Available Virtual Memory 1843 MB Data Cache 10 0 MB 24
93. Opacity Oo __ 100 of the gridlines using the Gridlines slider 9 You may alter the opacity of an individual channel using the Opacity slider control that corresponds to each color 10 Once you have explored this view you may close the slide by selecting the Xl in the upper right hand corner or by choosing File gt Close Slide For further information about volume rendering please see Displaying a 3D or 4D Volume View Volume Rendering on page 189 38 Chapter 2 Quick Tour 2 7 Creating Masks and Generating Statistics A mask 1s a two or three dimensional set of binary values that has the same extent as the image to which it is assigned Masks are often termed regions of interest ROIs in other imaging programs Masks are used for performing advanced selection and analysis of image data and can either be created and edited manually or generated automatically through threshold segmentation and other techniques A single image can contain several different masks We will now learn how to create masks using a variety of techniques 2 7 1 Creating a Mask using Threshold Techniques 1 Select File gt Open Slide and navigate to QuickTourWIN2 sldin the Manual folder within the S1ideBook directory A slide window will open that contains two images 2 Double click on the Fibroblast thumbnail to open a Main View of the image 3 Create a threshold mask by clicking on the mask menu icon E and selecting
94. Open User Preferences Folder Click inside of the Windows Explorer folder view that opens Right click and select Paste from the menu You will be informed that there is already a file in this location with the same name Select the option to overwrite all files or folders and click the box to apply to all files and continue Close all folder views and restart SlideBook 4 6 3 Exceptions Exceptions is a folder that is backed up with your system and user configurations This folder may contain a number of folders that begin with ExceptDump These files are created when a process is interrupted in SlideBook If your SlideBook session is interrupted and your data was not saved you can recover the data by looking in the Exceptions folder It may be useful to monitor this directory and delete unnecessary files 75 SlideBook 5 0 User Manual 4 7 Defining System Parameters You can adjust the z axis increment size the default interplane spacing the index of refraction of the oil you are using and the disk drive on which to put temporary files using Edit gt Define Optics gt System Parameters System Parameters Microscope step size um nl Default import spacing um 03 Oil index of retraction 1 518 Sistem Password Temporary File Storage Temp volume for Cl p Cancel For microscope automation to work correctly you need to enter the size of a single z axis increment of the stage in microns
95. P Displays the pixel of maximum intensity along the axis perpendicular to the viewing plane This type of rendering does not require an advanced graphics card however performance of certain functions will be improved with one X Ray Displays the summed intensity along the axis perpendicular to the viewing plane Choose File gt Open Slide and navigate to the Sample Files folder in the SlideBook directory Double click on QuickTourWIN3 sld The following slide window appears that shows a thumbnail of one image This slide contains one 3D image of a mouse mammary gland section a QuickTourWIN3 o O 3 Image Comments Capture Date Capture Type Channels Dimensions Size Masks Chapter 2 Quick Tour 2 Click anywhere in the area of the image in the list and select View gt 3D Volume View gt High Speed or High Quality depending on the desired mode NOTE If the image contains a marquee selection SlideBook will only render the portion of the image that 1s selected J 1 Bi QuickTourWIN3 Capture 8_Cropped Cl croppedk1 5 180 Y 002 00 degrees Red cya Dolly 1 000 Greer FITE a na Baja Blue O DAPI 10um grid To change the channels that are displayed use the channel menus as you would for the Main View Three View or Tile View To alter the shading grab the lighting tool by clicking on the icon in the upper right hand corner and rotating it to achieve the desir
96. Q TTL DAQ TTL DAQ TTL DAQ TTL DAQ TTL DAQ TTL DAQ TTL DAQ TTL DAQ Alternate Source Analog Input Ausilary Filter Brightfield Shutter Camera Ernission Filter Excitation Filter External Trigger Fluorescence Shutter FRAP Trigger LOD Filter ND Filter Y Stage Z Stage User Login Properties Enable User Login L Enable User File Names Volume Rendering avy Stage Yokogawa CSU Properties Zelis Asiolmager Heiss AvioObseryver Zels Asioplan 2 Asiovert 200M Zeiss MCU 28 Property Information Mame Enable User Login Categor User Login Properties Description Should SlideBook enable multiple uzer login mode Apply Change Default alue E Revert To Default Close 4 Restart SlideBook Upon startup you will see the following screen 55 SlideBook 5 0 User Manual h a slideBook User Login Default User Add User To add a user click Add User The following dialog will appear User Mame Cancel Enter the desired user name in the User Name in the edit field and click OK Upon restart choose your user name from the drop down list and then click Login Your capture preferences will be available in the Capture dialog 4 2 Configuring Hardware SlideBook has the ability to control a range of hardware including the following 56 CCD cameras Automated microscope components gt Objective turret gt Magnification changer turret
97. Remove Multiple Timepoints Current Image Untitled C Remove Timepaints O to E C Remove every 2 th timepaint starting at plane D to E C Keep every 2 th timepolnt starting at plane o to E f Remove timepoints Enter in a list of points such as 1 3 5 10 i Place result in new image Cancel 2 Enter the timepoint or group of timepoints that you wish to remove You may also choose to subsample the image by selecting Remove every nth timepoint or Keep every nth timepoint Alternately you may enter a list of points to be removed If you display timestamps you will see that the timepoints have been removed 3 Select the checkbox Place result in new image if you choose to do so then select OK If you do not choose to do so the timepoints will be irreversibly removed from the current image 9 4 2 Merging Timelapse Series If you have an experiment that was captured into multiple timelapse images you may merge these images by selecting Image gt Advanced Operations gt Merge Timelapse Images The following dialog box will appear 206 Chapter 9 Preparing an Image for Analysis or Export Merge 2D Timelapse Images Base Image Time Seres 1 Next Image Time Series 2 W Keep Original Images Cancel In the example dialog above Time Series 2 will be appended onto Time Series 1 and placed into a new image The original images will remain unchanged 9 5 Performing Flat Fiel
98. Slide ol SlideBook 5 0 User Manual 3 SlideBook Organization SlideBook was designed to seamlessly integrate microscope control and image operations such as capture display and analysis In this chapter you will learn about the organization of SlideBook elements The following topics will be covered e Hardware Control and Image Capture e Data Storage Display and Analysis 3 1 Hardware Control and Image Capture Chapters 5 6 and 7 In order to capture an image two distinct processes are performed First a sample is brought into view and focus using the Focus Controls Second image capture parameters are set and capture 1s initiated in the Capture dialog box Once the capture begins 1t can be monitored in the Capture Controls 3 1 1 Blrocus Controls In this window the microscope parameters are selected and moved into position if motorized A semi live stream of images are captured and displayed in the Focus Controls Additionally parameters for the XY stage such as points of interest and for the Z stage such as the depth for a 3D capture can be set in this window Once the hardware is set as desired and the sample 1s in focus the user will move to the Capture dialog box See Chapter 5 for further explanation of the Focus Window 3 1 2 capture Dialog Box Camera and capture parameters are set in the Capture dialog box The appropriate exposure time and capture sequence are determined in this window For instance
99. To open a Three View from an open slide 1 Click once anywhere on the thumbnail or within the shaded region that surrounds the thumbnail to highlight the image 2 Choose View gt Three View to display a new Three View 162 Chapter 9 Preparing an Image for Analysis or Export ed QuickTourWIN1 Metaphase B Cell 3 200 93 103 3 Hesla lali y Z A NOTE You may notice that your object may appear disproportionately small or squashed in the z direction When performing 3D imaging each plane that is captured is represented by a set of pixels in the image set Each pixel is a perfect cube You will notice that the z dimension in the 3D image set appears to be different than what was actually captured This is because the z step size that was selected during capture is not actually the same as the length of the z dimension of the pixel In the image set planes are simply stacked on top of one another with no regard for the actual z distance traveled this is done in order to preserve data fidelity You may interpolate between planes and expand your z dimension to represent the actual z distance traveled by the microscope by generating an isotropic image see page 213 8 2 3 Spawning a Tile View The Tile View can display many planes simultaneously Both the 3D Tile View and the Timelapse Tile View are designed to enhance interaction with 3D and timelapse data 8 2 3 1 Displaying a 3D Tile View The 3D Tile Vie
100. UIRED IN THE US AND CANADA 3I warrants that a the SOFTWARE PRODUCT will perform substantially in accordance with the accompanying written materials for a period of ninety 90 days from the date of receipt and b any Support Services provided by 31 shall be substantially as described in applicable written materials provided to you by 31 and 31 support engineers will make commercially reasonable efforts to solve any problem issues Some states and jurisdictions do not allow limitations on duration of an implied warranty so the above limitation may not apply to you To the extent allowed by applicable law implied warranties on the SOFTWARE PRODUCT if any are limited to ninety 90 days CUSTOMER REMEDIES 31 s and its suppliers entire liability and your exclusive remedy shall be at 31 s option either a return of the price paid if any or b repair or replacement of the SOFTWARE PRODUCT that does not meet 31 s Limited Warranty and which is returned to 31 with a copy of your receipt This Limited Warranty is void if failure of the SOFTWARE PRODUCT has resulted from accident abuse or misapplication Any replacement SOFTWARE PRODUCT will be warranted for the remainder of the original warranty period or thirty 30 days whichever is longer Outside the United States neither these remedies nor any product support services offered by 31 are available without proof of purchase from an authorized international source NO OTHER WARRANTIES To the max
101. WARE tdt lt ltda dls 56 4 2 1 General Hardware Configuration oooonnnnnnnoconnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnonennnnss 57 ADD Configuring a Motorized AY MATE ios 58 ADS Configuring CM Image Splitter sian zeis feo co pd Sal oiler hee elias a Sah hls x 60 4 2 4 Configuring Camera and Ocular Ports ooonnnnnnnnnnnunuooconoooonooannnononnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnininns 61 4 3 DEFNING OBJECTIVES expr Sees aca ea sad ag tae a ti 62 Jo SACAMOS iio N 62 4 3 2 Modifying Information for an Existing Objective ooooonnnnnnnnninocooooooooanoooooon nono nononnnnnnnnnnnnonnnnnnnnnnnnnnnnnnnnnns 63 Bede O 2 IPOD CCVE a etre A A acente e tieitee ee ais 63 4 4 ID ERINING FILTER CONEIGURA TIONS do del dedo 64 O O RA O ala ante aaa ta Do alc 64 442 Filter Configuration TOV AMAS o eal 65 4 4 2 1 A A A nee rete 65 4 4 2 2 A O 65 4 4 2 3 PEM Ct POSO a di di 66 4 4 2 4 CAM idos 66 4 4 2 5 Channel Types AA A EE 66 4 4 2 6 Detault Color Displays o a ds ln le la EE E 66 AAS ALAMILLO TAIANA ES 68 4 4 3 1 Example Filter Configuration for Fluorescence Channels occnnncnnnncnconnnoncnnnonnnnnnnnnnnnnnnononinoninnnnninnnnninncnns 69 4 4 3 2 Configuring Channels for Color Cameras or Color LCD sliders ooooonncccncnccnanananonananaoaonannononnnonnnrnnrr ono nn nnnnnnos 69 4 4 3 3 Defining Filters when usino an Image Split nba ee eee 12 4 4 4 Modifying Information for an Existing Filter CoONfIZUratiO
102. WARNING The background subtraction operation described above is an irreversible operation Please be sure to save a copy of your original data 9 8 Creating a Projection Image You may wish to view 3D data as a single 2D projection image To do so 1 Make the desired image the active image by clicking on 1t in the slide view You may also start from an open view of the image 2 Select Image gt Create Projection Image The following dialog box will appear 213 SlideBook 5 0 User Manual Create Projection Image Projection parameters Operation Als le Over Z asis Average Overa axis Sum 0 Overy axis Image range f Current image All images in slide Selected Images Capture 3 Timepoint 1 3 Select the Operation as described below e Maximum The brightest pixel throughout the chosen axis will be displayed e Average An average of all the pixels through the chosen axis will be displayed e Sum The sum intensity of all the pixels through the chosen axis will be displayed 4 Select the Axis you would like to compress to create the projection image 5 Select the Image Range as described below You may also select and deselect images using the checkboxes in the Selected Images list e Current Image Operation will be performed on the selected image only 214 Chapter 9 Preparing an Image for Analysis or Export e All Images in Slide Operation will be performed on all
103. a composite RGB image Users can also choose to display individual channels with their default coloring or to display multiple timepoints or z sections of the image To display a Multidimensional Channel View from an open slide 1 Click once anywhere on the thumbnail or within the shaded region that surrounds the thumbnail to highlight the image 2 Choose View gt Multidimensional Channel View to display a new Multidimensional Channel View 5 QuickTourWIN1 Metaphase B Cell 3 o 5 E 100 730 33 240 CY3 8 2 4 1 Multidimensional Channel View Settings The Multidimensional Channel View allows users the flexibility to create images for export from 2D 3D 2D timelapse or 4D data types This section discusses adjusting the Multidimensional Channel View Setting in order to maximize the information shown in the 167 SlideBook 5 0 User Manual exported image You may wish to choose a region before generating the Mulitidimensional Channel View in order to improve viewing ability see Making a 2D or 3D x y z or x y t Selection on page 186 To create a Multidimensional Channel View 1 Select an image in a slide by clicking on it 2 Select View gt Multidimensional Channel View A Multidimensional Channel View will appear 3 You may then alter the display of channels composite images and timepoints z planes by going to the View Settings icon in the View window show below or by going to View gt Multidime
104. ability the above limitation may not apply to you CONTRACTUAL STATUTE OF LIMITATIONS AND NOTICE To the maximum extent permitted by applicable law no action or claim relating to this Agreement may be instituted more than one 1 year after the event giving rise to such action or claim In addition you must notify 31 at least thirty 30 days prior to the instituting of any lawsuit under this Agreement with a full description of your claim and what you are seeking in damages DISPUTE RESOLUTION Any controversy or claim arising out of or relating to this Agreement or the breach thereof with the exception of injunctive relief sought by 31 for any violation of the proprietary terms of this Agreement shall be settled by arbitration in accordance with the rules of the American Arbitration Association Before entering into arbitration You and 31 shall each appoint an arbitrator and these two arbitrators shall select a third arbitrator to be a member of the panel Should the two arbitrators not be able to agree on a choice of the third then the American Arbitration Association shall make the appointment of a person who is neutral to the parties in controversy None of the arbitrators shall be officers or employees of the parties to this Agreement Such arbitrators shall be recognized experts in the computer software field The cost of arbitration including fees per arbitrator shall be borne equally by IV End User License Agreement the parties T
105. adapters with different relay lens magnifications you can also use magnification changer definitions to specify which relay lens is in place 4 6 Working with SlideBook Preferences and Hardware Properties SlideBook stores two different types of configuration files User Preferences and System Preferences User Preferences refers to settings that are user specific such as saved capture settings or saved particle tracking protocols System Preferences refers to system wide settings such as hardware configuration and hardware properties There are several tools built into the software to help you manage and backup preference files as discussed below 4 6 1 Backing Up SlideBook Configuration 1 To back up your SlideBook configuration go to Edit gt Setup Guides gt Backup Preferences the default behavior is to save the backup files file in C Program FilesiIintelligent Imaging Innovations Imc SlideBook 5 0 2 Select where you would like the backup files to be located If desired create and name a new folder 3 Click OK 4 Three folders containing system configuration will be placed in the directory designated The folders are as follows e Exceptions This folder contains backup data from interrupted sessions and will be discussed later in this section e Global Preferences This folder contains the system configuration files such as SlideBookHardwareProperties dat SlideBookPrefs dat PSFs dat and FlatFields dat e Use
106. age These limits prevent the microscope from being focused above or below certain z positions The Stage Limits radio buttons are used to enable or disable this feature 5 1 2 5 Lamp On microscopes with a computer controlled lamp voltage adjustment the intensity of the brightfield illumination can be controlled using this slider Moving the slider to the far left corresponds to turning the lamp off while moving the button to the far right corresponds to turning the lamp to maximum intensity 5 1 2 6 Condenser On microscopes with an automated condenser turret you may select condenser positions from the drop down menu 9 1 3 Z There are several possible ways to set up 3D capture using the Focus Window These methods are explained in further detail in Chapter 7 3D Capture on page 105 You must first choose the basis for your capture You may define your capture based on the current z position a selected reference position or by defining the top and bottom of capture Examples of using the Focus Window for 3D capture are as follows e Current z position Use the Focus Window to find a plane of interest for example the focal plane and then choose to range your capture around that position in the Capture dialog e Define a reference position in the Focus Window and then define a total range in microns or number of planes to capture in the Capture dialog 81 SlideBook 5 0 User Manual e Define a top and bottom position in th
107. ame HeLa Cells Edit Info Comments y mitotracker red svtox green r Cancel Capture Info Date 10 20 2001 17 52 Capture type 20 Capture Microns per piel par 0 319 Step size in microns Unknown Objective 20 Air Mag changer 1 0 Binning 1 1 Channel l FITC LP at 263 ms Independent Channel Cra at 25 ms Independent 3 Press the Edit Info button if you wish to change any of the optical configuration or collection information This brings up the following dialog box 102 Edit Image Info Capture Type 12D Capture Microscope Components Objective 20 Air Mag Chg 1 0 30 Collection Chapter 6 Image Capture and Import Interval mej 0 Optical Parameters F Microns pixel 0 648 Num aperature 0 5 Working distance 2000 Interplane spacing fum o Channels Filter Configuration Channel 1 Other FITCLP Channel 2 Other Exv 3 Lambda um 0 54 0 59 Channel 3 l i Channel 4 fF l I Channel 5 PF ol I Channel E f l 1 Channel 7 Do Channel 8 Dv Eta i Cancel TATE NOTE If you open a SlideBook file that was created on another computer or with a different SlideBook preferences version the filter and objective configurations will be displayed as Other To prevent this make sure that the SlideBook preferences match those that were used for capture 4 Select the Objective and Mag Changer used during captur
108. ame Mask Name Export Only FF Some advanced features require a secondary mask Secondary Mask None Remove objects smaller than 10 c c Clear 11 Check the Entire Mask radio button in the Mask Scope section and then expand the Intensity menu in the Features section of the Mask Statistics dialog box by clicking on the to the left of the checkbox Then check Mean Intensity and then click Display A table will appear that resembles the following f QuickTourWIN2 Fibroblast Image Fibroblast Mask Mask 1 Capture Date 07 31 2003 19 44 39 Mean Intensity ADU ch1 DAPI Mean Intensity ADU ch1 Cy3 Mean Intensity ADL ch1 FITC These statistics can be exported as a tab delimited file by selecting Export 12 To perform background subtraction choose Image gt Channel Operations gt Channel Math The following dialog box will appear Channel Math Scope f Current Image First Channel Coefficient Channel Offset ii ef DAPI 0 j Note Batch Operations only include images that match the channels of the selected image ie 4 D Ir de eo f no op Second Channel Result di DAPI Channel Hame oo Cancel 43 SlideBook 5 0 User Manual 13 Enter the mean background intensity value for the given channel DAPI to the offset data field for the first channel then select no op Enter a name such as DAPI Background for the new c
109. and a low pixel intensity for the second channel will appear as dim red An area with a low pixel intensity for the first channel and a high pixel intensity for the second channel will appear as bright blue 4 If you would like to gate the first channel by the intensity of a second channel select a second channel from the dropdown menu 8 3 2 4 Changing the Display to a User Defined Color Palette You can now display up to eight channels in colors of your choosing This is useful for applications such as fluorescence in situ hybridization FISH To use this feature 1 Open a Main View Three View or Tile View 2 Select View gt User Defined Color Your image will appear in the following format All channels that were captured will be listed in the channel list 175 SlideBook 5 0 User Manual A FISH example Image 6 1 lola 50x 6 1088 8 0 Y DAPI j f Ps ns ENTE 164 mV cy3 76 m V cys 3 To change the color of one of the channels click on the colored box next to the channel whose color you would like to change The following dialog box will appear Hue io Red 1179 Sat 240 Green o Lum e4 Blue 0 Add to Custom Colors 4 Tochange the color either click on one of the Basic colors in the upper left section of the dialog or click on one of the Custom colors in the lower left or create your own color on the right side If you would like to add a color to the Custom colors use the c
110. apture During 4D Imaging SlideBook offers the ability to capture up to three 2D mid volume images during each timepoint of a 4D capture This feature is useful for acquiring channels that often do not need to be imaged in three dimensions such as DIC or Fura 2 during a 4D capture To setup a 4D mid volume capture 1 Goto Capture Preferences dialog box by pressing the Advanced button in the Capture dialog box 125 SlideBook 5 0 User Manual 2 Select the 4D tab from the capture preferences dialog box The following window will appear Capture Preferences Spool Sequence Notes General TTL 4D Focus Periodic FRAP 4D Capture Capture midvolume plane at end of 3D acquisition Exposure ms ho Multi channel Z Series f Capture all channels at each z position AB move z AB move z AB C Capture one channel at a time for all z positions A for all z then E for all z r a 3 Select the Capture midvolume plane checkbox This will activate the edit box to the right allowing you to specify the plane at which a midvolume image will be acquired The midvolume plane must fall within the plane limits of the 4D capture as specified in the Capture dialog box 4 SlideBook defaults to capturing the midvolume plane during each 3D acquisition For instance consider the case of a 4D capture with a 15 plane 3D image being acquired at every timepoint If the midvolume capture plane is set to 10 SlideBook will acquire 1
111. ar larger than the size of the monitor which defeats the Tile View s purpose of allowing simultaneous viewing of multiple planes along the same axis So although a Tile View can be created directly from the slide window you will more likely be making a 3D selection in either a Main View or a Three View or even another Tile View and spawning a Tile View from there In order to spawn a Tile View from another view 1 Make the desired view the active window by clicking on it 2 Define the selection There are several ways to do this and they are discussed Making a 2D or 3D x y z or x y t Selection on page 186 3 Choose the desired Tile View from the View menu to display a new 3D or Timelapse Tile View of the selected portion of the image 4 To change the portion of the image that is displayed select the hand tool from the tool menu and click and drag in one of the panes see table on page 184 fora description of this function 165 SlideBook 5 0 User Manual You can create a Tile View that shows slices along any of the three axes simply by making your selection in a view or pane that 1s currently set to the desired invisible axis see description of the axis menu in the table below In other words if you make a selection in a Main View that has an invisible y axis and the selection spans ten units in the y axis the resulting Tile View will have ten panes that display the x axis horizontally and the z axis vertically
112. ard Time point A speed EU ome redmitochonea ed sdCY3 Time 00 00 04 0010 s 180 OOF 400 degrees Dolly 1 195 Greer sdFITC Total Time Blue None Tirmepoint 1 ajr i Timepoints 120 ALA A Loop y o Slaw Fast Time 00 00 000 Timepoint 1 10um and You may use any of the timepoint controls to move between timepoints or play the images like a movie Other controls such as rotate zoom hand axis and histogram function exactly as in the 3D Volume View 8 5 2 Displaying a 3D Surface View You may also display your image as a surface to do so click on a thumbnail in your slide and select View gt 3D Surface View A new window and corresponding renormalization dialog will appear 193 SlideBook 5 0 User Manual eo QuickTourWIN3 Capture 8 Cropped Cl cropped 1 E 180 7 00 4 00 degrees Dolly 1 000 e alo ja 10um grid This view has functions similar to the 3D Volume View The rotate tool hand tool zoom tool axis tool and lighting tool all function as described above in the section on 3D Volume View The 3D Surface View Settings function similarly to the Volume View Settings To change the intensity at which the surface is drawn adjust the bar on the histogram of pixel intensities 3D Surface View Settings Background Color 0 Black White Grid Lines 0 None Bright Outline Width 1 0 None 5 pixels Sutaces Threshold 4
113. as a manual objective turret you must make sure the proper Objective selected To view objectives in the focus window on a manual microscope go to Edit gt Hardware Configuration and select Manual Objective Turret from the Objective Turret dropdown menu Select the Auto Focus button from the Focus Window Z Stage Simulated 7 Stage 10 1 1 gt 45 429 urn 4 The Auto Focus dialog box is used to determine the optimal auto focus parameters for your particular sample Chapter 7 Advanced Capture Auto Focus Method Spectral O Templal E Total Search g Range Save TIFFs Save logfile Total Time 0 ms Post Focus p Offset Image Time 0 ma Peak Delta 0 20000001 Threshold Best 7 Objective 29 planes FD 0 2rum Filter at Threshold Cancel Exposure 300 5 Enter parameters into the edit boxes according to the following descriptions e Method The computational method used to evaluate and compare focal planes Changing the method can affect the success of the auto focus routine The default method of Spectral 2D Template will work best in almost all cases The other methods are experimental You should consult 31 1f you are not having success with the default method e Total Search Range The total distance in the z dimension that will be searched during the auto focus routine starting from half the specified distance below the current z position SlideBook will au
114. asks Perform Boolean Mask Operations Use Masks to Perform Smooth Curve Analysis Generate and Export Mask and Object Statistics Display Graphs 10 1 Displaying Image Histogram and Mean Intensity You may quickly get a statistical snapshot of your image using the Statistics gt Intensity Statistics function A dialog box will display a histogram of the intensities of the entire image by channel along with the mean intensity and standard deviation You must first open a view of your image before selecting the Intensity Statistics menu selection 10 2 Creating Masks and Objects A mask 1s a 2D or 3D set of binary values that has the same extent as the image to which it is assigned Once created a mask 1s permanently stored with its corresponding image Masks are used for performing advanced selection and analysis of image data They can elther be created and edited manually or generated automatically through threshold segmentation and other techniques Selecting Mask gt Create creates a mask with the first 223 SlideBook 5 0 User Manual method selecting Mask gt Segment creates a mask with the second These menua items can also be found under the mask icon in any View window A single image can contain multiple masks Often you may have several distinct objects that will have intensities that are very similar and thus will segment together A mask that contains several distinct entities may be divided into objects For instanc
115. at the eleventh plane is displayed 5 Close the Main View that you just created 2 4 Generating a Three View SlideBook allows you to view all three axes simultaneously using a Three View 1 Select View gt Three View to generate a new Three View 4 QuickTourWIN1 Metaphase B Cell 2 pu foe x io 200 182 2 0 00 Cra 150 HTE OO A new window will open with an xz pane on the top a yz pane on the left and an xy pane in the middle The most useful tool for exploring this view is the point selection tool 2 Choose the point selection tool icon in the tool menu 3 Click on the xy pane center pane of the Three View with this tool Notice that you update the xz and yz panes 33 SlideBook 5 0 User Manual 34 11 Hold the mouse down and drag 1t around the center pane to get a cutting plane effect Click and drag the mouse along the x axis of the center pane and note that the yz plane is rapidly changing while the xz pane 1s relatively still Next click and drag the mouse on the yz pane to the left and observe that the other two planes are being updated Now we will make 3D selections in the Three View using the marquee tool Select View gt Go To Plane Enter plane 20 and select OK Next choose the marquee tool from the tool menu in the info tool bar Select the chromosomes by clicking and dragging a rectangle around them Then go to View gt Define Selection Cube The followin
116. at you can also specify the direction of travel of the stage or nosepiece The stage and objective should always travel toward each other Thus the direction of the arrow should normally point upwards You may also choose to set either number of planes or the step size in the Capture dialog as shown in 3D Capture on page 105 9 1 4 XY If your system 1s equipped with a motorized xy and z stage the XY tab lets you specify store and visit multiple xy or xyz locations These points can be used for visiting multiple locations of interest in a sample and returning to them later as well as for specifying a sequence of locations to repeatedly visit during timelapse acquisition Scope Z xY Camera Stream ooo E Montage Extent 1803 0 1347 0 343 0 5409 0 4041 0 343 0 Visit Point Update e S 4 E Update Z Reset All Z Clear Point Clear All Edit Description Home e Set Point adds the current xyz location to the list e Clear Point removes the currently selected xyz location from the list 83 SlideBook 5 0 User Manual e Visit Point moves the stage and z focus to the selected xyz location e Clear All removes all locations from the list e Reset All Z updates the z positions in all points to the current z position e New Point Z updates the z position of the selected point to the current z position e Montage Extent sets the boundaries that will be used to create
117. aximum value for a pixel depends on the bit depth of the camera For instance on 12 bit cameras the pixel values can range from 0 to 4095 In order to take advantage of the full dynamic range of the camera it is important to try to get the maximum pixel value well into the four digit figures Pressing the Once button will automatically adjust the exposure time to try to achieve a maximum pixel value 91 SlideBook 5 0 User Manual of about 3000 In extremely dim samples this may not be practical but 1t 1s a good target value Conversely it is important for deconvolution that no pixel values are at the camera s maximum value as that means that the image is overexposed and past the linear range of the CCD detector Pressing the Once button will reduce exposure times if necessary to try to reach a lower maximum value 6 2 Selecting the Area to be Imaged Typical cameras contain either a 1392 x 1040 or a 512 X 512 pixel chip but often you will want to capture from a smaller portion of the chip in order to reduce the memory required for capture and or increase the camera frame rate You may select the area to be imaged in two ways 6 2 1 Using the Image Extent Menu The image area can be selected using the features in the Image Extent and Binning section of the Capture window 1 Select the x and y extent of the image from the Width and Height drop down menus You may also manually enter the desired values in the edit field Extent
118. ayed If left unchecked the movie will begin with a view with surfaces at right angles to the selected axis 198 Chapter 9 Preparing an Image for Analysis or Export o Axis relative to screen check this box if you d like the image to rotate around an axis relative to the screen If left unchecked the image will rotate around the image axis e Rotation Increment enter the desired rotation increment in the data field The rotation range that you selected is computed in the increments you provide Choosing a smaller increment increases the smoothness of the rotation and adds to the depth cue provided by moving the image Projection 1s an expensive computation so choosing a larger increment will make the projection process faster To generate a single head on projection of the entire image choose 860 e Rotation Range select 360 to rotate the data completely You may select a smaller range if desired e Show current timepoint only disable animation available for 4D images only Allows you to render a single timepoint to conserve time and memory e Timelapse Options select the timepoints you would like to render and how you would like your timelapse movie to proceed either step through time as the object is rotating or complete a rotation before proceeding to the next timepoint e Movie Dimensions allows you to change the dimensions of the movie o Keep Aspect Ratio keeps the current height width ratio o
119. ayed in a presentation or online publication You may export either as a QuickTime movie or as a QTVR QuickTime Volume Rendering file QTVR files can be rotated at arbitrary angles in QuickTime available at www apple com To export your Volume or Surface View 1 In the Slide View click on an open Volume or Surface View to make 1t the active view 2 Select View gt Export gt Movie The following dialog box will appear Movie Export Options Asie of Rotation Options Com fa CZ CO Both Y QTVA None Timelapse only I Start at current plot orientation i Axis relative to screen Rotation increment degrees i 5 Y Asis Range f 360 t From o i To oO Include Tool elements Show current time point only Disable animation Timelapse Options f All time points l Single rotation asis options C From l 1 f Rotate while stepping through time To EN f Cycle through time points then rotate Movie Dimensions W Export at recommended size 580 y ER Pixels Scale i i E qe Pixels Movie frames per second 4 canci The dialog has the following sections e Axis of Rotation Options you may generate either QuickTime movies where your volume or surface rotates around a single axis x y or z or QTVR movies where you may rotate your volume as desired using QuickTime o Start at current plot orientation check this box if you want to start your movie with the view that is currently displ
120. bed in Chapter 5 Using the Focus Window In order to bring the z stage of the microscope to the correct position focus on any irregularities e g dust or scratches on the blank slide Then move the xy position of the slide to a location where there are few irregularities and adjust the microscope so it is slightly out of focus This will minimize the contribution of irregularities with the slide to the flat field images Select Edit gt Setup Guides gt Flat Field Guide to bring up the flat field guide The following dialog will appear Flat Field Guide Current Flat Field Instructions Flat field correction removes constant flaws in the optical path The correction requires an image that contains only the constant flaws This is captured by focusing on an empty sample and collecting an image in the linear range of the camera Number of images to average 3 Exposure time ms 100g Scale 1 4 Optics Objective 636 Dil F Mag changer 4 py Filter configuration DIC En Cancel Capture Image 1 4 Specify an exposure time and number of images to take Later you will be able to adjust the exposure time so that the linear range of the camera is being used The number of images required depends on the user the more images the more accurately the flat field matrix will reflect only constant artifacts though the collection process will take more time Typically three will be sufficient Chapter 9 Prepar
121. below The timelapse composite channel shows the image data as accumulation over time This 1s useful 1f you wish to see the path of travel for migrating cells or other objects of interest 00 01 34 00 02 22 Timelapse Composite Channel 9 2 Cropping an Image Before analyzing an image you may wish to crop an image This 1s especially important to increase the speed of computationally intensive processes such as deconvolution Please see Cropping an Image on page 177 9 3 Aligning an Image Sometimes due to misalignments of filters or lack of mechanical repeatability data for one channel of an image may be slightly offset from data for another channel To solve this problem SlideBook offers an alignment guide that lets you adjust relative positions of any channel in the x y and z axes The alignment guide works alongside a Main View a Three 204 Chapter 9 Preparing an Image for Analysis or Export View or a Tile View see Using Data Views to Display Images on page 160 in order to shift how each channel lies in the image with respect to the other channels Changing channel alignment for an image makes a permanent change to the image itself and will be saved with the image In other words every view that you open from that point on will reflect the new alignment Of course you can re adjust the alignment later 1f you wish To align an image 1 Select the image in the Slide View by clicking on it 2 Open a Ma
122. box similar to the following will appear Color Basic colors i on Bee eal N A TT TT fees FF Ennu NN Custom colors E Ff i ie Hue 80 Red 0 a Ml MM MA fa Green 255 Cancel Add to Custom Colors Select one of the basic colors by clicking on the color block in the upper left hand color or design a custom color using the color spectrum on the right and choose Add to Custom Colors 67 SlideBook 5 0 User Manual 4 4 3 Adding a New Filter Configuration 68 Select Edit gt Define Optics gt Filter Configurations The Filter Configuration Parameters dialog box will appear To add a new filter configuration definition click on the Add button Enter the filter name in the data entry field Select the light source and enter the emission wavelength in um for fluorescent filters Also 1f the filter requires a UV objective select the checkbox under the emission wavelength data field The Light Source field permits sequential brightfield or Nomarski and fluorescence imaging on systems equipped with both a fluorescence shutter and either a brightfield shutter or electronic voltage control NOTE You may configure multiple transmitted light channels if you have a motorized condenser turret For instance you may define a DIC channel by selecting Transmitted light source and DIC from the Mode dropdown menu You could also define a phase channel by creating a new channel with Phase selected from the Mode dro
123. c image see page 213 4 Create a Volume or Surface View as discussed in the sections on pages 189 and 193 8 5 4 Creating Series Movies 8 5 4 1 2D Timelapse Images Scans through time lapse images can be converted into QuickTime movies To do so 1 Open a Main View of the image as discussed above 2 Alter the renormalization parameters and color display as desired see pages 171 and 174 You may also wish to crop the image see page 177 3 Select View gt Create Series Movie The Series Movie Properties dialog box will appear Series Movie Properties Frame Duration E E Cancel 195 SlideBook 5 0 User Manual 7 Enter the speed at which you would like the movie to play in the Frame Duration s edit field A value of 0 2 seconds generally yields acceptable results Click OK The following dialog box will appear Movie Export Options Export format Apple QuickTime Advanced Cancel Select the export format and quality that you prefer and then click OK Lossless has the highest quality and the largest file size A standard Save As dialog box will appear Type in a filename and click Save and you are finished 8 5 4 2 4D Images There are two ways to generate movies of 4D images 1 Open a slide that contains a 4D Capture 2 Generate a projection image as discussed in Creating a Projection Image on page 213 SlideBook will automatically generate a 2D timelapse image f
124. calibrated before then you can use the Restore button to load the previously set Upper Left Upper Right and Lower Right positions Caution Your xy stage MUST be Home d before you set the positions or they will not be saved see Setting Points on page 128 After you restore calibration positions you should use the corresponding Visit buttons to view and then the Reset button to adjust for changes in the plate placement 7 6 5 Selecting Wells for Capture 1 Select the wells you wish to image by clicking on the well and then clicking Select If you wish to select multiple wells first select a single well it will be gold Starting from the selected well click and drag over the wells you want to image The selected wells will turn gold 2 To unselect wells click on the well that you don t want to image and press Unselect To unselect multiple wells first unselect a single well then click and drag over the wells you wish to unselect The unselected wells will turn gray 7 6 6 Initiating and Monitoring Capture To begin imaging you will first make sure that the first well is in focus 1 Go to the first well that you will capture by clicking on the first well and using the Goto command 2 Open the desired light path by clicking Open Fluor or Open Bright 3 Select the desired filter from the Filter Configuration section 137 SlideBook 5 0 User Manual 4 Obtain focus on your sample in the camera view 5 If you wo
125. cally when Closing the Focus Window If you would like your fluorescence shutter to close automatically when you close the focus window select Edit gt Hardware Properties then expand the Focus Window section Click on ShutterUponClose 86 Chapter 5 Controlling the Camera and Microscope Hardware Edit Hardware Properties tea Property Information Dummy Hardware Name ShutterUponClose Filter Set Information Focus Window Shutter ponClose Camera Settings Pre Delay Camera Settings Post Delay EnableFLAutoS hytter EnableFLAutoT oggle EnablebF Autos Hutter Enables SAutoS hatter Ocular Photo Prism Pos 1 700 Eyes Description Ocular Photo Prism Pos 2 Description Ocular Photo Prism Pos 3 Description Camera Video Prism Pos 1 Description Camera Wideo Prism Pos 2 Description Camera Timeout StoprFocusDiunnags trearn Update Z Step Size Automatically Set Intensification Per Filter FRAP Parameters Hamamatsu Properties Hardware Properties Wersion Infa Apply Change Laser Launch Test Leica CTR Leica D0Me D00 e A Lifetime Ludi Into Marzhauser Corvus Revert To Default Cloze MATLSB Vernfication Category Focus Window Description Shutter fluorescence and alternate source Upon closing focus window or stopping camera Default Value E E E E E El E El E Set the value to Yes and then click on Apply Change Click Close and then restart SlideBook 5 2 3 Labeling the O
126. ce anywhere on the thumbnail or within the shaded region that surrounds the thumbnail to highlight the image Select View gt 3D Volume View or View gt 4D Volume View and select either High Speed or High Quality A new window will appear can QuickTourWINS Capture 8 Cropped Cl cropped 1 la E ES a 180 Y 00 Z 00 degrees Red Cra Dolly 1 000 Green FITC e lo f Blue DAPI 10um grid 3 D Dynamic Lighting Volume View To change the channels that are displayed use the channel menus as you would for the Main View Three View or Tile View To alter the shading grab the Lighting Tool in the upper right hand corner 0 and rotate 1t to achieve the desired lighting To zoom into the view click on the zoom tool and then click and drag up and down in your view to zoom in and out F To rotate the image click on the rotate tool and then click and drag in the direction that you wish to move the image You may either click and drag directly on the image or you on the coordinate image in the lower right hand corner To move the image in the field of view click on the Hand Tool icon E and then grab and move the image by clicking and dragging on it 191 SlideBook 5 0 User Manual 8 To instantly reorient your image you may choose to display a specific face of the volume by choosing the Axis Tool E and selecting Top Bottom Left Right Front or Back 9 To select a different rendering mode or to alte
127. cssccscccscccsccesccescceusccesccescceesscusccucceusceusceuees XVI 1 11 CHAPTER 9 PREPARING AN IMAGE FOR ANALYSIS OR EXPORT ocooccnoconccncccnccnncnnoconcconcnncconaconcnnaconccnncnnnos XVI Lads CHAPTER LO SIMAG EAN AE Y SIS A dad XVI 1 INSTALLING SETDEBOOR Usina aos 17 1 1 COMPUTER SYSTEM REQUIREMENT Soi ld ts ao heed ak atenm a puscaceu ace 17 LLE TAS A OD Soe eS E eS Nene ee eee eee 17 TAD E A A a TA AEAT 17 11 21 PROCESS OS A A A tit da iaa 17 115262 Memory Requiem iii 17 1 1 2 3 A O A e mo Soe Oe a eS On eae 17 1 2 CONNECTING THE HARDWARE KEY cta a da dd 17 1 3 ABOUT SLIDEBOOK INSTAEGERS dao 18 1 4 COPYING THE SOFTWARE FROM CD ROM uu icc ce cc ccecccecccscccsscccscccscccssccesccescccusccescceccessceesscessceceeesseesecenscss 18 1 5 COPYING THE SOFTWARE FROM YOUR DOWNLOAD SITE cscccsecccscccscccsccesccescccusceusccesccessceusccuseceusceuscss 18 1 6 INSTALLING SLIDE BOOK 9 Dia A a 18 Ly INSTALCING UPDATE S setts sacs cll ces ener earsetac aide AS 22 2 QUICK TOUR lt a E EEEE Re 23 Zad OPENING SLIDES AND IMAGES rer talas 23 2 2 DISPLAY NGOA MAIN VIE W cera a a E E A TE a A A 23 2 3 USING THE MO OOE BAR ato 25 2 3 1 Channel Menus and Data Values a tl dt dias adan bis 25 2 3 2 Up Down Arrows Scrolling through a 3D image oooonnninnncinuncuunooaaoannnnanannn nono nono nono nono nn nan nn nn nn n nn nnn nan nano 27 Ar AA E N E A Ee 27 2 3 3 1 The Marduce Odia 27 23 32 A EREI EE ET EE E EEE II ELE E AE P 28 2 3 3 3
128. ction tool allows you to easily maneuver around the Three View and it is the default tool for the Three View Click the point selection tool somewhere in the pane where z is the invisible axis the x y pane and the x axis y z pane and y axis x z pane will both scroll to the same coordinates that you selected in the z axis pane Clicking and dragging around any of the panes will continuously update the other two panes 8 4 3 Making Distance or Velocity Measurements This tool can be used in any of the views to measure a desired distance 2D 3D images or velocity 2D timelapse images In all cases it is critical that both the optical parameters of the objective those that determine um pixel are accurately defined see Defining Objectives on page 62 and the z or t interval is specified in the image info see Getting and Editing Image Information After Capture on page 101 The ruler tool makes one measurement at a time and each measurement replaces the previous measurement In other words the ruler tool is not used for permanent measurements but it can be used to make scale bars or to make quick and easy measurements 8 4 3 1 Making a Measurement on a 2D Image This measurement can be made in any of the views 1 Select the ruler tool from the view s tool menu 2 Click and hold the mouse button on the image at the point where you would like to begin measurement 3 Drag the mouse to the desired end point and release the mouse
129. cular Photo Prism Positions You may also provide custom labels for the emission selection positions ocular photo prism positions that appear in the Scope tab of the focus window To do so select Edit gt Hardware Properties then expand the Focus Window section Click on the Ocular Photo Prism Pos you wish to edit and enter the label in the edit field Click Apply Change Close and restart SlideBook 5 2 4 How to Perform Streaming Capture The streaming capture function allows you to run your Hamamatsu or Photometrics camera at the fastest possible speed There are limitations with Streaming Capture When a camera is recording in this mode you may only alter other hardware via the Focus Window Streaming Capture does not work using the Capture dialog box so you may not perform automated multi channel 3D 4D multipoint or montage capture This capture mode is ideal for rapid 2D timelapse capture To run your camera in streaming capture mode 1 Open the Focus Window and select the Stream tab 87 SlideBook 5 0 User Manual 88 Scope Fa XY Camera Stream Number of frames to Camera Settings average i xl Width 1392 Hapsed Time 00 00 01 0 eis sea F cape 0 Mean 13596 65535 Image Name Stream to disk E 2 Select the Start button A new image capture window will open with a counter for number of images captured ke Stream 49 4 o E 3 Your camera will continuously generate images with the expos
130. cus your sample using precise movements To use this window press Start open the desired shutter select the appropriate filter and focus your sample When you are finished close the shutter press Stop and then Continue Alternately you can press Snap to check a single focal plane then adjust the z stage then Snap again to update the view The later approach may minimize exposure to the sample 7 2 5 3 Viewing Previous Timepoints You may choose to view previously captured images while your 2D timelapse or 4D experiment is in progress To do so 1 Set up your capture as you would normally in the Capture dialog box Make sure that you are saving images to memory or to memory and disk see page 149 122 Chapter 7 Advanced Capture You can do this by selecting Advanced to bring up the Capture Preferences dialog Then select Spool and be sure that one of the first two radio buttons 1s selected 2 Once you have set up your capture click OK The Capture Controls will appear 3 Select the Live tab Capture Controls status FRAP Notes Live stages Z Stage XYZ Position da 0 25 um Current Position Updating the 7 position is not possible during a 6D capture Goto XYZ Update 2 Update xv Next Capture 00 00 07 Time Remaining 00 01 27 Elapsed Time 00 00 13 Capturing channel DAPI timepoint 2 of 10 plane 2 of 5 Graph Channels Regions a Individual z Show Y 0 By Type Select All Set
131. d Correction As with any optical system specks of dust or pieces of lint will find their way into the microscope s optical path There may also be some variation in the level of illumination across the field of capture SlideBook offers an operation called flat field correction which eliminates many of these constant artifacts from an image Flat field correction is particularly useful in brightfield and DIC Nomarski imaging where such artifacts are usually more evident than in fluorescence imaging Flat field correction or shading correction is not the same as background correction background subtraction 1s strictly subtraction When flat field correction 1s applied the desired image is multiplied by a matrix of coefficients that 1s determined from a series of flat field images see Collecting Flat Fields below To generate this matrix flat field images are collected and the average of each pixel for the set of images is calculate to generate a single averaged image flat field image The average intensity of all the pixels in the resulting averaged image is then calculated Finally a coefficient matrix pixel by pixel map is calculated so that when the intensity at any pixel location is multiplied by its coefficient the generated pixel intensity matches the average pixel intensity of the flat field image If you are planning to perform both background subtraction and flat field correction perform flat field correction first 9 5 1 Co
132. date the image 6 Enter 400 and 1000 in the Low and High data entry fields and click on Apply to register the change Renormalize Image lu O Channel z Range Lowy 400 High hooo Gamma i C Intensity E Reset to Selection Min Max Reset all to Image Min Max so cose 7 7 Choose OK to exit the Renormalize Image dialog box NOTE You may also bring up the Renormalize Image dialog box by selecting View gt Renormalize 2 3 6 Al Thumbnail Button The thumbnail button lets you set the default view settings for a particular image color assignment of particular channels renormalization information and the z position of a newly opened image If you close the Main View that is currently open without setting a default the changes that you just made using the renormalize button will be lost 1 Select View gt Go to plane and enter 10 in the edit box Note that plane number 10 is actually the eleventh plane as plane numbering starts at zero 2 Select OK 3 Click on the thumbnail button to select your current display as the default 32 Chapter 2 Quick Tour Now all subsequent views of the image will default to the same display You have also changed the thumbnail image that is displayed in the slide window 4 Open another Main View of the image by double clicking on the thumbnail Note that the new settings that you entered previously apply to this new Main View Also notice th
133. deBook user may access a private download site in order to obtain current installers and updaters Please contact 31 support intelligent imaging com or 303 607 9429 to obtain your user name and password To access your download site L 2 Go to www intelligent imaging com Go to Downloads and then Customer Login and click on link Click Here to Login Enter your username password case sensitive Click on the desired link to download the software When prompted select Save and then navigate to the desired destination for the file Click OK to save the file 1 6 Installing SlideBook 5 0 The SlideBook installer allows you to install the program drivers adjunct programs the manual and tutorials If you are installing SlideBook for the first time continue with this section If you already have SlideBook installed on your machine please see Installing Updates 18 Chapter 2 Quick Tour 1 Double click on the InstallSlideBook icon to start the setup utility The following dialog box will be displayed ie slideBook 5 0 InstallShield Wizard 2 Select Next and you will be prompted to review and accept the terms of the license agreement 45 SlideBook 5 0 InstallShield Wizard END USER LICENSE AGREEMENT FOR 31 SOFTWARE IMPORTANT READ CAREFULLY This 31 End User License Agreement EULA is a legal agreement between you either an individual or a single entity and Intelligent Imaging Inn
134. different functions when a mask is displayed see Chapter 10 Allows the user to create and or display a mask or region of interest as a blue overlay on the image It is usually best to display image data in only red and green while displaying or editing masks Chapter 9 Preparing an Image for Analysis or Export Allows the user to choose the invisible axis in Main Views of 3D and Axis Menu 2 timelapse data and in Tile Views of 4D data The up and down buttons are tied to the invisible axis Allows the user to alter the default view of a particular image Once the thumbnail button is selected subsequent views and the slide thumbnail Thumbnail Button A image will retain channel settings renormalization information and invisible axis plane tl Allows the user to alter the look up table or renormalization parameters Renormalize Button for each channel of the view Now we will explore several data view manipulations that require features of the info tool bar 8 3 1 Altering the Renormalization Parameters Lookup Table You will often want to highlight a particular range of data values in an image Perhaps a particular feature is dim relative to other features so you will want to saturate the mapping between the data and the display so that this feature will become more apparent Or perhaps in one channel you have a histological marker that might otherwise overpower the data and want to dim its contribution to the color valu
135. dividual colors 4 4 2 4 Camera SlideBook supports use of multiple cameras If you have more than one camera you may select the camera that will be used for capturing the given filter configuration 4 4 2 5 Channel Types All fluorescent filter configurations that are not used for ratio imaging should be set to Independent Configurations for ratio and FRET channels are discussed in the Ratio FRET Module manual Brightfield channels that use a color camera should be set to RGB Color Brightfield channels that use an LCD color filter or three separate color filter cubes should be set to Virtual RGB red green or blue 4 4 2 6 Default Color Display You can always change the colors or color display type of data captured with different filter configurations The Default Color Display however is a convenient way to choose how data from different filter configurations is displayed in the capture status dialog box as well as the initial view created for an image Channels may be set to standard RGB pseudocolor monochrome user defined or set to the emission wavelength 4 4 2 6 1 Pseudocolor Pseudocolor Color and Pseudocolor Intensity together determine the look of a pseudocolored image A filter configuration that has Pseudocolor Color as its default will set the hue value of the display according to the normalized 1 e scaled from minimum 66 Chapter 4 Configuring Your System to maximum value of each voxel A filte
136. e FirstlrageD elay Subsequent mageD elay FlipSecondlamera Split apture Split Capture Number of Frames Channel Registration Parameters Less Magniied Camera SlideBook Info Statistics Properties SuperPro Network Settings Sutter 10 2 Settings Sutter 10 3 Settings Sutter OG 4 Settings TIFF pooling TILL Folpchrome M Ho TILL Polvchrome TTL Filter Wheel TTL Shutter Properties TTL DAQ Alternate Source Epa CASTE TTL DAGQ Analog Input TTLADAQ Ausilarny Filter TTL DAQ Brightteld Shutter m ie TTL DAQ Camera TTLDAO Emission Filter H TTLIDAD Excitation Filter Revert To Default Close H TTL DAQ Categor Simultaneous Capture Description Split camera frame into multiple frames e g when using a Photometric DualYiew or Quad View Value Default Value E E E E E E E E E E E E E E E E E H External Trigger 3 Select Yes Apply Change and then Close 4 Restart SlideBook to register the changes To use the image splitter you must define filters appropriately see Defining Filters when using an Image Splitter on page 72 To align your image splitter use the Test Dual View function in the Focus Window as described on page 85 To capture images using your image splitter use the Simultaneous Capture checkbox as described on page 133 60 Chapter 4 Configuring Your System 4 2 4 Configuring Camera and Ocular Ports If your system has motorized ocu
137. e if an image contains a field of many cells performing threshold segmentation of a nuclear stain such as Hoechst will result in a mask that contains distinct islands Defining objects permits functions such as counting of objects and generating statistics for each subregion We will now learn how to create masks using a variety of techniques 10 2 1 Creating a Mask using Threshold Techniques Threshold techniques allow the user to perform a global selection based on image intensities SlideBook s segmentation process generates regions of interest that persist throughout the entire image in all dimensions 1 Click on an open view to make it the active view 2 Create a mask by selecting the mask icon gt Create Segment Mask or by going to Mask gt Segment A dialog box similar to the following will appear Note that it resembles the Renormalization dialog box Segment Image Channel CYS f Manual e Lowe o w High 14261 C Automatic Pidler Calvard f Current image io f Current capture type C All images with same channels Mask Name Mask 1 Apply Close 224 Chapter 10 Using Masks for Image Analysis 3 Select the channel that you would like to segment from the dropdown menu 4 Entera name for your mask 5 Select the scope of the operation using the radio buttons For any operation that segments more than one image you must select OK to segment all of the desired images If
138. e Focus Window and choose to use those positions in the Capture dialog You may define the step size in either the Focus Window or the Capture dialog The Z tab contains controls that let you make an interactive selection of a z axis range that can be imported into the Capture dialog when performing 3D acquisition It is available on all systems that have motorized z focusing Scope Z xy Camera Stream Top Set Top Capture Inftormatior 99 60 um Number of Planes 1 Total Travel ur 0 05 pr Center 33 57 Go Step size um 0 27 m 63 Oil Clear All Reference Sel 99 55 urn Go 5 J _ Bottom 99 55 um Set Bottom oO m NOTE You can also select a z axis range directly in the Capture dialog box see 3D Capture on page 105 The Z tab allows you to interactively define the 3D capture range and is generally used under circumstances where the precise z extent of the sample is unknown 5 1 3 1 Set Top Set Bottom Set Reference Center The Set Top and Set Bottom buttons let you specify the end points of a 3D capture range You can select these in either order For instance suppose you have found a cell that you want to capture and are in the middle of the focus range To set the boundaries of capture in the z direction first move the stage or nosepiece up until the focal plane is at the top of the volume that you wish to capture This can be done either by repeatedly clicking on one of the up but
139. e Optics gt Objectives and looking at the available objectives in the dropdown list If the objective 1s not available add the objective definition as described on page 62 It is extremely important that the microns pixel data field is reported accurately If a magnification changer or relay lens was used make sure that its definition exists by selecting Edit gt Define Optics gt Mag Changers and looking at the available magnification changers in the dropdown list If the magnification changer is not available add a new magnification changer definition as described on page 73 Add the objective and magnification changer information to the image by selecting Image gt Get Info pressing the Edit Info button and then selecting the objective and magnification changer information from the dropdown menus You will also want to select the capture type from the dropdown menu Select OK to exit the Edit Image Info dialog box then OK in the Image Info box to save the image information Use the ruler tool as discussed above to perform distance and velocity measurements 8 4 3 4 Using the Ruler Tool to make a Scale Bar 1 188 Open a Main View or Three View of the image Chapter 9 Preparing an Image for Analysis or Export 2 Select the ruler tool as discussed above to draw a line of the desired length observing the distance reported in the info tool bar 3 To preserve the line export the view of the image as discussed below
140. e from the drop down menus NOTE If you have imported an image from another program you must first enter the objective and magnification changer definitions as discussed in Chapter 4 Then you may select the proper objective and magnification changer from the drop down menus 5 6 exposure times for each channel image Enter the Interplane Spacing um for 3D images Select the filter configurations from the dropdown menus and enter the Select OK when you are finished The data that you entered is saved with the NOTE Even if you are working with 2D images it is a good idea to provide a value for Interplane Spacing as this will be used in the no neighbors deconvolution algorithm for the theoretical spacing value That way you will not have to change any values that appear 103 SlideBook 5 0 User Manual in the deconvolution dialog box As a guideline you should use the approximate z resolution for the objective used 6 7 2 Imported Images You may also add information to images that are imported into SlideBook from other programs 1 Import the image as discussed above in Importing an Image on page 96 2 Make sure that the objective definitions exist by selecting Edit gt Define Optics gt Objectives and looking at the available objectives in the dropdown list If the objective is not available add the objective definition as described on page 62 3 Ifa magnification changer optovar or relay lens was
141. e image as discussed in Scrolling through the Invisible Axis in a 3D or Timelapse Image on page 180 and Scrolling through a Three View on page 187 Note that the mask persists throughout the entire 3D extent of image 8 If you are satisfied with your mask click on OK If you are not repeat step 6 225 SlideBook 5 0 User Manual NOTE Once you have created a mask using threshold techniques you may use editing tools to add or remove pixels in the mask see Editing the Mask on page 227 10 2 2 Creating Masks Manually SlideBook also offers a variety of tools that can be used to manually select regions of interest An example demonstrating the creation and use of a manual mask is described in the Quick Tour section Creating Masks Manually Background Subtraction Example on page 41 10 2 2 1 Creating an Empty Mask To make a mask manually you will first create an empty mask then you will select your regions of interest using editing tools 1 Click on an open view to make it the active view 2 Create a mask by selecting the mask icon gt Create Empty Mask or by going to Mask gt Create The following dialog box will appear Create New Mask Name mE f In the curent image m C In all images with the curent capture type C In all images with same channels Cancel 3 Enter a name for the mask in the edit field 4 Choose to create the mask for the current image or for multiple images using the radio b
142. e regions by creating a binary overlay called a mask Masks are generated from the Mask menu Once you have made a mask you may obtain information such as average intensity and number of pixels in the region of interest by using the Statistics menu NOTE Screenshots used in this manual were taken with SlideBook running in Windows Vista Appearance may vary slightly with different operating systems xvi Chapter 2 Quick Tour 1 Installing SlideBook 1 1 Computer System Requirements Your computer must meet the following system requirements in order to run SlideBook 5 0 1 1 1 Operating System Windows XP Professional for computers driving hardware also known as online systems Windows 32 bit or 64 bit Vista for computer running analysis only also known as offline systems 1 1 2 Hardware 1 1 2 1 Processors Recommend at least dual quad core Xeon 3 2GHz processors 1 1 2 2 Memory Requirements RAM recommended 3 GB minimum 2GB Hard drive minimum 200 GB required for installation 1 1 2 3 Graphics Card for Interactive Volume Rendering only VRAM minimum 256 MB recommended 512 MB or more Minimum models NVIDIA GeForce or NVIDIA Quadro FX Recommended models NVIDIA GeForce 9800 NVIDIA Quadro FX 3540 or higher Please contact your 31 support intelligent imaging com or 803 607 9429 with questions regarding hardware compatibility 1 2 Connecting the Hardware Key SlideBook comes with a hardware key or dong
143. e the file enter a filename and select Save 8 1 1 2 Opening Slides To open an existing slide 1 Select File gt Open Slide 2 Navigate to the desired slide and select Open 8 1 1 3 Closing Slides To close a slide 1 Click on the slide that you wish to close to make it the active slide 2 Select File gt Close Slide or click on the XI in upper right corner 8 1 1 4 Deleting Slides Slides can be deleted using standard Windows operations for deleting files You may not delete a slide from within SlideBook however you may delete the images that are present in the slide 157 SlideBook 5 0 User Manual 8 1 1 5 Changing the Slide View Display SlideBook offers several ways to view your Slide To change your Slide View display go to View gt Slide View Display and select one of the following options 4 QuickTourWIN2 a QuickTourWIN2 KA BES Fibroblast ES HeLa Cells Fibroblast HeLa Cells Large Icons List The default display is the Details display shown at the beginning of the chapter 8 1 2 Working with Images As discussed above each slide may contain several images Using the same clipboard metaphor as other applications SlideBook lets you remove images from a slide duplicate images and insert images You can cut or copy an image from one slide and insert it into another NOTE There is currently no mechanism for multiple selection you can only manipulate one image at a time 8 1 2 1 Selecting an
144. ect for photobleaching in 2D timelapse images The photobleach correction operation takes the mean intensity at each timepoint and fits a line or curve through the mean intensity versus time data Then 1t determines a multiplication factor for each timepoint that is used to adjust the intensities at each pixel resulting in constant intensities over time Before performing photobleach correction you should examine your data to determine what type of fit is most appropriate To do so you need to view the intensity time profile of your data set You can do this by drawing a box over the entire field for a single plane then selecting Image gt Statistics gt Ratio Timelapse Data If the intensities decrease linearly over time you may perform a direct fit If they seem to decrease exponentially choose exponential fit To perform photobleach correction 1 Select Image gt Photobleach Correction The following dialog will appear Photobleach Correction Source Data All Images with Same Capture Type f All Images with Same Channels C All Images Fit Type f Direct Fit f Single Exponential Fit Regions to Use and Modify f Entire Image e e Timepoints to Use and Modify f All Time Points C From To Time Pointe Enter time points or time point ranges separated by commas For example 1 3 5 12 Results f Create New Image C Replace Existing Data Cancel 2 Select the fit and the scope of operation a
145. ed for 3D masks Output Grouped By these choices are only activated if you are performing object statistics on 2D timelapse or 4D images Sample output 1s shown below Output Grouped By Statistic atest testr2 2101 x Image best Mask Mask 4 Capture Date 12 03 2003 14 26 04 Mean Intensitp FP Elapsed Tim Clock Time 0 00 000 14 26 04 176 65 182 52 183 79 150 46 1 01 051 14 26 05 177 63 181 38 183 36 12318 E 02 052 14 26 06 177 44 191 43 183 71 179 10 3 03 053 14 26 07 177 67 150 36 103 78 179 45 4 04 053 14 26 08 17731 150 68 183 29 180 03 5 05 054 14 26 09 177 63 180 90 183 29 180 43 E 06 055 14 26 10 177 97 181 45 183 25 150 43 T 07 056 14 26 11 177 32 180 91 183 37 179 88 g 08 056 14 26 12 172 04 191 38 183 38 180 67 3 03 056 14 26 13 176 55 191 33 193 32 150 43 10 10 056 14 26 14 1739 21 181 57 183 01 180 49 11 11 057 14 26 15 173 36 181 87 183 22 180 27 12 12 057 14 26 16 179 29 182 10 183 30 13 13 057 14 26 17 12311 182 40 182 98 14 14 057 14 26 18 179 51 182 45 183 47 15 057 14 26 19 179 18 182 30 183 12 14 90 90 Chapter 10 Using Masks for Image Analysis Output Grouped By Object al x Image test Mask Mask 4 Capture Date 12 03 2003 14 26 04 wa E Of 4 total objects Elapsed Tim Clock Time Standard D Standard D 0 00 000 14 26 04 176 85 10006 33 4 53 17 62 1 01 051 14 26 05 177 63 10005 54 455 18 76 E 02 052 14 26 06 177 44 10004 51 4 33 10
146. ed lighting To zoom into the view click on the zoom tool a and then click and drag up and down in your view to zoom in and out F To rotate the image click on the rotate tool and then click and drag in the direction that you wish to move the image You may click and drag either directly on the image or on the coordinate image in the lower right hand corner To move the image in the field of view click on the hand tool icon Ni and then grab and move the image by clicking and dragging on it 91 SlideBook 5 0 User Manual 6 To instantly reorient your image you may choose to display a specific face of the volume by choosing the axis tool E and selecting Top Bottom Left Right Front or Back 7 To select a different rendering mode or to alter the colors displayed select the renormalization icon The Volume View Settings window will appear NOTE The Volume View Settings window automatically opens when you open a 3D Volume View Several features are offered in addition to the ability to renormalize the image change the lookup tables 8 Alter the background color using the Background slider and change the brightness Volume View Settings Background Color 20 Black White Grid Lines 20 None Bright Style Dynamic Lighting 86 CS 12882 16002 D pacity _ 100 403 403 1579 37721 iach r 100 imina kullin a iati TT g oe o a 67 67 27687 27687
147. el TIL 4D Focus Periodic FRAP Spool Sequence Notes Spool Mode Capture images to memory only Spool Directory C Program Files intelligent Imaging Innovations Inc SlideBook 5 0 2 Select the desired spool capture mode e Capture images to memory only Images are saved in memory and only become permanent after capture if Save Slide is selected e Capture to memory and save to spool file after each timepoint Images are both kept in memory and saved to disk This feature allows 153 SlideBook 5 0 User Manual you to permanently save images as they are captured and immediately view the images post capture no need to import a spool file This is only useful for captures that are not larger than the memory avallable 3 Select the location for storage of spool files by selecting Browse and then navigating to the desired location 4 Click OK to save these preferences and then set up for your desired capture sequence Once your capture is finished you may import it for viewing as discussed in the section Importing SlideBook Spool Files on page 97 7 12 Saving Capture Parameters You can store and recall frequently used capture parameters The following parameters can be saved Image extent Binning Capture type Channels and exposure times Camera intensification and gain settings if applicable Capture preferences e g Periodic 4D Spool features To do so 1 Begin a new capture by s
148. electing Image gt Capture The Capture dialog will appear 2 Select your capture parameters To access advanced parameters such as periodic capture select the Advanced button The Capture Preferences dialog will appear 3 Configure the desired preferences and select OK 4 To save these preferences select Save As from the Capture Settings drop down menu in the Capture dialog If you choose Save the default parameters will be overwritten Capture settings Capture settings Current Default mod Advanced The following dialog will appear 154 Chapter 7 Advanced Capture Save Preferences As Description Experiment Name Cancel 5 Enter a name for these preferences and select OK 6 If you would like to load a different set of parameters select one from the dropdown list If you modify your capture parameters the name of the currently loaded parameters will say mod next to the name You may choose to save them under the same name by selecting Save or under a different name by selecting Save As as discussed above 155 SlideBook 5 0 User Manual 8 Image Display and Manipulation Views SlideBook stores images in slides and displays images using views There are two kinds of views data views and display views Data views are interactive the user can display any combination of different channels and masks as well as change lookup table parameters renormalization and edit
149. en subtracted from the original image leaving objects that are more likely to be in focus 9 11 1 Running No Neighbors Deconvolution 1 Select the image that you would like to correct by clicking on 1t You may also start from an open view 2 Make sure that the image information is correct as described in Getting and Editing Image Information After Capture on page 101 219 SlideBook 5 0 User Manual 3 Select Image gt Deconvolve gt No Neighbors The following dialog box will appear Mo Neighbors Deconvolution f Current Image m m Microscope Components Objective 35 Oil Mag Chg 1 0 x Channels Clear Select All Optical Parameters Micronspret 0 106 Num aperature 1 32 Working distance rU Index of refraction 1 518 Advanced Options Options Subtraction Constant Theoretical Spacing Il Edge padding e 67 ne 62 4 Select the scope of the deconvolution operation e Current Image Operation will be performed on the selected image only e Current Capture Type Operation will be performed on all images in the slide with the same capture type 2D 3D 4D etc as the selected image e All Images with Same Channels Operation will be performed on all images in the slide with the same channels as the selected image 5 Select the Channels that will be deconvolved by toggling the checkbox next to the channel name 6 Select the deconvolution Options according to
150. enter lt calculated from top and bottom positions Bottom Required 0 00 KB Available 1 61 GB 3D Capture gt Use current Range um s C position es oa e Use reference Planes 5 position Step Size um fi Use top and l AER bottom positions Offset um Range around center Return to center of volume after capture Fig 7 1 Method 1 is accomplished by setting top and bottom positions in the Focus Window and then setting the capture window parameters as shown Current position Required 0 00 KB Available 1 61 GB 3D Capture Use current R um ls i positio am pe y Use reference Planes J postion Step Size um 1 y Use top and Siem bottom positions Offset um 5 Y Range around current Return to reference position after capture Current position Available 1 61 GB Required 0 00 KB 30 Capture A AS Range um 5 position Planes 5 gt Use reference position Step Size um fi y Use top and k E bottom positions Offset um lo Range around current Return to reference position after capture Fig 7 2 Method 2 can be accomplished using either the current position or a chosen reference position This figure shows how capture 1s performed when using the current position of the z stage with the given capture window parameters 111 SlideBook 5 0 User Manual Reference i
151. er does its job marked by white specks appearing after applying the flat field 9 5 1 3 Displaying Existing Flat Fields You can get a list of the existing flat fields and their corresponding optical combinations by selecting Edit gt Setup Guides gt Display Flat Fields To display the flat field click on the desired flat field icon in the table and press the Display button 209 SlideBook 5 0 User Manual Flat heki Flat Field Dimensi Date Display FF Flat 1 Eas Oil 1 0 Ary 139328 I Remove 9 5 2 Applying Flat Fields If you wish to flat field correct an image that was captured using an optical combination that has a corresponding entry in the flat field database you may choose to apply the flat field correction during or after image capture 9 5 2 1 Applying Flat Field Correction During Image Capture 1 210 Bring a sample into view and focus as discussed in Using the Focus Window on page 85 Open the Capture dialog box by selecting Image gt Capture or by selecting the Capture icon in the SlideBook toolbar Set all other capture parameters as discussed in Chapters 6 and 7 Select the Flat Field Correction checkbox which is located below the exposure information Chapter 9 Preparing an Image for Analysis or Export Capture Current Parameters Default modified Capture Settings Extent Offset and Binning pixels Default Image Bin Factor Width 1392 y Height 1040
152. er than 10 E a Some advanced features require a secondary mask Secondary Mask Hone Remove objects smaller thar 10 e e Select All Clear The dialog box has the following sections 243 SlideBook 5 0 User Manual 244 Image Scope allows for generation of statistics in multiple images If you are working with a 2D or 3D image the first radio button will be selected by default If you are working with a 2D timelapse or 4D data set the second radio button will be selected by default You may select the third radio button if you have multiple images with the same mask name Mask Scope allows you to select whether you want statistics for the entire mask or on a per object basis If you have chosen to obtain statistics for multiple images the Object option will be unavailable Primary Mask defaults to the mask that is currently displayed but can be altered via the dropdown menu The primary mask will automatically be made into objects You may also remove objects under a specified pixel size gating by size see page 229 To get statistics for all pixels in the mask leave this box unchecked Secondary Mask can be selected to obtain additional statistics see below Features lists all available statistics that may be calculated Note that the list of features will depend on the dimensions of the mask For instance area will be displayed as a statistic for 21 masks while volume will be display
153. erT oggleM ode TTL Count Pulses during Streaming gt File Size Warming Limit Automatically Reopen Focus Capture Dialogs Automatically renormalize capture data Automatically renormalize test captuer MightAndD ay MightAndD aylycleLength Might ndO ay umCucles MightAndD ay O feet H DiffusionM SDs Automatically renormalize test capture Advanced Z Stage Parameters Analog Y Stage Control Properties Analog Z Stage Control Properties Andor Properties ASI Siege Apply Change ASI Z Stage Camera Camera 2 Camera 3 Camera 4 E a DeconLive Settings Revert To Default Close Diagnostic Instruments Camera Properties Description Should the brighthield shutter close between brightheld timelapse Images Default Value E a Ee a a a a E E E a a E 3 Select Yes and then Apply Change 4 Select Close and then re launch SlideBook to register the changes 7 2 1 8 Changing the Status Update Frequency You may change the frequency with which the elements in the Capture Status window are updated by choosing Advanced in the Capture dialog box to bring up the Capture Preferences dialog and then entering the desired update frequency in the edit boxes in the General tab An update frequency of 3 would update the given element at the conclusion of every 3rd capture Reducing the update frequency 1s primarily useful for increasing capture rate with single processor computers 114 Chapter 7 Advanced Capture
154. es To alter the range of data that is displayed you will choose the minimum and maximum data values that correspond to the absence or full saturation of the color associated with each channel You also have the ability to apply nonlinear lookup tables to your view 8 3 1 1 Changing Renormalization Parameters for a Single View To alter renormalization parameters 1 Create a Main View Three View Tile View or Channel View of an image If you have several views open make sure that the desired view 1s the active window by clicking on 1t 2 Click on the renormalize button ih or select View gt Renormalize The Renormalize Image dialog box will appear 171 SlideBook 5 0 User Manual 172 Renormalize Image 0 Channel Range Low 334 High 7074 Gamma C Intensity C Reset to Selection Min Max Reset all to Image Min Max Apply Close A histogram is displayed showing the relative number of pixels on the y axis and intensity values on the x axis for the specified channel The x axis value shown on the left is the lowest data value across the entire image The x axis value shown on the right 1s the highest data value In the dialog box above the CY3 channel intensities range from 0 to 13067 Select the channel that you wish to renormalize from the Channel dropdown menu The red and green bars allow you to select the minimum and maximum intensities that correspond to the absence black or full sat
155. eset to Selection Min Max button You can also set all channels to the global min max the lowest and highest values of any channel present in the image by pressing the Reset all to Image Min Max This is particularly useful when working with transmitted light color images Remember that renormalization is a property of a particular view not the entire image It is possible to have multiple views on the same image each with their own renormalization values If however you decide that a particular selection of values is best for most viewings of an image you can press the thumbnail button in the corresponding view and make those values the default for that image see Changing the Default Display on page 180 8 3 1 2 Changing Renormalization Parameters for a Group of Images in a Slide You may apply the default renormalization parameters of an open view to all images in a slide by selecting Image gt 4D Operations gt Renormalize All NOTE You MUST set the display as default using the thumbnail button before you apply the settings across the images in a slide You may also set the renormalization to the global minimum and global maximum for each channel for all images in a slide To do so select Image gt 4D Operations gt Renormalize All to Global Min Max This function finds the lowest minimum and highest maximum pixel value for the group of images in the slide on a per channel basis and then uses those values to set the renormalization o
156. espond to intensities for a given channel e Notes formerly called Annotations in previous SlideBook versions can be used to record experimental actions see Creating Notes on page 116 e Object IDs corresponding to objects in a displayed mask e Regions selections made using ROI tools 180 Chapter 9 Preparing an Image for Analysis or Export eae ratiosample Untitled 1 fs foe x 200 y 315 110 5 117 _Fura 2 3401 ian 212 _Fura 2 3801_ Timestamp 1260 open ONA 5 j FPS 100 Lookup Object ID Seale Bar 8 3 8 1 Displaying Annotations To display any of these annotations on an open view 1 Open a Main View Three View or Channel View of your data 2 Select the item you wish to display from the Annotations menu J SlideBook ratiosample File Edit Image View Timestamps Ctrl Shitt T Scale Bars Ctrl S5hrft 5 Lookup Tables Ctrl Shift L Notes Ctrl Shitt N Object IDs Ctrl Shift 0 Regions Ctrl Shift G Settings Set Default Settings Graph Rectangle Tool Ellipse Tool Polygon Tool Freehand Tool 181 SlideBook 5 0 User Manual 3 When an item is selected for display a checkmark will appear next to the item on the menu 8 3 8 2 Formatting Annotations Several formatting options for annotations are available Once you have displayed your annotations you may format by doing the following 1 Open the Annotation
157. f all images in the slide 8 3 1 3 Applying Nonlinear Lookup Tables SlideBook allows you to apply nonlinear lookup tables using a gamma correction This feature is particularly useful for emphasizing dim features in an image To apply a non linear look up table simply click and drag the gray line that crosses the histogram diagonally This adjusts the value of gamma Alternatively you may enter a value for gamma manually and select Apply If you wish to return the gamma to 1 no gamma correction simply click and drag the line off of the histogram and it will return to 1 173 SlideBook 5 0 User Manual Renormalize Image Channel ap Range Low 221 High i da Gamma 0 471 mal C Intensity E Reset to Selection Min Max Reset all to Image Min Max som ce 0 8 3 2 Changing the Display Colors Images may be displayed using a variety of color schemes including RGB monochrome inverted monochrome pseudocolor and user defined color SlideBook also supports a mixed display that blends a monochrome background image e g DIC with an RGB image The default display 1s determined by the filter configuration definitions see Defining Filter Configurations on page 64 though you may easily change the colors that are displayed for a given view Generally the filter configurations will be set to R G or B However monochrome display may be useful for visualizing a single channel Pseudocolor display may be useful f
158. ffset Alternatively draw a selection in the Live View and press the Update button For more information on defining image subregions refer to the Selecting the Area to be Imaged section of Chapter 6 on page 92 Chapter 7 Advanced Capture 7 10 Varying Capture Rates During Timelapse Capture Sequences You may choose to capture at various capture rates throughout your experiment when performing a multi channel 2D or 3D timelapse capture The capture rates may be changed either automatically in a preset protocol or manually during capture 7 10 1 Setting Capture Preferences To use this feature you must first set up capture preferences To do so 1 Open the Capture dialog Image gt Capture and press the Advanced button 2 In the Capture Preferences dialog select the Sequence tab Capture Preferences General TIL 4D Focus Periodic Spool Sequence Notes E a Stages Up Down Duration Add Remove Timepoints Stage Details Stage Name Timelapse Interval lo Duration of Stage lo Comment The elements of the dialog box are described below A sequence 1s made up of a series of stages Each stage may have a specified duration and timelapse interval time between timepoints e Begin capture with first event in sequence if checked capture will start with the first event in the sequence e Loop to first stage when done if checked capture will return to first stage e Stages each sta
159. fo tool bar Note that the coordinates in the upper left corner give you the x y z position of the mouse pointer As you scroll through the image stack the z position will update to your current location 2 Select View gt Go To Plane Enter 2 in the data entry field and select OK The value of the z coordinate is now 2 which corresponds to the third plane in the image as the count starts at 0 Go to plane 2 of 49 Cancel 3 Next select View gt Previous Plane and View gt Next Plane commands observing the value of the z coordinate in the upper left corner of the info tool bar 4 If your mouse is equipped with a scroll wheel it can also be used to move through the planes of the image 2 3 3 Tool Menu The tool menu lets you choose the function the mouse cursor performs 2 3 3 1 The Marquee Tool The marquee tool is the default tool of the tool menu and allows you to select a two or three dimensional region of the image 1 Click and drag the mouse over a region in the Main View Note that the cursor appears as cross hairs Release the mouse button 27 SlideBook 5 0 User Manual de QuickTourWIN1 Metaphase B Cell 1 faz 180 26 2 30 4 dak ee E 2 You have just selected a 2D region using the marquee tool In the visible axes x and y the marquee tool works as 1n any drawing program but because SlideBook is designed around the 3D images a marquee must be manipulated
160. formed on all images in the slide with the same capture type 2D 3D 4D etc as the selected image e All Images with Same Channels Operation will be performed on all images in the slide with the same channels as the selected image 5 Select the channels that will be used for the mathematical operations from the dropdown list For instance if you would like to subtract the FITC channel from the CY3 channel you would select the FITC channel from the second channel menu and the CY3 from the first channel menu 6 Select the mathematical operation that you would like to perform using the radio buttons From left to right the operations are addition subtraction absolute difference division and no operation If you select no op the second channel selection will become unavailable 7 Enter any desired Coefficients or Offsets in the edit fields The coefficient will be multiplied by the selected channel and the offset will be subtracted 203 SlideBook 5 0 User Manual 8 Enter a Channel Name in the edit field and select OK to generate the new channel This new channel will now appear in the channel selection drop down box in the image views 9 1 4 Creating a Timelapse Composite Channel To create a timelapse composite channel select Image gt Channel Operations gt Create Timelapse Composite Channel Select the desired channel from the dropdown channel and then OK An example of a timelapse composite channel is shown
161. fset Y Offset ER CO white Balance ji a Full Chip Timelapse Capture Available 69 14 GB of Time Points 1 ae Pont Range um 1 Estimated Duration ls gt position Planes 4 AS Ise reference 54 Correct C H e Interval 1000 ms ositic Timelapse sa sl Step Size um Jo 27 m A Use top and Li A Ze Eo E 0 play Renormalize tot 0 a bottom positions Offset um Jo Multiple XY Location Capture Range around center CurrentLocation Entire List Montage Simultaneous Stereology Return to fcomputed ref current location Adjust Exposure ND Current Expose wo ms Once Aux ND Current Test Find Best li OS Intensify Current Correct 7 Dark Field 7 Flat Field Offset Mowe Up Move Down Onti Objective 63x oi Mag Changer 1 0 x 2 First select the binning factor in the upper left drop down menu Binning lets you group several pixels together to increase sensitivity at the cost of resolution For instance selecting 2 x 2 binning effectively gives you a camera with half the horizontal and vertical resolution but four times the sensitivity Note that as you increase binning you will need to reduce the exposure time 3 Select the desired Filter Set from the dropdown menu 4 Click on the checkbox of the channel that you would like to expose The channel that you select will be highlighted in blue 90 Chapter 6 Image Cap
162. ft corner of the selection and click on the button The x y and z coordinates of that position will appear in the XY tab s list of points with a LL designation next to it 131 SlideBook 5 0 User Manual Scope E xY Camera Stream askas aoaea aa mE Montage Extent 1235 9 1159 9 839 2 UR et Point MET R F A Visit Point I 3x3 gt l Update 1 Le Update Z Reset All Load Save Clear All Edit Description Home Selecting these two corners lets SlideBook automatically compute how many camera fields need to be captured in order to stitch together a montage image that will cover the entire region When a montage extent has been successfully defined the number of vertical and horizontal camera fields needed to cover the region will be displayed in this case a 2 x 3 grid of 6 images If X axis or Y axis is displayed SlideBook cannot compute a montage extent because the selected coordinates are not correctly orientated with respect to one another For instance Y axis would be displayed if the selected lower right position is actually located above the selected upper left position If you continue to see these errors please check that your xy stage is configured properly page 58 If you want to change the montage extent you can simply go to a different location and click on one of the buttons again as appropriate The previous coordinates will remain on the list of po
163. g gt Manual Tracking The following dialog box will appear 238 Chapter 10 Using Masks for Image Analysis M Automatically proceed to the next time point LID Start tt End tt T Es E Delete Path New Path OR Cancel 3 Select either the medium or large pencil tool from the tool menu The view will go to the first timepoint automatically 4 Click near the center of the first object you would like to track The view will automatically move to the next timepoint 5 Continue clicking on the object you would like to track until the path 1s complete When complete either choose New Path to go back to the first timepoint and start over or OK to close the dialog You may now generate object statistics as described in Generating Statistics for Masks and Objects on page 243 10 3 Displaying or Deleting a Mask If you have just issued either the Mask gt Create or Mask gt Segment commands the new mask is already selected and displayed If not you must select the mask you want to edit using the mask pop up menu Yin an open view of the image Note that while you are editing from within a particular view the mask is owned by the image not just the particular view Any changes you make to the mask can be seen in any other view in which the same mask 1s displayed To delete a mask 1 Select Mask gt Delete The following dialog box will appear 239 SlideBook 5 0 User Manual Delete Mas
164. g capture while a negative number corresponds to the stage or objective moving down during capture e Offset um The distance above or below the current stage location that the stage or objective will move before starting image collection For example on an inverted microscope a positive number corresponds to the stage moving up while a negative number corresponds with the stage moving down Thus on an inverted microscope you will need to use a positive spacing and negative initial offset If your sample is already in focus you may select the Use current position radio button and choose the Range around current checkbox and simply enter in the total range of travel Range and either the number of planes or the step size The remaining capture parameters will be calculated for you If you have set a reference position you may choose to use the reference position as the basis for capture 6 If you would like your z focus to return to the current position or reference position after capture check the Return to current position or Return to reference position checkbox as appropriate 7 Click OK to begin capture The Capture Controls window will appear and display each plane as it 1s captured You may choose to pause cancel or stop your capture any time after it has started as described above in Method 1 A comparison of these capture situations are shown in the figures below 110 Chapter 7 Advanced Capture Top C
165. g dialog box will appear Define Selection Cube Extent OF Cancel a it 80 20 pu The Define Selection Cube dialog box allows you to manually enter coordinates for the selection box Change the Beginning Z value to 10 and the End Z value to 30 Notice that Extent Z has updated to indicate 20 planes are selected Leave the x and y coordinates unchanged and press OK Chapter 2 Quick Tour A QuickTourWIN1 Metaphase B Cell 4 o eae l200 180 95 27 235 Lys 304 FITC 330___DAPI None Notice that the z extent of the marquee selection only spans the 20 planes that were selected Next we will spawn a Tile View that contains the data defined by the selection cube 2 5 Spawning a Tile View SlideBook can generate a plane by plane mosaic called a Tile View from any Main View or 3D selection Now we will spawn a Tile View of the selection cube that we just created 1 Select View gt Tile View A large a mosaic of the chromosomes is displayed with each panel representing one of the 20 different z planes that was contained within the marquee selection 3 QuickTourWIN1 Metaphase B Cell 5 o 5 E l1o0x 8066 27 1345___CY3 FITC lalt m a fro 82 1358 DAFI None 2 Select the hand tool from the tool menu in the info tool bar 3 Click and drag around a single pane of the mosaic This adjusts the position of the display cube within
166. g of the first plane of an image the default or of some portion of an image if you have specified a particular thumbnail using the thumbnail button see Changing the Default Display on page 180 The information includes the following e Image name automatically generated in the case of captured images or corresponds to a file name in the case of imported images e Comments entered by the user either before or after capture e Capture date and time e Capture type 2D 3D 2D timelapse or 4D 156 Chapter 9 Preparing an Image for Analysis or Export e Number of channels number of fluorophores e Dimensions image size in pixels and planes can be X Y Z T or X Y Z T e Image size image size in MB e Masks number of masks stored with the image 8 1 1 Working with Slides The Slide sld file is SlideBook s native document type Opening closing and saving slide files are performed in a manner similar to other software programs 8 1 1 1 Saving Slides To save a newly created or updated slide 1 Click on the slide that you wish to save to make it the active slide 2 Select File gt Save Slide If you have not previously saved the slide you will be prompted for a file name 3 Enter a filename and select Save To save a copy of a slide 1 Click on the slide that you wish to save to make 1t the active slide 2 Select File gt Save Slide As 3 Navigate to the location where you would like to sav
167. g through the Invisible Axis in a 3D or Timelapse IMA oonnnnnnnnnnnnnnnnncconccccoaconn nono no nono nononons 180 OS CHAMANES o a de o 180 Is a eee nied cect aes eae aie ee 180 8 3 8 1 Display ne Anno OMS 52a ce ee assk E EE A 181 8 3 8 2 Formatia A TMObAL OMS a A OREA 182 8 3 8 3 Settino Default AnnotInon SCUINGS ii 184 8 4 USING THE LOOM MENU scot Sth de eel ces le do e dd Seana ie teehee ee cust teay 184 Oat Making a 2D 0F 3D EZONE 186 8 4 1 1 Makine a 2D Selection tmeany Dala Vies 186 8 4 1 2 Making a 3D Selection in a Main Three and Channel VieW coccccncnnnnnnnnnnnnnononnnnnnnnnnnnnnnonininonininininininnn 186 8 4 1 3 Makine a 31D Selection im any Data Vie Wars is 186 8 4 1 4 Selecting an Entre MACS ci is 187 G42 SONO Moucha Three VIEW td dd adi da 187 8 4 3 Making Distance or Velocity Measurements A cia 187 8 4 3 1 Makine a Measurement ona 2D mao esti dr aa li e Li aan Died Loli da ites ala 187 8 4 3 2 Making a Measurement on a 3D or 2D Timelapse lmMage occccccnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnannnnns 188 8 4 3 3 Making a Measurement onan Imported Mir ib 188 8 4 3 4 Using the Ruler Tool to make a Scale Batt sairaan aana raned dana KEA a a a KADETE a aa 188 8 5 USING DISPLAY VIEWS CREATING RENDERINGS AND MOVIES ccccceeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 189 8 5 1 Displaying a 3D or 4D Volume View Volume Rendering oooononnnnnnnnnonoccooooocanoonnnnnnnnnnnnnnnnnnnnnnnnnnnnns 189 8 5 1
168. ge 1 Open the slide that contains montage data 2 Select Image gt Generate Montage SlideBook will attempt to create a montage image using only stage coordinates and pixel size information A dialog box similar to the following will appear Montage Creation Guide Images Image Mo Capture 1 Position 1 Bow Position 2 Position 3 Colurnr Position 4 Position 5 4 Offset Position 6 Position 7 Y Offset Position E Z Offset Fit Error NA m m im Hold down shift key to Destination Montage move all images below 2 and to the right of the selected image Auto Align Cancel 3 You may choose to manually align the images by choosing an image from list and then pressing any of the arrow buttons Only the selected image will move You may also hold down the shift key to move all of the images to the right and below the selected image If you click on an image in the montage display the appropriate image will be highlighted in the image list Alternatively you may choose to perform an auto alignment by pressing the Auto Align key 4 Once you are satisfied with the alignment select OK to close the dialog box and save your alignment settings 8 3 Manipulating Data Views Using the Info Tool Bar All Data Views have at the top a set of buttons menus and readouts collectively called the info tool bar 169 SlideBook 5 0 User Manual X Y Z T Position Adjust Renormalization
169. ge 169 Displaying a Montage 7 6 MultiWell Capture Multi well capture uses a different interface than other forms of capture In this section you will learn how to Create a Layout Manipulate Hardware and Focus on the Sample Calibrate the Stage Position Select Wells for Capture Initiate and Monitor Capture 133 SlideBook 5 0 User Manual 7 6 1 Creating a Layout In order to perform multi well capture you must first create a layout or template that defines capture parameters These layouts may be saved and applied for future captures To create a layout 1 Goto Image gt Advanced Operations gt Capture Multi well Plate The Multi Well Capture dialog will appear Multi Well Capture Layout E New Delete p Camera Browse eriment Name AA Comments Filter Configuration x jo um Jie o um Transform Goto xY Stage 3 Z Stage A Auto Focus Step Z 99999 99 fio _Set UR mb mb mb MN Exposure Time AMS I Color A E O O IV Guides Stop Open Fluor Close Bright 0 H BDO ms EE a a m Bin Factor Renorm Min Max Edit cicacrcar 7 Progress E Live picture during Current Well Current Image capture V Save Images 00000000 00000000 ort Po nts i Select Unselec 2 Goto the upper left corner and select New to generate a new layout The following dialog will appear 134
170. ge will be automatically performed in the order they are displayed from top to bottom 149 SlideBook 5 0 User Manual 150 Not in Sequence these stages must be manually selected in the Capture Status dialog they will not be performed automatically Stage Name defined by the user Timelapse Interval the delay between the beginning of one timepoint and the beginning of the next timepoint Available units are milliseconds seconds minutes and hours Duration of Stage how long capture will occur at the defined rate You may define the length as timepoints e g 5 timepoints a time period e g for 10 min or continuous ending when manually selecting the next stage An example of a completed dialog box is shown below Capture Preferences General TIL 4D Focus Periodic Spool Sequence Notes Begin capture with first stage in sequence Loop to first stage when done Not in Sequence Rapid capture a z Up Domi Duration 00 02 00 000 Add Remove Timepoints 124 Stage Details Stage Name Rapid capture Timelapse Interval 11 ims Duration of Stage 20 timepoints Comment For fast events click here To add a stage select Add Enter the stage information described above The stage will appear in the Stages list To add a stage that will be started manually in the Capture Controls click on the Not in Sequence list to activate it Chapter 7 Advanced Capture 5 Add a
171. ght to open the desired lightpath Next you will need to bring the sample into focus This may be accomplished by elther viewing the sample through the eyepieces or viewing the sample in the focus window If your microscope is equipped with a motorized emission light path the image can be directed to the eyepieces or the camera using the Scope tab Moving the xy stage and changing the z focus can be accomplished by either using the manual controls on the microscope or the computer controls see above Using the slider set the exposure time so that the image 1s not overexposed Using the controls in the Focus Controls window or joystick or manual controls adjust the x and y position of the stage and z position of the stage or nosepiece to find and focus on an area of interest TIP If you double click on an object in the image display the xy stage will move so that the object is centered Once the sample focus 1s satisfactory you may wish to close the lightpath for light sensitive samples and then close the focus window by pressing the X button in upper right corner If desired the Focus Window and Capture dialog box may be open simultaneously 5 2 1 Snapping an Image You may find that you want to capture a single image as you are using the Focus Window To do so simply press Snap once you are happy with the image in the live view The image will be stored into the open slide file 5 2 2 Closing the Fluorescence Shutter Automati
172. gth E in timepoints 3 Track and Continue Track This operation tracks particles in either 2D timelapse or 40 images Particles must have been properly cy identified prior to tracking op Menu Mask gt Basic Particle Tracking me 5 Select Track and the tracking algorithm will be executed When completed the paths will be displayed on the image You may click on the image and scroll through the timepoints to see the paths The first point in the path appears blue and the last appears red with a gradation of color between At this point you may wish to view the object IDs by selecting View gt Annotations gt Object IDs Also you may wish to perform the tracking algorithm using new parameters You may enter those parameters and then press Track again If you already know the parameters in advance you may select Track and Continue The tracking algorithm will be executed and the protocol will automatically advance to the next step NOTE You may turn path display on and off by selecting Mask gt Particle Tracking gt Display Paths 6 Once tracking 1s complete select Next The protocol will advance to the next step 235 SlideBook 5 0 User Manual Particle Tracking Protocol Path 5 x SavevLoad Settings Fath Statistics Defaut Endpoint Displacement _ Endpoint Speed Done Rega OperationName Average Speed ud x Path Creation Total Displacement Path Statistics Path Length Object Stat
173. hannel that will be generated The dialog box will look like this Channel Math Scope f Current Image First Channel Coefficient Channel Offset DAPI 200 57 Mote Batch Operations only include images that match the channels of the selected image Ca es 16 Di ee f no op Second Channel Result nO aa o Channel Name DAPI Background cue 14 Select OK to create the background subtracted channel This new channel will now be present in the channel pull down menu within the Main View 47 QuickTourWIN2 Fibroblast 1 E 00 368 0 7 455 Cuz ao ___ FITC _ ie None Y DAPI Cy3 FTE DAPI Background 15 You may repeat steps 12 14 for each channel by selecting the appropriate channel in the Channel Math dialog box and subtracting the relevant background intensity value When you are finished close the Main View of the Fibroblast image 44 Chapter 2 Quick Tour 2 7 3 Defining Multiple Objects Often you may have several distinct objects that will have intensities that are very similar and thus will segment together A mask that contains several distinct objects may be separated into objects as we will now demonstrate 1 Open a Main View of the HeLa image within the QuickTourWIN2 sld by double clicking on the thumbnail 2 Create a new threshold mask by selecting Create Segment Mask from the mask menu The following dialog box will appear Segment Image 0 187
174. he arbitration shall be held in the City of Los Angeles in the County of Los Angeles in the State of California in the United States of America SlideBook 5 0 User Manual Table of Contents END USER LICENSE AGREEMENT FOR 31 SOFTWARE cccsscsssccsscccscccssccsscccscccsccccscccscccsscccssccscceees II TABLECECONTENES cicssveccanssnsccucccvccesosdecescccsssudctelscbecsedsaedsncicedacsdccandsaadcavecucucsdducscesseuasasudesedescacccdcasdsneicswcousdds VI MANUAL CONVENTIONS oic hei oiicck ec sncs bidet O is bas suchicaheteuiccb dec vawsbedelsencecboncbesettaes XIV 1 1 IPOGRAPOIO ONIN DIG NG et te tuno daa eet tate ol eos ao ta duck Sule Dae do XIV 1 2 OE PIRES AND V OXEUS ses teseec dto XIV USING THIS MANTA atea XV 1 3 CHAPTER 1 INSTALLING SLIDE BOOK Mitad XV 1 4 CHAPTER OU a TOR lola tada ae ee XV 1 5 CHAPTER 3 SLIDEBOOK ORGANIZATION cccccseccsssccscccsscescccsccesscesccesscessceesccesscesseesseesccesseesceesceescs XV 1 6 CHAPTER 4 CONFIGURING YOUR SYSTEM cccccsssccsscccsccessccsccesccessccusseusccesseeusseusccecceusceusecuscseeeceuceuecs XV 1 7 CHAPTER 5 CONTROLLING THE CAMERA AND MICROSCOPE ccscccssccscccscccesccesccescceusccusccescceesceesceuecs XV 1 8 CHAPTER 6 IMAGE CAPTURE AND IMPORT ccccscccssccsccesscessccusccescccusceusccessseusceusccuscceusceusccuceeeeceusceuecs XV 1 9 CHAPTER 7 ADVANCED CAPTURE 0 ti did XV 1 10 CHAPTER 8 IMAGE DISPLAY AND MANIPULATION cccscc
175. he following will appear pera New white balance factors R 1 00 G 1 00 E 6 48 Previous factors R 1 00 6 1 00 B 1 00 95 SlideBook 5 0 User Manual 11 12 13 14 15 16 NOTE If you are getting inconsistent color maximums when performing capture and the RGB factors are varying significantly each time you perform a white balance then you should adjust your illumination Bright dim or inconsistent lighting may cause this phenomenon Select OK to close the dialog box You may now capture an image by selecting OK in the Capture dialog box The white balance settings will remain in the system memory until the slide file is closed If you would like to manually enter white balance settings go to Edit gt Define Optics gt Filter Configuration and select the red filter configuration for an LCD filter or to the color filter configuration for a color camera In the default Color Display section select the Settings button The following dialog box will be displayed Default Color Display E Color Balance f Red es Gain Factors C Blue veel C Pseudocolor Color Eo i C Pseudocolor Inter it a RGE Settings Blue i Monochrome a St ie Enter the white balance gain factors In general the green factor will be the largest Click OK to save the settings 6 6 Importing an Image You may also use SlideBook to display and analyze images captured using other programs The
176. hecking the desired channels in the Channel list Select the destination of the filtered channel e Replace each channel The selected channels will be replaced with the filtered data e Create a new filtered channel New channels containing the filtered data will be created within the image Select either a 3x3 or a 5x5 kernel size Enter the desired Coefficient and Offset in the edit field The output value of the kernel will be multiplied by the coefficient and added to the offset These parameters can be used to scale the convolved data to a desired range and to account for negative kernel output values Enter the desired matrix elements in array of edit fields Select OK to perform the convolution 217 SlideBook 5 0 User Manual 9 10 2 Gaussian Hot Pixel Mean and Median Filters SlideBook offers a set of standard filters for digital image processing Gaussian A blurring filter that utilizes a Gaussian kernel of a given radius Useful for removing noise from an image Gaussian Derivative An edge detection filter that uses a first derivative Gaussian kernel of a given radius Mean or Median Blurring filters that replace the intensity value of a pixel with the mean or median intensity value of pixels within a given radius Hot Pixel A nonlinear filter used to remove hot pixels in an image If a pixels intensity value is some factor greater than the mean of its neighbors it is replaced with thi
177. held 1003 Eves Condenser 100 Oil Magnification Aperture Changer O Cames O ideo Position Pas 5 Camera Test Camera 0 r Exposure 100 ms Bin ixi Ta E a Filter Set Widefield Open Fluor 2 Open Bright Open Alt stage Neutral Density ral Xi 5 903 mm Simulated Z Stage Primary e de 100 y 328 1 pm da aair a gt Y 4 041 mm _Auto Focus Y 100 10 i gt 5 00 um 5000 set 77 SlideBook 5 0 User Manual The Focus Controls dialog box always has at least two tabs Scope and Camera that control and report the status of automated microscope components and cameras Typically systems also have either a Z tab or an XY tab or both Additionally there are a number of permanent controls in the lower half of the dialog box that are displayed no matter which tab 1s selected 5 1 1 Permanent Controls The lower half of the Focus Controls dialog is present regardless of the tab selected Camera Old om gt 060 saco aora Ss E Open Fluor TxRed FitC l 1 OPEN Open Bright Open Alt XY Stage Z Stage Neutral Density X 0 00 pm Simulated 7 Stage sd Primary p r A 100 x Loot mm Auxiliar E Y El l ENE Auto Focus E a rs Epon ee Y joo 10 1 1 gt 5 000 m 5000 set Filter Set Widefield 5 1 1 1 Camera and Display Settings The Exposure time slide bar allows you to select the duration of a single camera ex
178. his in Chapter 1 1 4 Chapter 2 Quick Tour This chapter will take you on a step by step guided tour of the basic elements of SlideBook This is the perfect starting point for the beginning user 1 5 Chapter 3 SlideBook Organization This chapter describes how hardware control image capture data storage and data analysis are organized into SlideBook menus windows and objects 1 6 Chapter 4 Configuring Your System If your system was installed by 31 or a 31 reseller then you will not need to configure your system prior to use However 1f it was not installed by 31 or a 31 reseller or you need to make changes to your system configuration you may do so by selecting Edit gt Define Optics or Edit gt Hardware Configuration Chapter 4 teaches you how to configure hardware and define optics 1 7 Chapter 5 Controlling the Camera and Microscope Next you will learn how to set your microscope to the proper optics and bring your sample into view and focus Any motorized aspect of the microscope can be controlled through SlideBook s Focus Window You may bring up this window by selecting Window gt Focus Window or clicking on the focus window button in the toolbar 1 8 Chapter 6 Image Capture and Import Parameters for image capture will be set in the Capture Window Here you will set camera parameters such as exposure and binning by performing test exposures You may bring up this window by selecting Image gt Cap
179. hite balance function in the capture dialog 1s sufficient please see Performing Auto White Balance for Color Capture on page 95 70 Light Source C Fluorescence Emission wavelength urm Transmitted Mode Unknown Filter Configuration Parameters Filter Configuration New Fluoroprobe kal Parameterz Hame Red 0 5 Prompt if mode ig not available hor current objective C Alternate Source Position Filter set widefield Excitation wheel position Unmaunted Internal turret position Unmaunted i Emission wheel positiorr Unmounted E LCO4BF filter position Ausiliary filter position Unmaunted Camera Any Unmountk Associated Channel Type FRET Conor FRET Acceptor FRET Transter Virtual AGB Green Virtual AGB Blue FRET Donor Prebleach Default Color Display C None Red Green Blue Pseudocolor Color cos le e e Monochrome E ee eo Pseudocolor Intensity RGE Settings OF Cancel Chapter 4 Configuring Your System Filter Configuration Parameters Filter Configuration New Fluoroprobe ka Parameters Remove Associated Channel Type Mame G Green FRET Donor OK FRET Acceptor Light Source FRET Transfer E Cancel f Fluorescence Virtual RGB Green Emission fp 5 Virtual AGE Blue wavelength um FRET Donor Prebleach Ps ver Transmitted Default Color Display Mode Unk
180. hows through you must raise the minimum intensity threshold on all of the RGB channels See Altering the Renormalization Parameters Lookup Table on page 171 4 To change the display so that the DIC and fluorescent channels are blended at 50 opacity select Blend Background from the View Settings button or select View gt Blend Background You will not need to alter the minimum intensity threshold on the RGB channels to see DIC 8 3 3 Cropping an Image You may wish to crop an image in order to reduce its size or center on a specific portion of the image CAUTION Cropping is an irreversible process You may wish to make a copy of the image before cropping see Copying an Image on page 159 or select Crop to New Image To crop an image 1 Open a Main or Three View of the desired image 2 Define a 2D or 3D selection as described in Making a 2D or 3D x y z or x y t Selection on page 186 177 SlideBook 5 0 User Manual 3 Select Image gt Crop to replace the existing image or Image gt Crop to New Image to generate a new cropped image 8 3 3 1 Extracting Timepoints from a Timelapse Image You may wish to extract only a subset of timepoints from a 2D Timelapse or 4D data series To extract timepoints 1 Select an image in an open slide or open a Main View of the desired image 2 Select Image gt Extract Subseries to New Image The following dialog will appear Extract subseries Starting time point
181. iating and Monitoring Timelapse Capture ooooninnnnccuooconaoaoooonnnnonnnnnonnnnnnnnnnnnnn nono nn nono nono nono nnnnnnnnnnnnos 119 LRD Creating Graphs to Monitor Regions of Interest ccccccseesesseeeeeeseeeeeeseeeeseeeeeeeeeeseseeseeeeeeeeeeeesseeeeegs 120 f252 Pocas Durna Capre est he cra ce decenas 121 12 53 Viewme Previous LimEpOIM So emi 122 F3 JOC ATTORE oaa a a a a Aa a a OI 124 Tad Shifting the Volume During DIMAS a o SA 125 7 3 2 Mid volume Capture During 4D Imagine occ ccc nooo a a a E a 123 Vso MAA CARR A ES TUS a ad a e da 127 7 4 MULTPON CAPTURE aaa 127 TAL SCENE ADUCE FENCES tid ht relate tina ate keds 127 TAD Sene FOCUS Window Parameters 2 A O tada 128 7 4 2 1 O 128 TA 22 Saving and Loading a Multa 129 7 4 35 Setting Capture TNA WITT ai 130 7 4 3 1 5D Multiple Gocattom AA EEE E EN NA 130 7 4 3 2 Timelapse Multiple Location Cap Mres naa e e A a E A E 130 7 5 MONTAGE CAPTURE rira A R AE See N E OT EE E E 131 kdd SCAN SE OCUS WINGOW F AIQINCLCTS oeira E a S E E AE ane Beaten teacate ek 131 Lha Sene Capture Window LILA Saa 132 7 6 MULTIWELE CAPTUR D it cae late AE ee ee A Rae 133 A AFAN GC LGV OUT aires pete ces RN Gece iterate Sines Sac A Ne Gl eee ae aniseed 134 7 6 2 Manipulating Hardware Focusing on the Sample oooononnnnnnnnnonnonococooocnnnnnonnnnnononnnnnnnnnnnnnnnnnnnnnnnnnnnns 136 RD CALO TIC SIAC CT OSU ONS SS A See en ence ea ace 136 LOA Savednd Restore Cara OSTIAS eros i inn Sneed brie A
182. ick Tour slides have been installed If not please go to Chapter 1 Installing SlideBook and follow the procedures for installing the software and the Quick Tour slides 2 1 Opening Slides and Images To begin the tour we will first open a slide file SlideBook s native file type sld A slide file corresponds to a single file on the disk and can be thought of as SlideBook s document type It can contain many multi plane multi channel images in any combination 1 Launch SlideBook by pointing to Programs gt SlideBook from the Start menu An empty slide will appear in the window under the SlideBook menus 2 Close the new slide by choosing File gt Close Slide or by clicking on the l in upper right corner 3 Choose File gt Open Slide and navigate to the Sample Files folder in the SlideBook directory Double click on QuickTourWIN1 sld The following slide window appears that shows a thumbnail of one image This slide contains a single 3D image of a dividing B cell 6 4 QuickTourWIN1 1 o O a UE PO Capture Type e osi l M 2 2 Displaying a Main View SlideBook can display any image using a variety of views Each data set can have as many views open at one time as you desire SlideBook has seven kinds of views that display individual images 23 SlideBook 5 0 User Manual Main View Three View Tile View Channel View Multidimensional Channel View 3D Volume View 3D Surface View
183. ide of the dialog box Edit Hardware Properties Ad Property formation F vanced Capture Settings Advanced Z Stage Parameters Mame StaticSeralMoveDelay H Capture Direction Up S StaticS erialMoveDelay Static T TLMoveDelay Maximum StageSizeMicrons Enable Position Query Analog Y Stage Control Properties Analog Stage Control Properties Andor Properties SOTF Properties AST Hy Stage AS Z Stage Camera 1 Camera 2 Camera 3 Camera 4 DeconLive Settings Diagnostic Instruments Camera Properties 0 Dongle Info Durnmy Camera Dummy Camera 2 Dummy Camera 3 SPIE Enza Durnin Hardware Filter Set Information Focus Window ee FRAP Parameters Hamamatsu Properties Hardware Properties Version Info Fevert To Default Che Leica CTR ya Category Advanced lt Stage Parameters Description The number of milliseconds to Walt after a serial lt stage move during capture ms Default Value ES E El E e E E e E e E El E E E El E E E E E E a 4 Click on the next to Polytech PI to expand the section 5 Click on ShortMoveSerialDelay 6 Enter 0 in the edit field and then select Apply Change Edit Hardware Properties Leica CTR Leica 0M 000 Lifetime Lud Info Marzhauser Corvus Memory Cache NAI Fiber Switcher Mikon TE2000 Ocular Lightpath Olympus OP Camera Olympus OSU Settings Olympus MAL Olympus TTL TIRF
184. idual users to access their capture preferences see Saving Capture Parameters on page 154 Hardware filter and objective configurations are shared among all users To configure user logins 1 Goto Edit gt Hardware Properties and scroll down until User Login Properties is visible 94 Edit Hardware Properties TIFF Spooling TILL Polpchrome M TILL Polchrome W TTL Filter heel TTL Shutter Properties TTLDAO Alternate Source TTL DAG Analog Input TTLAOAG Ausiliary Filter TTL OAQ Brightheld Shutter TTLDAD Camera TTLDAD Emission Filter TTLOAG Excitation Filter TTLOAQ Extermal Trigger TTLDAO Fluorescence Shutter TTL DAD FRAP Trigger TTL D40 LCD Filter TTL DAG HD Filter TTL DAQ 1 Stage TTLADAQ Z Stage User Login Properties Enable User Login Enable User File Names Volume Rendering ey Stage Yokogawa CSU Properties Zeiss Amol mager Zeiss Amol bserver Zeis 4vioplan 2 Asiovert 200M feiss MCU 28 Chapter 4 Configuring Your System Property Information Mame Category Description Apply Change Default alue Revert To Default Clase 2 Click on the next to User Login Properties to expand the section and then click on Enable User Login Edit Hardware Properties 3 Click Yes and then Apply Change TIFF Spooling TILL Polychrome M TILL Polychrorme W TTL Filter Wheel TTL Shutter Properties TTL DAG TTL DAG TTL DAG TTL DAG TTL DAG TTL DA
185. ified in the default sld view as shown below 159 SlideBook 5 0 User Manual A Capture Types fo Comments Capture Date Capture Type 2D Capture 3 10 2009 13 12 33 2D Capture 2 1392 x 1040 5 5 MB 0 3D Capture 3 10 2009 13 22 29 3D Capture 2 1392 x 1040 x 7 38 7 MB 0 Bg 2D Timelapse 3 10 2009 13 23 30 2D Timelapse 2 1392 x 1040 x10 55 2 MB 0 4D Capture 3 10 2009 13 26 19 4D Capture 2 512x512x7x10 70 0 MB 0 a t Capture Capture Timelapse Capture ThreeView Y Y Yo sDTileView Y AAA IO O Multidimensional Channel View V 3D Volume View A ee a 4D Volume View 3D Surface View 2D Timelapse Intensity Plot Next we will discuss how to generate data views and manipulate those views using the info tool bar Finally we will discuss display views Views 8 2 Using Data Views to Display Images This section describes how to display data views SlideBook allows multiple data views of a single image to be open simultaneously 8 2 1 Displaying a Main View The Main View can be used to display any type of data The Main View displays a single plane at a time where the z or t axis can be scrolled using arrows or a slider To generate a Main View 1 Choose File gt Open Slide and navigate to the desired slide file Double click on the slide file to open it A slide window will appear 160 Chapter 9 Preparing an Image for Analysis or Export A QuickTourWIN1 Commen
186. ilable from the SlideBook toolbar at the top of the application window 184 Zoom Tool E Point selection Tool Ruler Tool Sl Chapter 9 Preparing an Image for Analysis or Export Allows you to draw elliptical ROIs on an open view by clicking and dragging Allows you to draw closed polygons by clicking to draw a series of straight connected lines Double clicking closes the shape Allows you to draw freehand by clicking and holding Shapes close automatically when mouse button is released Main and Three Views lets you move the displayed portion of an image in a window smaller than the dimensions of the displayed axes performs the same function as the horizontal and vertical scroll bars Tile View that displays a portion of an image adjusts the position of the display cube within the image therefore scrolling the portion of the image that is visible in all planes Main and Three Views grows or shrinks the size of the window and image by a factor of two Tile View the overall size of the window and its panes remains constant but the zoom factor of the displayed image is adjusted by a factor of two Clicking on a view will zoom in double the size and clicking while holding down the shift key will zoom out halve the size Three View updates the displayed position in the other two panes by clicking or dragging in any of the three panes Dragging over a pane will continuously update the other two panes
187. ill appear that will allow you to choose the file destination Select a destination and a file name then select Save The file will be saved as a tab delimited text file txt file which can be easily importable into a spreadsheet program such as Excel 10 7 Displaying Graphs SlideBook allows you to view intensity profiles of selected data You may view line intensity or timelapse intensity profiles 10 7 1 Viewing a Line Intensity Profile SlideBook allows you to generate a line intensity profile using the Statistics menu To view the intensity profile of a line do the following 248 1 From an open view use the ruler tool to draw a line see Making Distance or Velocity Measurements on page 187 You may draw this line in two or three dimensions Select Statistics gt Line Intensity A dialog box similar to the following will appear Chapter 10 Using Masks for Image Analysis Line Intensity 5 Select the channel that you would like to view from the Channel menu The line intensity profile displays pixel distance on the x axis and intensity on the y axis To export the graph as a tiff click on the t1ff icon a You will be prompted to enter a filename To export the data as a text file click on the notepad icon 2 You will be prompted to enter a filename Click Close to close the dialog box 10 7 2 Viewing Timelapse Intensity Profiles SlideBook also allows you to view timelapse intensity profiles There a
188. images in the slide 6 Click OK The projection images will be added to the bottom of the slide The type of projection will be displayed in the Comments section of the slide view 9 9 Creating an Interpolated Isotropic Image You may notice that your object may appear disproportionately small or squashed in the z direction especially when using the Three View When performing 3D imaging each plane that is captured is represented by a set of pixels in the image set Each pixel is a perfect cube You will notice that the z dimension in the 3D image set appears to be different than what was actually captured This is because the z step size that was selected during capture is not actually the same as the length of the z dimension of the pixel In the image set planes are simply stacked on top of one another with no regard for the actual z distance traveled this 1s done in order to preserve data fidelity Generating an interpolated image will expand your z dimension to represent the actual z distance traveled by the microscope Creating interpolated images can also help smooth the z axis when less than optimal z step size 1s used To create an interpolated image 1 Select the image that you would like to expand by clicking on it You may also start from an open view of the image 2 Select Image gt Create Interpolated Image The following dialog box will appear Image Interpolation Image Range f Current Image Tour image has
189. imum extent permitted by applicable law 31 and its suppliers disclaim all other warranties and conditions either express or implied including but not limited to implied warranties of merchantability fitness for a particular purpose title and non infringement with regard to the SOFTWARE PRODUCT and the provision of or failure to provide Support Services This limited warranty gives you specific legal rights You may have others which vary from state jurisdiction to state jurisdiction LIMITATION OF LIABILITY To the maximum extent permitted by applicable law in no event shall 31 or its suppliers be liable for any special incidental indirect exemplary or consequential damages whatsoever including without limitation damages for loss of business profits business interruption loss of business information or any other pecuniary loss arising out of the use of or inability to use the SOFTWARE PRODUCT or the provision of or failure to provide Support Services even if 31 has been advised of the possibility of such damages In any case 31 s entire liability under any provision of this EULA shall be limited to the greater of the amount actually paid by you for the SOFTWARE PRODUCT or U S 5 00 provided however if you have entered into a 3I Support Services Agreement 31 s entire liability regarding Support Services shall be governed by the terms of that agreement Because some states and jurisdictions do not allow the exclusion or limitation of li
190. in View Three View or a Tile View of an image see Using Data Views to Display Images on page 160 3 Select Image gt Align Channels The following dialog box will appear Alignment Guide Align all we timepoints Deltas 4 Select the channel that you wish to align from the dropdown menu 5 Use the left and right arrows to move the selected channel in the x direction and the up and down arrows to move in the y direction or enter an offset for any axis directly into the corresponding edit controls NOTE The view will update automatically when you use the arrow buttons but you must press the Apply button if you manually enter the offsets Clicking Cancel will ignore any changes to the edit field that were not previously applied 6 Click OK when you are satisfied with the image alignment NOTE If you find it necessary to align certain filters consistently your system may need repair Please contact 31 support intelligent imaging com or 303 607 9429 205 SlideBook 5 0 User Manual 9 4 Manipulating Timelapse Series SlideBook allows you to remove timepoints from a timelapse series or merge two timelapse series into one series 9 4 1 Removing Timepoints from a Timelapse Series To remove timepoints from an image 1 Click on an image s thumbnail in the Slide View or have an open view and select Image gt Advanced Operations gt Remove Multiple Timelapse Planes The following dialog box will appear
191. increase the lower threshold or decrease the upper threshold then select OK The number of objects generated will decrease This is particularly useful when working with noisy data that may contain a very large number of objects NOTE You may not decrease the lower threshold to increase the number of objects If you have removed too many objects then you will need to remake your mask 10 2 4 Splitting Objects If you have two or more objects that are touching but exist under one contiguous region of the mask a mask object you may choose to split the mask into multiple objects This can be achieved either manually by drawing lines along which to split the object or in an automated fashion 10 2 4 1 Manually Splitting Objects To split objects manually use the line ROI tool to draw lines where you would like to separate the objects Then select Mask gt Advanced Operations gt Split Objects Along Lines To see the split objects turn the ROI display off by going to Annotations gt Regions You may undo the split by selecting Mask gt Advanced Operations gt Undo Split Objects Along Lines 10 2 4 2 Automatically Splitting Objects To split objects automatically SlideBook employs a watershed algorithm This is useful to separate objects that may be joined by a thin connector as shown below Raw Data Initial Mask After Split Objects To perform a watershed operation 1 Generate a mask that contains distinct regions either u
192. ing Image Information After Capture on page 101 9 10 Applying Filters SlideBook offers a variety of filtering options including a user defined kernel and semi defined Gaussian Gaussian derivative hot pixel median and mean filters These filters perform local rather than global calculations on a pixel by pixel basis In other words each pixel is altered based on the intensities of the neighboring data 9 10 1 User Defined Convolution Kernels This filtering option allows you to define a custom kernel To apply a custom kernel 1 Select the image that you would like to correct by clicking on 1t You may wish to copy the image before applying the filter as it is an irreversible operation see Copying an Image on page 159 of Chapter 5 You may also start from an open view of the image 2 Select Image gt Filter gt Convolve The following dialog box will appear 216 Chapter 9 Preparing an Image for Analysis or Export Convolution Filter Filter Description Convolution Filter a general purpose filtering tool m with a user entered kernel Channel Parameterz For the following channels Coefficient Offset O C 5x5 o 7 Destination o f Replace each channel 6 Create a new filtered channel Cancel Select the Batch as to determine if you will apply the filter to the current image or all images with the same channels Select which channels will be filtered using the convolution kernel by c
193. ing an Image for Analysis or Export 5 Select Capture Image 1 SlideBook will capture an image and display 1t in the upper left corner of the guide Adjust the exposure time such that the maximum intensity shown in the histogram is 3000 3500 for 12 bit CCDs on a 0 4095 scale or 48000 56000 for 16 bit CCDs on a 0 65535 scale 6 If necessary make an appropriate adjustment to the exposure time and click Recapture Image 1 Otherwise move the xy position of the slide slightly and capture the next image by clicking Capture Image 2 After the first image it s unlikely you ll need to recapture any of the subsequent images but you will still have that option 7 Acquire the remaining images making sure to move the slide slightly between capture 8 Once you ve captured all the images click on the Finish button to complete the process 9 Repeat the process for other optical combinations 9 5 1 2 When to Update the Flat Field Database If you try to add a flat field matrix for optics where one 1s already defined SlideBook will alert you and ask if you want to replace the existing one SlideBook does not keep multiple flat field matrices for identical optics As time passes 1t 1s possible for a flat field matrix to become outdated Specks may have changed position or other constant artifacts have either appeared or disappeared A telltale sign that it is time to update a flat field is when performing the flat field operation no long
194. ing microscope parameters in the Focus Window and then setting capture parameters in the Capture dialog box In order to perform 3D capture your system must have motorized z control There are two procedures for performing 3D capture In Method 1 you will use the focus window both to bring the sample into view and to interactively set the capture extent in the z dimension These z parameters will then be imported into the capture dialog box In Method 2 you will use the focus window to bring the sample into view and focus but then set the z parameters in the capture dialog box You may wish to use Method 2 if you already know the z range of capture For example if you are imaging an object that you know to be 30 um in height you may bring the cell into focus and then capture 20 um above and below the plane of focus However if you are unsure of the extent of the object of interest in the z dimension you should use Method 1 since it allows the range of capture to be interactively defined Both methods are outlined below 7 1 1 Method 1 7 1 1 1 Setting Focus Window Parameters 1 Open the Focus Window by either selecting Window gt Focus Window or clicking on the focus window button in the toolbar The current camera image will begin updating as soon as the Focus Window is opened 2 Select your objective magnification changer and filter configuration and bring your sample into view and focus as discussed on page 85 in Chapter 5
195. ints but the LL or UR designation will move to the new point Once you have added the locations to the list you may visit or adjust them To perform these operations first highlight the location by clicking on it and then click on the appropriate button to perform the following actions e Visit Point moves the stage and z focus to the selected location e Clear All clears all locations from the list e Reset All Z resets the z coordinate of all locations to the current z position e New Point Z resets the z coordinate of the selected location to the current z position 8 Once you have selected two corner boundaries close the lightpath for light sensitive samples by choosing Close Fluor 7 5 2 Setting Capture Window Parameters 1 Once you have set the boundaries of the montage image in the Focus Window open the Capture dialog box by selecting Image gt Capture or by selecting the capture icon in the SlideBook toolbar cn 132 Chapter 7 Advanced Capture 2 Select channels and set exposure times as described in the section Selecting Channels and Setting Exposure Times in Chapter 6 on page 89 Enter image information and settings as described in the section Entering Image Information and Optical Parameters before Capture in Chapter 6 on page 93 NOTE It is critical to verify that the Objective and Mag Changer fields under Optical Parameters are set correctly SlideBook uses its knowledge of the size of
196. istics Path Lifetime Apply to Batch Path Start Point Path End Port MSO Select All Clear All Calculate and Continue Theze statistics report information pertinent to the path as a whole such as lifetime or total distance ct traveled aop Menu Mask gt Path Statistics Nest gt Close Here you may choose to calculate the following statistics for each path Fig 10 1 Schematic of tracked object Blue circles represent centroids e Endpoint Displacement the distance traveled between the first and last timepoints of the path as represented by the length of the red line in the figure above e Endpoint Speed the distance represented by the length of the red line divided by the time required to travel that distance e Average Speed the total distance traveled the sum of distances d1 to d7 as shown in the figure above divided by the time required to travel that distance e Total Displacement the total distance traveled by the object the sum of distances d1 to d7 as shown in the figure above e Path Length number of timepoints in the path e Path Lifetime the total elapsed time for a given path 236 Chapter 10 Using Masks for Image Analysis e Path Start Point centroid of the first point in the path e Path End Point centroid of the last point in the path e MSD mean square displacement coming soon NOTE The Path Statistics dialog box can also be found b
197. itals Notes that you should pay particular attention to are prefaced CAUTION 1 2 Of Pixels and Voxels In other contexts image typically connotes a two dimensional entity However SlideBook was specifically designed to operate on three dimensional data as easily and intuitively as two dimensional data In light of this a two dimensional data set can be seen as a special case of a three dimensional set where data has been collected for only one value of the z axis An image corresponds to all data collected during a particular capture To simplify this manual we always refer to a single data element as a voxel and a region as a volume regardless of the depth of the image These terms map directly to pixel and area respectively for a two dimensional set XIV Using this Manual Using this Manual The SlideBook Manual describes all of the steps necessary for performing digital microscopy from installing the software to analyzing images The chapters are outlined below If you are new to SlideBook you may wish to start by taking a step by step Quick Tour as outlined in Chapter 2 This will familiarize you with basic elements and commands in SlideBook 1 3 Chapter 1 Installing SlideBook If your system was installed by 31 or a 31 reseller then you will not need to install software on your system However if it was not installed by 31 or a 31 reseller or you need to upgrade your software you can learn how to do t
198. iven by the equation below The value T threshold is considered to be zero intensity Thus adequate background subtraction must be performed Y Y bh 1 gt T ls gt al b Pel po i eS Sp All T All b Mi SlideBook offers the following statistics to describe relationships between objects that are present in two different masks You must first set the Mask Scope to Object then select a secondary mask to view these statistics Cross Mask e Mask Overlap objects for each object in the primary mask this returns a count of the number of overlapping objects in the secondary mask e Mask Overlaps voxels for each object in the primary mask this returns the number of overlapping pixels voxels in the secondary mask 247 SlideBook 5 0 User Manual e Mask Overlaps largest object volume for each object in the primary mask this returns the area or volume of the largest overlapping object in the secondary mask e Mask Contains Other Masks returns the IDs of the objects in the secondary mask that overlap with the primary mask 4 Select Display to generate a new window that shows the selected statistics Alternatively you may directly export the data by pressing Export see below If you have generated object data you may click on any of the object numbers in the table and the corresponding object will be highlighted in pink in the data view To save the data select the Export button A dialog box w
199. k Delete from all images in current slide Delete mask Mask 1 C Delete all masks Cancel 2 Choose the mask s you wish to remove using the radio buttons and dropdown menu You may also choose to delete the mask from the current image or from all images in the slide 3 Choose OK to remove the mask 10 4 Boolean Mask Operations Masks can be combined or altered using Boolean operations For instance the intersection of two masks can be generated by an AND operation The Mask Operations dialog lets you either generate a new mask that contains the result of a Boolean operation or replace an existing mask with the result of an operation To perform Boolean mask operations 1 Open a view that contains the masks you would like to manipulate 2 Select Mask gt Mask Operations The following dialog will appear Mask Operations f Perform operation on selected masks in all images in current slide Lett Mask Operation Right Mask 9 ehu Mask 2 C MINUS t NOT t OF C SOR Mask 2 Result C Left Mask 6 Mew Mask C Right Mask Mask 3 240 Chapter 10 Using Masks for Image Analysis Using the radio buttons select whether the operation will take place for the current image or the entire slide Select the masks to be used from the Left Mask and Right Mask lists Select the operation to be performed using the radio buttons e AND selects pixels that are in both masks e MINUS removes any pixe
200. lar photo and CCD Video prisms as well as multiple cameras you may define the positions of the prisms that correspond to each camera To do SO L 2 6 Select Edit gt Hardware Properties and expand the section titled Camera 1 Click on Prism1Pos The right side of the dialog will be updated Edit Hardware Properties Ad Property Informatica vanced Capture Settings Advanced Stage Parameters Mame Prien Pos Analog 1 Stage Control Properties Analog Stage Control Properties Andor Properties ADTF Properties o B ASI gY Stage a i i hor light to ASIZ Stage e directed to camera Bitflow Camera 1 S PriemlPos Preme Pos Camera 2 Camera 3 Camera 4 DeconLive Settings Diagnostic Instruments Camera Properties Dongle Into Dummy Camera 2 Category Camera 1 Dummy Camera 2 Dummy Camera 3 Dummy Hardware Filter Set Information Focus Window FAAP Parameters Hamamatsu Properties fo Hardware Properties Version Info Laser Launch T est Leica CTR Revert To Default Close Leica DM s000 Apply Change Default Value E E E E H E H H E H E H H E H E En Enter the value of the ocular photo prism Prism 1 position that corresponds to the position of Camera 1 in the edit field Select Apply Change Repeat steps 4 and 5 for Prism2Pos CCD Video prism Repeat steps 2 through 5 for any other cameras and the ocular lightpath Once you have defined the prism positions you can click
201. le that permits the application to run on the machine that has the key attached The key is a Sentinel series black with purple accent USB device manufactured by SafeNet Before you begin the software installation please connect the dongle to a USB port in the computer NOTE Given the hardware key system it is possible to install the SliideBook software on several different machines However the software will only run on the machine that has the key attached Networked computers may run SlideBook if one of the computers on the network has a network key 17 SlideBook 5 0 User Manual 1 3 About SlideBook Installers SlideBook installers are available as compact or complete Compact installs SlideBook program files Sentinel dongle driver user manual and QuickTime Complete installs all SlideBook program files user s manual QuickTime program files and Sentinel dongle drivers Also allows the option to copy camera driver files Edgeport driver files and QuickTour sample files to the Intelligent Imaging Innovations Inc directory 1 4 Copying the Software from CD ROM Insert the SlideBook CD in your CD ROM drive Navigate to the CD ROM and copy the SlideBook setup program InstallSlideBook exe to your desktop Double click on the icon to begin installation and proceed to the section Installing SlideBook 5 0 below 1 5 Copying the Software from your Download Site Every Sli
202. lider PolyTech Pl Prior PYCAM i go Clearlycles Apply Change Flip PY OAM Camera 0 Default alue Flip PCS km Camera 1 Flip PY OAM Camera 2 p Revert To Default Close E E E E E E E E E E E E E El E E E Flip PCS km Camera 3 Number of PYOSM Free Run Butter Set PY OAM Camera Cooling Temperature PY CAM Camera 0 Cooling Temperature Set PY OAM Camera 1 Cooling Temperature PY CAM Camera 1 Cooling Temperature Set PY YOOM Camera 2 Cooling Temperature 4 Enter 0 in the edit field and then Apply Change 5 Click Close and then restart SlideBook to register the change 6 Before performing an experiment make sure to capture a test image immediately before beginning the experiment This will help reduce the effect of charge build up in the first frame of the image capture 7 8 2 Sutter DG 4 You may increase the switch time on your Sutter DG 4 by employing TTL control You must purchase the SlideBook TTL Synchronization Module to enable TTL control please contact 31 at support intelligent imaging com or 303 607 9429 if you wish to purchase this module Note that in order to use TTL control the exposure times of the various channels to be captured must be equal Please see the TTL module documentation for details on configuring a system for TTL control The TTL module for SlideBook includes software plus a National Instruments multifunction DAQ board and breakout box 7 8 3 Z Stages The
203. llecting Flat Fields In order to perform flat field correction SlideBook needs to know what an image with no sample in focus looks like This image should contain all the constant artifacts but little that will change from sample to sample From this image SlideBook can compute a coefficient matrix that can be applied to an image to eliminate most of its constant artifacts SlideBook in fact can average several empty images to better ensure that the flat field contains only constant artifacts of the optical system and not some peculiarity of a single empty image Since constant artifacts might exist anywhere along the optical path a flat field matrix is necessary for each combination of objective filter and magnification changer setting To keep things simple SlideBook stores all collected flat fields in a single database and keeps track of the optical parameters for each one 207 SlideBook 5 0 User Manual 9 5 1 1 Adding or Replacing a Flat Field If you want to add a flat field matrix to the database or replace an existing entry you will need to capture a series of images on an empty sample using the desired set of optics including the filter objective and magnification changer To add or replace a flat field do the following 208 1 Place a blank slide containing no sample or a uniformly fluorescent slide 1f collecting a fluorescent flat field on the stage Bring the desired optics into place as descri
204. lready displayed 3 Select the z axis from the axis menu to return the view to the xy plane 2 3 5 ii Renormalize Button The renormalize button brings up the Renormalize Image dialog box This tool allows you to change the range of intensities that are displayed for any channel that you choose 1 Click on the renormalize button The following dialog box will appear Renormalize Image 0 Channel Cra Range Low 934 High 7074 Gamma i C Intensity O Reset to Selection Min Max Reset all to Image Min Max so ce oR A histogram 1s displayed showing the relative number of pixels on the y axis and intensity values on the x axis for the specified channel In this image the CY3 channel intensities range from 0 to 14281 2 Click on the drop down channel menu and select FITC 3 Click on the red and green bars in the histogram window and drag them to the left or right observing the change in the display of the green data The red and green bars allow you to select the minimum and maximum intensities that correspond to the absence black or full saturation of color in the display respectively Again the underlying values in the image are not affected l SlideBook 5 0 User Manual 4 Now enter the numbers 300 and 800 in the Low and High data entry fields Note that the image does not automatically update as it does when moving the red and green bars 5 Click Apply to register the change and up
205. ls that are present in the right mask from the left mask e NOT selects all pixels that are not marked in the left mask e OR selects pixels that are in either of the two selected masks e XOR adds both masks and removes any common pixels Select the destination for the mask operation using the radio buttons If you want the result to replace the contents of elther the left or right mask select the corresponding radio button for that mask If you want to generate a new mask select the New Mask radio button and give the new mask a name in the edit field Click OK to perform the Boolean operation You may display the new or edited mask as described above 10 5 Using Masks for Smooth Curve Analysis Kymograph You may use masks to identify regions to be used for smooth curve analysis also known as kymograph analysis Smooth curve analysis allows you to follow time dependent lateral intensity changes along a static object For example an action potential moving along a neural spine could be displayed as an x vs t image using smooth curve analysis You could then extract velocity and displacement information from this image To perform smooth curve analysis L 2 Open a Main View or Three View of your timelapse data Create a new mask as discussed above in Creating an Empty Mask on page 226 Select the single pixel pencil tool from the tool menu and draw a line along the desired object Copy the mask to all planes in the
206. m lo 27 m Use topand bottom positions Offset um 425 2 Return to center of volume after capture your capture any time after it has started e Pause pauses the capture until you press Continue e Cancel cancels capture and does not save any images e Stop stops capture and saves a truncated sequence 7 1 1 3 Setting Capture Preferences to Open and Close Shutter during Capture If you are performing single channel 3D capture the default fluorescence shutter action in SlideBook is to leave the shutter open during capture If you would like the shutter to open and close after every capture select the Advanced button in the Capture Settings section of the Capture dialog box Select the 2D 3D capture checkbox labeled Open and close shutter on single channel or simultaneous 3D capture in the General tab The default action for multiple channel capture is to open and close the shutter after every capture 108 Chapter 7 Advanced Capture Capture Preferences Spool Sequence Notes General TTL Focus Periodic FRAP 20 30 Capture Altemate two channel mode ABBAABBA instead of ABABABAB Live Capture Captured Image Updates Suppressed When Possible C Always SS EET TET TT doves if Update histogram every h frames W Update displayed frame every i frames W Update curent plane every i frames Suppress TTL optimization of hardware devices Automatically restore focus and image capture dialog
207. mage Desktop Uy Moni f A Computer A Network wae I Files of type TIFF Files tif tiff x Cancel 7 Open as read only 4 Navigate to the file and select Open The following dialog box will appear if you have selected TIFF If you have selected TIFF sequence proceed to step 6 99 SlideBook 5 0 User Manual Import TIFF There i one plane in this file Select which planes will be imported into one or more channels of an image Note that all plane numbers are zero bazed L e plane number 0 12 the first plane First Range Import i planes starting at plane 0 as channells Taed for red data Fit for green DAPI for blue data Second Range a E planes starting at plane p as channel c5 Third Range E E planes starting at plane p as channel C 5 Select the planes you would like to import as well as the channels to be used for each color in the image and select OK For example 1f you have an image that displays FITC as green data you will designate the green data as FITC in the dropdown menu 6 The following dialog box will appear 100 Chapter 6 Image Capture and Import Image Info Image Name Edit Info la Cancel Capture Info Date Unknown Capture type Unknown Microns per pel pr Unknown Step size in microns Unknown Objective Mag changer Binning 1x1 Channel TsAed at 0 me Independent Channel 2 FitC at O
208. mask in current z plane to f all planes f all subsequent planes C the nest i plane s C planes p through 5 Timepaints i Copy mask in current timepoint f all timepoints Call subsequent timepoints f the next h timepointe s t timepoint o through 20 Cancel 2 Select the desired data you would like to copy the mask to and select OK You may also copy the mask to all of the images of the same size in the same slide To do so from an open view of an image 1 Display the mask that you wish to copy by selecting 1t from the mask menu in the info tool bar 2 Select Mask gt Copy to all images in slide The mask will be copied to all of the images in the slide provided they are the same size 10 2 3 Defining Objects Objects are defined when you wish to get data for individual objects in a mask The mask statistics dialog box allows you to automatically define objects when generating statistics However in order to view timelapse graphs of individual objects or to obtain a quick object count you will need to define objects To do so 1 Generate a mask that contains distinct regions either using manual editing or segmentation techniques 2 Select Mask gt Define Objects in Mask The following dialog box will appear 229 SlideBook 5 0 User Manual Define Objects Size Filter Generate for all similarly named masks in current slide Cancel The dialog box allows you to set upper and
209. melapse or 40 images Particles must have been properly c identified prior to tracking top Menu Mask gt Basic Particle Tracking The dialog box contains the list of protocol steps on the left along with a dropdown menu for saving and loading settings The list of steps is underneath Required steps must be completed before moving to the next step in the protocol Steps that are not required may be skipped Once a step 1s complete an X will appear in the Done column The right side of the dialog will update as you progress through the protocol 3 Select Tracking Parameters as described below e Mask Name Select the Mask that contains the objects you would like to track The menu will default to the mask that is currently displayed on your open view 233 SlideBook 5 0 User Manual 234 Remove Objects smaller than Check this box to selectively remove objects smaller than a given size Enter the size in the edit field and choose either microns or pixels as the unit Track with Select the parameter that you wish to track from the dropdown list Center of Area the coordinates of the center of the object unweighted by intensity values Center of Intensity the coordinates of the center of the object weighted by intensity values If you select this feature you will also need to select the channel that is used for weighting Mean Adjusted Center of Intensity the coordinates of the center of
210. mepoints 1 2 and 3 9 1 1 Inserting an Image as a Channel You may wish to insert an image containing a single channel such as a background image into another image that you have collected To do this 1 Make sure that the image you would like to insert is in the same slide as the destination image see Copying an Image on page 159 2 Select the destination image the image that will contain the newly inserted image by clicking on 1t You may also start from an open view of the image 3 Select Image gt Channel Operations gt Insert Channel The following dialog box will appear 200 Chapter 9 Preparing an Image for Analysis or Export Insert Image as Channel Image Capture 3 Timepoint 1 Collapse all channels into destination Cancel Position Pos fp Y Fos 2 Pos Montage Image Infer Position Width Height 4 Select the image that you would like to insert as a channel from the dropdown menu If the image that you select contains more than one channel the channel that is listed as channel 1 see Getting and Editing Image Information After Capture on page 101 will be the channel that is inserted 5 Select the x y and z coordinate positions for insertion in the X Pos Y Pos and Z Pos edit fields If left at 0 O O the single channel image will be inserted in the upper right hand corner of the image in the first plane if the destination image has multiple planes Images tha
211. mplete the following box will appear noting the number of objects generated slideBook 7 Click on OK and now position the cursor over one of the objects in the Main View Note that the object number appears to the right of the cursor location coordinates in the info tool bar at QuickTourWIN HeLa Cel 100 M0b 52 O 8 SlideBook can generate individual statistics for each object Select Statistics gt Mask Statistics The following dialog box will appear 4T SlideBook 5 0 User Manual Mask Statistics Image Scope m Features Compute Category e C Date Of Capture none amaan Side anih Same Mask 7 Morphometry none Name Export Only H C Intensity none Description H C Cross Channel none E Under Development none Mask Scope C Entire Mask Object Export Primary Mask M ask 1 Cancel Remove objects smaller than 10 O Microns Pixels Some advanced features require a secondary mask Secondary Mask None X Remove objects smaller than 9 Expand the Morphometry and Intensity statistics selections by clicking on the signs to the left of the checkboxes Mask Statistics Image Scope Features e Current 2D 3D Image Compute Category O ames 0 Tre loss Did LE Morphomety none All Images in Slide with the Same Mask i oi C Area pixels r i C Name Export Only Area escription be Perimeter L
212. ms interval and have checked the Begin capture with first stage in sequence checkbox in the Capture Preferences dialog In the Capture window you select a timelapse capture with 100 timepoints and a 1000ms interval When you click Start capture will start by executing the sequential events in the displayed order in the Stages list After the total sequence has been captured 22 timepoints the remaining 78 timepoints 100 22 78 will be captured 152 Chapter 7 Advanced Capture with the interval set in the Timelapse Capture section of the Capture dialog box 1000ms 7 10 3 2 Example 2 Suppose you have set up capture as in Example 1 but did not check the Begin capture with first stage in sequence checkbox Capture will begin with 1000ms interval between timepoints To execute the stage click on the stage to be executed and click Execute Stage Twenty two timepoints will be captured The remaining timepoints will be capture at 1000ms interval until the total of 100 timepoints have been performed 7 11 Saving Images to Disk Spooled Capture SlideBook allows you to save your images directly to disk This 1s especially important when performing long timelapse captures that will exceed your computer s memory limit To save images to disk 1 From the Capture dialog box select Advanced to bring up the Capture Preferences dialog box 2 Select the Spool tab The following window will be displayed Capture Preferences Gen
213. my Camera 3 Dummy Hardware Filter Set Information Focus Window Property Information Mame Mad City Static Delay Categor 451 Z Stage Description Should the 45 Mad City 2 stage use a static delay 1 wait for controller gt O delay in millizeconds beyond serial port write delay i Apply Change Default Value p Revert To Default Close 6 Enter the number 1 in the edit field and then select Apply Change 7 Select Close and then restart SlideBook to register the change 1 Select Edit gt Hardware Properties acceptable results You may find that you need to increase the delay above Oms in order to achieve scientifically 7 8 3 2 Physik Instrumente PIFOC Piezoelectric Focusing Collar The PIFOC collar can be operated in TTL mode see TTL module documentation or in serial mode In order to operate in TTL mode you must purchase the TTL module for SlideBook You should perform the following procedure to increase speed in serial mode whether you are configured for TLL or serial mode There may be instances where the TTL mode will not be activated so you will need to increase the performance in serial mode To increase the speed of this device in serial mode perform the following steps 141 SlideBook 5 0 User Manual 142 2 Click on the sign to expand the section next to Advanced Z Stage Parameters 3 Click on StaticSerialMoveDelay and enter 0 in the edit field on the right s
214. n A pixel will be replaced with the mean or median intensity value of pixels within a designated Radius e Hot Pixel A pixel whose intensity value is higher than the mean of its neighboring pixels by more than the designated Factor will be set to the mean of its neighboring pixels 5 Select OK to filter the image 9 11 Performing No Neighbors Deconvolution Deconvolution is the process by which SlideBook mathematically removes out of focus information from a fluorescence image set SlideBook can implement three deconvolution approaches constrained iterative nearest neighbors and no neighbors No Neighbors deconvolution is incorporated into the base SlideBook package Nearest neighbors and constrained iterative deconvolution are available as an additional SlideBook module For more information about nearest neighbors and constrained iterative deconvolution refer to the supplemental SlideBook 3D Deconvolution Module manual or contact 31 support intelligent imaging com or 303 607 9429 No neighbors is the least computationally intensive method of deconvolution offered in SlideBook and the only method that can be used on 2D images While no neighbors deconvolution can be applied to three dimensional data sets it does not use information across multiple planes to calculate and remove out of focus information Rather 1t functions by creating a theoretical blurred image from the original two dimensional image This blurred image is th
215. nannnnonnnnnnncncnnnnnnncncnnnnnnnss 169 8 3 1 Altering the Renormalization Parameters Lookup Table 171 8 3 1 1 Changing Renormalization Parameters for a Single View cccccccceeeseseeeeeeeeeeeeeeeeeeeeeeeseeeeeeeeeeeeeeeeeeees 171 8 3 1 2 Changing Renormalization Parameters for a Group of Images in a Slide oooooonnccnccununuuanononononooororronnnnnnnnnoos 173 8 3 1 3 Applyime Nonlinear Lookup Tables cai tdt id tdt ea 173 Ge Chanoine tHe IS PICOS sn att da a ia et cda ed eel depa peine de 174 8 3 2 1 Changing the Channels Displayed on an RGB ImMag6 ooocnnnnnncccononononnnnanonnnnnonnnnnn nono noo nono nn non non naar nana nnn nana nss 174 8 3 2 2 Changing an Image Display to Monochrome ooooooccnnccnoonooooooonononono nn noo non ono nn nnno nono non nono nono nn nono non nn nn n nn nn nnnnnnnnnos 174 8 3 2 3 Changing an Image Display to Pseudocolor oocnnnnnnnninininnnonnnnnnnananananannnnnnnnnnno nono non nono anar nano nr rn rr nr nn nn nro nnnnnos 73 8 3 2 4 Changing the Display to a User Defined Color Palette oooocnnnnnnnccnnnnnnnnonanananaannanonorrrrrrrrono nono non nnonnnnnnononos 175 8 3 2 5 Displaying a DIC image as Background of an RGB Image nnnnnnnnnnnnnnnnererrerrrererrrrrrrrrrrrrrrrrrrrrrrrrrerreen 177 Oaa 10 0 01 SEA AIMA O nera T EEE a ee E 177 8 3 3 1 Extractine Timepoints from a Timelapse mace uti A 178 3d ROAN AR IMOT SEA ah Hea ada ane A did 178 OS AA a a a a a a a eeneeete 179 8 3 6 Scrollin
216. nd the list Edit Hardware Properties TTL DAQ TTL DAQ TTLADAGQ TTL DAG TTL DAG TTL DAQ TTL DAQ TTL DAGQ TTL DAG TTLDAC TTL DAQ TTL DAQ TTL DAGQ TTL DAG AY Stage E H E H E E E E H E E E E E H Alternate Source Analog Input ulla Filter Brighthield Shutter Camera Emission Filter Excitation Filter External Trigger Fluorescence Shutter FRAP Trigger LOD Filter ND Filter Y Stage Z Stage User Login Properties Volume Rendering MoveFieldRightSign H MoveFieldD ownSign StereclogyMontagel verlap FulllmageMontagel verlap Enable Y Position Query i Rotate Computed Montage 90 Degrees L Enable Home Command Override Yokogawa CSU Properties Zelzs Asiolmager feist Aol beerver Zeiss Asioplan 2 r Asiovert 200M Zeis MCU 28 Chapter 4 Configuring Your System Property Information Mame Category Description Apply Change Default value Revert To Default Close 6 Click on the item in the list titled MoveFieldRightSign The left side of the dialog box will be updated Edit Hardware Properties TTLADAG TTLDAL TTLDAL TTLDAD TTLDAD TTLDAO TTL OAQ TTLDAL TTL BAGQ TTL DAG TTL BAG TTL OAQ Alternate Source Analog Input Alla Filter Brightheld Shutter Camera Emission Filter Excitation Filter External Trigger Fluorescence Shutter FRAP Trigger LCD Filter HD Filter TTLADAQ
217. nformation and Optical Parameters before Capture You may specify the following image information and optical parameters in the Capture dialog box e Image Name and Comments can be entered in edit fields e Objective and Mag Changer can be selected or will automatically be selected 1f they are either encoded or automated using the dropdown lists The channels to be captured and their exposure times are automatically stored with the image Although these settings don t immediately affect the microscope settings or capture they are stored with the image and are important for correct deconvolution and measurement You can always go back and add or alter this information by selecting Image gt Get Info which is described in the section Getting and Editing Image Information After Capture on page 101 6 4 Capturing a Single or Multi Channel 2D Image The default mode of the capture window is for a single 2D image The 2D image may be either single or multi channel depending on the number of channels that you have checked If you wish to capture 3D timelapse 4D multipoint or montage images please see Chapter 7 Advanced Capture 93 SlideBook 5 0 User Manual 94 1 Once you have set all of the parameters channels exposure times binning etc in the Capture dialog box select OK to begin capture If your system does not have automated filter control you will be prompted to move the filters into the correct position
218. ngle sample and it is the native file type saved by SlideBook By collecting related images into a single slide SlideBook makes managing files easier Slides appear on your file system with a sld extension 53 SlideBook 5 0 User Manual 4 Configuring Your System In order for SlideBook to control any motorized aspects of your microscope system and to perform deconvolution and analysis of images it must know the optical and system parameters of the microscope used to collect the image By the end of this chapter you will know how to Designate User Logins Configure Hardware Define Objectives Define Filters Define Magnification Changers Work with SlideBook Preferences and Hardware Properties Define System Parameters Once the hardware is configured and the optical parameters are defined they can be utilized in several different ways If the optics are motorized they may be selected and brought into the light train using the focus window Once an image is captured using the selected motorized optics the capture information is automatically stored with the image If the optics are not motorized they may be selected in the capture window and then automatically stored with the image information Also optics information can be added or edited post capture which is especially useful for imported images 4 1 Designating User Logins You may define multiple user logins to separate user preferences The user login will allow indiv
219. nown f None e Prompt if mode is not available for Red current objective Green C Alternate Source Positions Unmaunt Filter set widefield Excitation wheel position Unmounted Internal turret positions lUnmounted Emission wheel position lUnmounted gt LCD BF filter position E Auxiliary filter position Unmounted Camera Any Blue Pseudocolor Color Pseudocolor Intensity RAGE Monochrome a ci CF Uy 0 oan 71 SlideBook 5 0 User Manual Filter Configuration Parameters Filter Configuration New Fluoroprobe ka Parameterz Mame Blue Light Source f Fluorescence Emission 0 5 wavelength um f Transmitted Mode Unknown Prompt if mode ig not available for current objective Alternate Source Position Unmaunt Filter set widetield gt Excitation wheel position Unmounted Internal turet positions lUnmounted Emission wheel position Unmounted gt LCDVBF filter position Auxiliary filter position Urmounted Camera Any Remove Associated Channel Type FRET Donor Uk FRET Acceptor FRET Transfer Cancel Virtual RGB Red Virtual AGB Green FRET Conor Prebleach Default Color Display f None Red Green Blue Pseudocolor Color Pseudocolor Intensity RAGE Monochrome a m m m m e A 4 4 3 3 Defining Filters when using an Image S
220. nsional Channel View Settings gt QuickTourWIN1 Metaphase B Cell 3 bol e ex Oli00 6 181 2 33 192 BE 189 FITC 48 DAPI None View settings Multidimensional Channel View Settings Annotation Settings Blend Background 4 The Multidimensional Channel View Settings dialog will open Adjust the following parameters to achieve your desired display Display Parameters are shown below Channel View Options e Select which DAR channels will be iaa displayed and select which channel will display the order in annotations such as Lookup Tables which they are shown Move Up Move Down spilt ares le A individual channels are Select if the individual Annotated Channel composte shown in monochrome EE iiaes UE kin or as the Channel horizontally as columns Channel Layout Horizantal vertical default setting ic ku vertically as i Component Image Col Channel Defaut 5 Monochrome RGB User defined Time pomts will array in Color etc the other coordinate Number of time points to show For images with time points select Ca the number of time p o points to display 5 Once the display parameters are selected as desired click OK 168 Chapter 9 Preparing an Image for Analysis or Export 8 2 5 Displaying a Montage SlideBook allows you to generate montages from images captured in SlideBook using the montage feature see page 131 To generate a monta
221. nuously will scroll e Using menu commands View gt Prev Plane View gt Next Plane View gt Go to Plane and View gt Loop will move the invisible axis location one plane down one plane up to a specified plane or in a continuous loop respectively See page 27 for instructions on how to use the Go to Plane feature NOTE To scroll around Three View images see the section titled Scrolling through a Three View on page 187 8 3 7 Changing the Default Display Once you have an image that has the color scheme and renormalization parameters that you desire you may save those parameters so that all subsequent views retain those parameters To do so 1 Using a Main View Three View or Tile View adjust the color display and renormalization parameters as described in the sections above 2 Click on the thumbnail button Al in the info tool bar to set new default display parameters The thumbnail image in the slide view and all subsequent views will display the new default parameters NOTE When using a Main View you may wish to scroll to a desired plane in the invisible axis before clicking on the thumbnail The selected invisible axis plane will be displayed in subsequent Main Views 8 3 8 Displaying Annotations The following annotation features are available in SlideBook 4 2 e Timestamps available for timelapse captures e Scale Bars bar showing relative scale size of image e Lookup Tables range of colors that corr
222. o capture 3D stacks at each location set up for 3D capture as discussed in 3D Capture on page 105 5 Click on the 3D checkbox in the Capture Type section 7 4 3 2 Timelapse Multiple Location Capture 130 6 If you would like the points to be visited repeatedly click on the Timelapse checkbox in the Capture Type section of the Capture dialog box 7 Enter the number of timepoints to be collected and the interval between capture in the appropriate edit fields NOTE In multiple location timelapse capture the timelapse interval corresponds to the time that elapses between the completion of capture for the last point in the list and the beginning of the capture for the first point in the list 8 Click OK to begin capture The Capture Status Window will appear and display each plane as it is captured You may choose to pause cancel or stop your capture any time after 1t has started Chapter 7 Advanced Capture 7 5 Montage Capture Often you may wish to capture an image that 1s larger than a single field of view SlideBook can stitch multiple fields together into what is called a montage image It does this by taking successive camera fields and moving the motorized xy stage a computed amount The procedure for capturing a montage image 1s described below NOTE Before attempting to perform montage capture make sure your xy stage 1s properly configured as discussed in Configuring a Motorized XY Stage on page 58 7 5 1 Setting
223. of time the giving of notice or otherwise constitute a violation of any applicable law or a breach of any provision contained in the Articles of Incorporation By Laws Articles of Partnership or similar document or contained in any agreement instrument or document to which You are now or hereafter a party or by which you may become bound SEVERABILITY If any provision of this Agreement is held to be invalid or unenforceable in whole or in part such holding will not affect the validity of the other provisions of this Agreement unless the unenforceable provision is essential to this Agreement FAIR MEANING The language in all parts of this Agreement shall in all cases be construed according to its fair meaning and not strictly for or against either party It is agreed that if any provision of this Agreement is capable of two constructions one of which would render the provision void and the other of which would render the provision valid then the provision shall have the meaning which renders it valid LIMITED WARRANTY LIMITED WARRANTY FOR SOFTWARE PRODUCTS ACQUIRED OUTSIDE THE US AND CANADA YOU HEREBY AGREE THAT THE UNIFORM COMMERCIAL CODE UCC OF CALIFORNIA AND THIS AGREEMENT SHALL CONTROL ALL WARRANTIES UNDER THIS LICENSE AGREEMENT AND EXPRESSLY WAIVE ANY RIGHTS YOU MAY HAVE UNDER INTERNATIONAL LAW OR TREATY INCLUDING BUT NOT LIMITED TO THE INTERNATIONAL CONVENTION ON THE SALE OF GOODS LIMITED WARRANTY FOR SOFTWARE PRODUCTS ACQ
224. olor picker on the right to select a color then select Add to Custom Colors 176 Chapter 9 Preparing an Image for Analysis or Export 5 To add a channel to display simply select the checkbox next to the channel You may choose to always display a channel using a color of your choosing by specifying it in the Filter Definition see page 66 8 3 2 5 Displaying a DIC image as Background of an RGB Image In an RGB image the first three popup menus control which channel is visible in red green and blue respectively The last popup menu labeled Bkgnd selects a background display channel In multi channel images one channel can be chosen to be displayed in monochrome wherever the other channels would not otherwise be visible This can be particularly useful for showing DIC information alongside fluorescence To display DIC alongside a fluorescent RGB image 1 Create a Main View Three View Tile View or Channel View of an image that contains a DIC channel and at least one fluorescent channel 2 Make sure that the fluorescent channels are displayed as RGB If they are not select View gt RGB Color to change the display to RGB and select the fluorophores to be displayed from the channel menus 3 Set the Bkgnd channel menu to DIC The DIC image will show through in any pixel that has RGB intensities that are below the minimum intensity threshold of the fluorescent channels If you would like to increase the amount of DIC that s
225. on for no neighbors deconvolution 9 12 Exporting Images If you would like to perform intensity analysis or image processing in a program other than SlideBook you may want to export the image from the Image menu This will export a 16 bit file that retains all of the intensity data from the original SlideBook image In contrast exporting an image from the View menu will generate an 8 bit file This will result in a loss of data assuming the images were captured on a 12 bit or higher digital camera To export an image from the Image menu perform the following steps 1 Select the image that you would like to export by clicking on it You may also start from an open view of the image 2 Select the appropriate file type from the Image gt Export hierarchical menu A dialog box similar to the following will appear 221 SlideBook 5 0 User Manual Tiff Export by image Image Range f Current Image C Curent Capture Type C All Images with Same Channels Selected Images Metaphase B Cell Output Directory CAU ser Monica Desktops anual York lf Write Log File Advanced Separate File For Each Plane Filename Format String ZN YP _Z Z_T v1 CAC EN Image Name EL Channel Index 22 E Plane Index ac Channel Mame ET Timepoint Indes lt P Unique Position Cancel 3 Select the Image Range as described below You may also select and deselect images using the checkboxes in the Selected Images list e C
226. ons close the lightpath for light sensitive samples by choosing Close Fluor 6 Return to the brightest field that you are planning to capture by selecting the location from the list in the XY tab and choosing Visit Point It is important that the brightest field is in view when setting exposure times in the Capture dialog box 7 4 2 2 Saving and Loading a Multipoint List This feature allows you to save the xyz multipoint list so that you may visit the same points in future experiments To do so 1 Before placing your sample on the stage open the Focus Controls and go to the XY tab Scope 7 xY Camera Stream Montage Extent Set Paint FA A Visit Point Update Y m 4 gt Update Z Reset All Load Save Clear Point Clear All Edit Description Home 2 Before setting points you will need to home the stage Some stages do not maintain an absolute coordinate system but rather reset to zero at whatever position the stage is in when it is turned on If it is your first time performing the home operation with the stage make sure that your objectives are in the load position as far from the stage as possible Once you have all possible obstructions cleared select Home If Home is grayed out you must manually home the stage by taking the stage to its limit at any of the four corners then resetting your stage to zero for instance on an ASI stage you would take the stage to the limit
227. onsult the SlideBook TTL Module Manual NOTE There are instances when you will need to select your objective and filter in the Focus Window even if these components are not motorized For example if you would like to perform auto focus you will need to select the objective and filter you are using In these instances you will define Manual Filter Turret or Manual Objective Turret COM port set to OFF Make sure that you specify the position of your objectives in the Objective Definition section see page 62 4 2 2 Configuring a Motorized XY Stage In order for your motorized xy stage to work properly when collecting montage images you will need to specify the relationship between the stage and camera l 58 To do so you will first need to bring a sample into view and focus in the focus window see Using the Focus Window on page 85 Using the joystick move the stage so that the image on the screen moves to the left Gn other words pan and move the image field to the right When panning to the right notice whether the x coordinate position in the XY Stage section of the Focus Controls window increases or decreases Repeat for the y axis by panning down so that the image appears to move up Note the change in the y axis coordinate position increasing or decreasing when panning Select Edit gt Hardware Properties The following Edit Hardware Properties dialog box will appear Click on the sign next to XY Stage to expa
228. oprobe v Add Parameters Remove TER Car SSSS S S Associated Channel Type Light Source TN ee Ratio 2 Numerator i Ratio 2 Denominator Emission oF 3 wavelength um FRET Donor fe Transmitted Default Color Display Mode Unknown v C None F Prompt if mode is not available for Red current objective Green C Alternate Source Blue Position Unmount C Pseudocolor Color C Pseudocolor Intensity Filter set Widefield v RGE Settings Excitation wheel position lUnmounted hd Monochrome c O Internal turret position Unmounted c Emission wheel position Unmounted hd LCD BF filter position Unmounted y Auxiliary filter position Unmounted v Camera Any 69 SlideBook 5 0 User Manual If you have a LCD slider such as that made by CRI you will need to configure three separate channels however these three channels will appear as one channel in the capture dialog You must define one channel each for red green and blue For the color LCD slider the filter definitions must appear as follows Create the definitions in the following order red blue green This is because you will most often perform autofocusing with the green channel and autofocusing will be performed with the last channel captured For the red channel you may also choose Red as the Default Color Display RGB Settings allows you to manually enter white balance values You may find that using the w
229. or any other damages or fees arising out of or attributed directly or indirectly to your negligent reckless or intentional conduct operations or performance of the SOFTWARE PRODUCT MISCELLANEOUS If you acquired this SOFTWARE PRODUCT in the United States this EULA is governed by the laws of the State of California If you acquired this SOFTWARE PRODUCT in Canada unless expressly prohibited by local law this EULA is governed by the laws in force in the Province of Ontario Canada and in respect of any dispute which may arise hereunder you consent to the jurisdiction of the federal and provincial courts sitting in Toronto Ontario If this SOFTWARE PRODUCT was acquired outside the United States then local law may apply Should you have any questions concerning this EULA or if you desire to contact 31 for any reason please contact 31 at Intelligent Imaging Innovations Inc 5124 Washington Street 303 607 9429 Denver CO 80216 303 607 9430 Facsimile AUTHORITY TO BIND COMPANY INSTITUTION The individual signing this Agreement hereby warrants represents and covenant that they have the right power and capacity and are and will be duly authorized and empowered to enter into execute deliver and perform this Agreement on behalf of the company 111 SlideBook 5 0 User Manual organization institution or other legal entity and that the execution delivery and or performance by of this Agreement shall not and will not by the lapse
230. or visualizing one or two channels 8 3 2 1 Changing the Channels Displayed on an RGB Image 1 Make the desired view the active window by clicking on it 2 Select the fluorophore that you would like to display from the corresponding drop down channel menu in the info tool bar NOTE If your image is displayed as something other than RGB you may change the display to RGB by choosing View gt RGB Color 8 3 2 2 Changing an Image Display to Monochrome 1 Make the desired view the active window by clicking on it 2 Select View gt Monochrome to change the display to monochrome 174 Chapter 9 Preparing an Image for Analysis or Export 3 Click on the channel menu and select the fluorophore that you would like to display from the dropdown list 8 3 2 3 Changing an Image Display to Pseudocolor 1 Make the desired view the active window by clicking on it 2 Select View gt Pseudocolor to change the display to pseudocolor Two channel menus will be available The first channel will be displayed as a pseudocolored image ranging from saturated red to saturated blue known as Pseudocolor Color 3 Click on the first channel menu and select the fluorophore that you would like to determine the pseudocolor hue from the dropdown list The second channel menu will act to gate the pseudocolor image based on intensity This second channel is called Pseudocolor intensity Thus an area with a high pixel intensity for the first channel
231. or your single COMPUTER You may not RUN the other medium on another COMPUTER You may not loan rent lease or otherwise transfer the other medium to another user except as part of the permanent transfer as provided above of the SOFTWARE PRODUCT 7 BACKUP COPY After installation of one copy of the SOFTWARE PRODUCT pursuant to this EULA you may keep the original media on which the SOFTWARE PRODUCT was provided by 31 solely for backup or archival purposes If the original media is required to use the SOFTWARE PRODUCT on the COMPUTER you may make one copy of the SOFTWARE PRODUCT solely for backup or archival purposes Except as expressly provided in this EULA you may not otherwise make copies of the SOFTWARE PRODUCT or the printed material accompanying the SOFTWARE PRODUCT 8 U S GOVERNMENT RESTRICTED RIGHTS All SOFTWARE PRODUCT provided to the U S Government pursuant to solicitations issued on or after December 1 1995 is provided with the commercial rights and restrictions described elsewhere herein All SOFTWARE PRODUCT provided to the U S Government pursuant to solicitations issued prior to December 1 1995 is provided with RESTRICTED RIGHTS as provided for in FAR 48 CFR 52 227 14 JUNE 1987 or FAR 48 CFR 252 227 7013 OCT 1988 as applicable 9 EXPORT RESTRICTIONS This SOFTWARE PRODUCT has been classified by the US Government as exportable under License Exception TSU Therefore the following terms apply You agree that you will not expo
232. oscope controls Returning to the brightest point in the sample is necessary so that the correct exposure times can later be defined in the capture window Close the lightpath for light sensitive samples by choosing Close Fluor Enter the step size that you would like to use for capture in the Step Size um edit field of the Capture Information group The number of planes that will be Chapter 7 Advanced Capture required to capture the entire z range will be calculated and displayed above the step size Note You may wish to alter the capture step size so that the size of the captured image 1s not excessive Alternately you may decrease the total distance traveled by resetting the top and bottom parameters 11 Close the focus window by pressing the Close button 7 1 1 2 Setting Capture Window Parameters L Once you have set the z parameters in the Focus Window open the Capture dialog box by selecting Image gt Capture or by selecting the capture icon in the tool bar A Select channels and set exposure times as described in the section Selecting Channels and Setting Exposure Times in Chapter 6 page 89 NOTE It is important that z parameter is set at the middle of the focus range brightest when setting exposure times so that the brightest planes are not overexposed Select the area to be imaged as described in the section Selecting the Area to be Imaged in Chapter 6 page 92 Enter image information and settings a
233. ou can select filters move x y and z positions and open the fluorescence or transmitted light shutters Before proceeding to the next section make sure that your sample is in view and focus 7 6 3 Calibrate the Stage Positions 136 Go to a column towards the left of the plate and find the top of a well in the calibration row as defined by Calibration Row in your layout generally row A Focus on the upper middle edge of the meniscus of the well and center it in the camera image see picture below and click Set UL Go to a column towards the right of the plate and find the top of a well in the calibration row as defined in your layout generally row A Focus on the upper middle edge of the meniscus of the well and center it in the camera image see picture below and click Set UR Go to a row towards the bottom of the plate in and find the right of a well in the calibration column as defined by Calibration Column in your layout column 11 by default Focus on the right middle edge of the meniscus of the well and center it in the camera image see picture below click Set next to Lower Right Test calibration by clicking on a well Gt will turn white and selecting Goto Chapter 7 Advanced Capture OO0000000080 OOOOOOOOOO _ OO0000000000 DIEL 00000000000 Gato Select Unselect Goto Restore 7 6 4 Save and Restore Calibration Positions If the currently selected layout has been
234. outine Using the zoom button to enlarge the image often aids in determining the best plane of focus see page 78 Once an acceptable set of auto focus parameters has been determined an auto focus routine can be added to the capture sequence 7 9 2 Adding Auto Focus to an Image Capture Sequence 1 To include an auto focus routine in an image capture sequence go to Capture Preferences dialog box via the Advanced button in the Capture dialog box and select the Focus tab The following window will appear 146 Chapter 7 Advanced Capture Capture Preferences Spool Sequence Notes General TTL 4D Focus Periodic FRAP Autofocus i Autofocus during timelapse multipoint captures Update focus every 5 image capture s Channel TxRed Total Search oo Peak Delta 0 2 Range ra Threshold Post Focus p Offset Uncaptured autofocus channel 2 Select the Auto focus during timelapse multipoint captures checkbox This will enable specification of several auto focus parameters 3 Enter the autofocus frequency for the image capture series in the Update focus every _ image capture s edit box The autofocus routine will always be initiated before the first image of a capture series Specifying the autofocus frequency will determine at which subsequent timepoints the autofocus will occur For instance setting a frequency of 2 would cause an auto focus to occur every other timepoint NOTE SlideBook keeps track of
235. ovations Inc sl for the 31 software product identified in the software section of the system registration sheet which includes computer software and a hardware component i e the hardware key and may include associated media printed materials and online or electronic documentation SOFTWARE PRODUCT The SOFTWARE PRODUCT also includes any updates and supplements to the original CricTiaAr nc Mini T nr ala al 12218 Inn H A mist m eS A Senn af al ml m mN I do not accept the terms in the license agreement Installshield 19 SlideBook 5 0 User Manual 3 Select Next and the following window will appear ie SlideBook 5 0 InstallShield Wizard Install SlideBook 5 0 to C Program Files Intelligent Imaging Innovations Inc SlideBook 5 04 Installshield 4 Ifthe default destination directory is acceptable select Next If you would like to install to a different directory click Change and navigate to the desired location and then select Next to continue installation The following dialog box will appear qH SlideBook 5 0 InstallShield Wizard Please select a setup type InstallShield 20 All program features will be installed Requires the most disk space Choose which program features you want installed and where they will be installed Recommended for advanced Users Chapter 2 Quick Tour 5 Select Complete then Next The following dialog box will appear
236. ow A unless you cannot move your stage to focus on the top edge of the top row and must use row 2 e Calibration Column column used for setting right side of lower right well see below You can generally specify this to be the rightmost column of the plate For a 96 well plate often you cannot reach the right edge of the wells in the 12th column and must set this value to the 11th column 6 Select the channels that you wish to capture by clicking in the appropriate checkboxes You may choose to perform an autoexposure or you may set the exact exposure time 135 SlideBook 5 0 User Manual 7 6 2 7 Select the type of capture that you wish to perform for each well as shown in the Capture Parameters section of the dialog You have the following choices e Images at the center of the well you may choose the size of the grid and you may generate a montage of the grid e Single center frame e Image pattern for the predefined grid pattern you may enter a separation value which will allow you to sample at various points in the well 8 Select autofocus parameters You may wish to determine these parameters first using the Focus Window see Autofocus on page 144 9 Click Save to exit the Multiwell Setup dialog box Manipulating Hardware Focusing on the Sample Once you have determined your layout you can interface with hardware in the Multi Well Capture dialog This works very similarly to the Focus Window Y
237. p down menu Then if you performed capture with either of these channels the condenser turret would move to the appropriate position automatically You must define the condenser turret positions when defining objectives for this feature to work properly see section Adding a New Objective Select the desired Filter Set from the dropdown menu If the exciter is located in an external excitation wheel select the position of the filter from the Excitation Wheel Position dropdown menu If it is mounted in a cube in the internal turret or if it is not currently mounted on the system select Unmounted Select the internal turret position of the dichroic mirror from the Internal Turret Position dropdown menu If the dichroic mirror is not currently mounted you should select Unmounted If the emitter is located in an external emission wheel select the position of the filter from the Emission Wheel Position dropdown menu If it is mounted in a cube in the internal turret or if it is not currently mounted on the system select Unmounted Repeat steps 1 through 8 for any additional filter configurations that you wish to add 10 Select OK when you are finished 11 Restart SlideBook to register the changes that you have made Chapter 4 Configuring Your System 4 4 3 1 Example Filter Configuration for Fluorescence Channels Example Quad Pass and Dual Pass sets on a system with filter wheels and motorized turret Some systems wi
238. plitter If you have configured an image splitter such as the Dual View from Optical Insights you should define two filters that have identical excitation wheel positions if applicable and identical internal turret positions Then depending on the position of the image splitter select one channel Camera position as Camera 1 and the second channel as Camera 2 For instance if you are performing CFP YFP FRET you may wish to define four filter configurations 1 YFP excitation YFP emission 2 YFP excitation CFP emission this is a dummy filter that must be configured but will not contain information 3 CFP excitation YFP emission 4 CFP excitation CFP emission In this example filters 1 and 3 will use Camera 1 while filters 2 and 4 will use Camera 2 Also filters 1 and 2 will use the same excitation position as will filters 3 and 4 You must define the filters in order so that the channels within each pair that share an exciter are adjacent In the example above channels would be defined in order of 1 2 3 4 72 Chapter 4 Configuring Your System 4 4 4 Modifying Information for an Existing Filter Configuration 4 5 Select Edit gt Define Optics gt Filter Configurations The Filter Configurations Parameters dialog box will appear Select the appropriate filter using the Filter Configuration dropdown list Modify the appropriate fields as desired Select OK to save the changes Restart SlideBook to
239. ply with the terms and conditions of this EULA In such event you must destroy all copies of the SOFTWARE PRODUCT and all of its component parts 3 UPGRADES If the SOFTWARE PRODUCT is labeled as an upgrade you must be properly licensed to use a product identified by 3I as being eligible for the upgrade in order to use the SOFTWARE PRODUCT A SOFTWARE PRODUCT labeled as an upgrade replaces and or supplements and may disable the product that formed the basis for your eligibility for the upgrade You may use the resulting upgraded product only in accordance with the terms of this EULA If the SOFTWARE PRODUCT is an upgrade of a End User License Agreement component of a package of software programs that you licensed as a single product the SOFTWARE PRODUCT may be used and transferred only as part of that single product package and may not be separated for use on more than one COMPUTER 4 PROPRIETARY RIGHTS All proprietary rights in and to the SOFTWARE PRODUCT including but not limited to patent copyright trade secret trademark or other proprietary rights any images photographs animations video audio music and text incorporated into the SOFTWARE PRODUCT the accompanying printed materials and any copies of the SOFTWARE PRODUCT are owned by 31 or its suppliers All title and intellectual property rights in and to the content which may be accessed through use of the SOFTWARE PRODUCT is the property of the respective content owner and may be
240. posure between 1 and 500ms This exposure is repeated continuously to form the semi live image display You can also select between four bin factors all of which are based on square pixel eroupings 1 e 1x1 2x2 etc Binning increases camera sensitivity and readout speed This is useful in the focus window because it allow shorter exposure times and faster image refresh rates ultimately making it easier to find a sample and bring it into focus Importantly the focus window exposure time and bin factor adjustments are used only for optimizing image display in the focus window Adjusting the camera settings for capturing an image is covered in Chapter 6 on page 89 On systems with multiple cameras and motorized camera port selection the Camera drop down menu lets you direct the light to the desired camera and display the resulting image On systems with two cameras running simultaneously from a single port these buttons let you select which camera s output will be displayed in the Focus Window The Start Stop button starts or stops continuous camera capture This can be particularly useful if you have a mechanically shuttered camera since the focus window can be left open without taking unnecessary camera exposures You may also press Snap to grab a single image that will be stored in an open slide 78 Chapter 5 Controlling the Camera and Microscope Hardware The gt and Al buttons in the bottom left corner are used to zoom in and
241. protected by applicable copyright or other intellectual property laws and treaties This EULA grants you no rights to use such content If this SOFTWARE PRODUCT contains documentation which is provided only in electronic form you may print one copy of such electronic documentation You may not copy the printed materials accompanying the SOFTWARE PRODUCT 5 CONFIDENTIAL INFORMATION You agree that the SOFTWARE PRODUCT contains proprietary information including trade secrets know how and confidential information that is the exclusive property of 31 During the period this Agreement is in effect and at all times after its termination you and any of your employees agents partners associates etc shall maintain the confidentiality of this information and not sell license publish display distribute disclose or otherwise make available this information to any third party nor use such proprietary information concerning the SOFTWARE PRODUCT including flow charts logic diagrams user manuals and screens to any person s not an employee without the prior written consent of 31 In addition You hereby agree to protect 31 s proprietary information and take appropriate action against employees independent contractors or others who violate any of 31 s proprietary rights 6 DUAL MEDIA SOFTWARE You may receive the SOFTWARE PRODUCT in more than one medium Regardless of the type or size of medium you receive you may use only one medium that is appropriate f
242. pse capture Open and close shutter between exposures for single channel capture Change the rate of live updates to increase capture speed Autofocus during capture Change capture frequency Save images to disk All of these options can be accessed in the Capture Preferences dialog This dialog box 1s displayed when you select the Advanced button in the Capture Settings portion of the Capture dialog Capture Settings Default X Current none gt Advanced 112 Chapter 7 Advanced Capture 7 2 1 1 Performing Periodic Capture You may choose to capture a single channel at a slower rate than other channels when performing a multi channel 2D timelapse capture You may do so by bringing up the Capture Preferences dialog and selecting the Periodic tab Select the check box the channel that you would like to capture at a slower rate and the frequency at which you would like to capture that channel For example a frequency of 5 indicates that you would like to capture a channel every 5th image If you would like to copy the image of the selected channel to timepoints where you are not collecting the channel select the check box to Fill uncaptured timepoints with data from last captured timepoint Capture Preferences x Spool Sequence Notes General TTL 4D Periodic FRAP Periodic Capture if Capture one timelapse channel at slower rate than other channels Channel DAPI Frequency 5
243. r Properties ADTF Properties AS Sy Stage ASI Z Stage Camera 1 Camera 2 Camera 3 Camera 4 DeconLive Settings Diagnostic Instruments Camera Properties Dongle Info Dummy Camera Dummy Camera 2 Dummy Camera 3 Dummy Hardware Filter Set Information Focus Window ee FAAP Parameters Hamamatsu Properties Hardware Properties Version Info Revert To Default Close Leica CTR Apply Change Default Value E E E E E E E E E E E E E E E E E E E E En 3 Click on StaticSerialMoveDelay and enter 0 in the edit field on the right side of the dialog box 4 Click on the next to ASI Z Stage to expand the section 140 Chapter 7 Advanced Capture 5 Click on Mad City Static Delay and the right hand side of the dialog box will up Edit date Hardware Properties F Advanced Capture Settings Advanced Z Stage Parameters El E E E E E E E E E E E En Z Capture Direction Up Statics eral oveD elay Static TLMoveDelay Maxinuni Stage SizeMicrons Enable Position Query Analog Y Stage Control Properties Analog Z Stage Control Properties Andor Properties ADTF Properties AST Stage ASI Z Stage me had City Static Delay Enable CAIFF Control CRIFF Focus Limit Penodic Ausillary lt dive update Camera 1 Camera 2 Camera 3 Camera 4 DeconLive Settings Diagnostic Instruments Camera Properties Dongle Info Dummy Camera Dummy Camera 2 Dum
244. r configuration that has Pseudocolor Intensity as its default will gate the intensity of the pseudocolored image according to the normalized value of each voxel If a captured image contains a channel with a filter configuration set to Pseudocolor Color but not Pseudocolor Intensity the corresponding view will show a range of hues at maximum intensity 1 e ranging from bright blue to bright red If the image also contains a channel with a filter configuration set to Pseudocolor Intensity the corresponding view will show a range from dark blue black to bright red If the image only contains a channel with a filter configuration set to Pseudocolor Intensity and no Pseudocolor Color counterpart the default setting will be treated as if it were set to None Pseudocolor color types cannot be displayed along with red green or blue channels Monochrome settings may only be used for a background channel when displayed with RGB channels In multiple channel images the pseudocolor setting will override any red green or blue settings Red green or blue settings will in turn override a monochrome setting Again this doesn t mean you can t easily get back to viewing any other channels but simply determines what will be visible initially 4 4 2 6 2 User Defined Color You may elect to set the default color to one of your choosing To do so select the User Defined radio button and then click on the color block to the right A dialog
245. r the colors displayed select the Renormalization icon al The Volume View Settings window will appear NOTE The Volume View Settings window also automatically opens when you open a 3D Volume View Volume View Settings Background Color 0 Black c hte Grid Lines 0 None A Bright Style Dynamic Lighting cra ities aly BE E 7802 16002 hack _ _ _ a mamumu 100 FITEA 403 2598 af rel Opacity 100 DAPI TTT Ree tet ia lil lla Ef 67 i qe 27687 hack _ MMM dA 100 Several features are offered in addition to the ability to renormalize the image change the lookup tables 10 Alter the background color using the Background Color slider and change the brightness of the gridlines using the Gridlines slider 11 You may alter the opacity of an individual channel using the corresponding Opacity slider control 12 Select the rendering mode from the Style drop down menu 192 Chapter 9 Preparing an Image for Analysis or Export 13 You may also change the channels that are displayed just as you would in other standard views 8 5 1 2 Working with 4D Volume Views The 4D Volume View has the same functions as the 3D Volume View plus a timepoint navigation feature To display a 4D Volume View click on a thumbnail in your slide and select View gt 4D Volume View Move time point oe forward backw
246. ran aran 206 042 Merone TIMES O AVES A A A A iS 206 9 5 PERFORMING FLAT FIELD CORRECTIONS A A eee 207 OSA A yh aaa cle SI ciate Seance ed Dee dtc neti te Dee nce fee A ieee 207 9 5 1 1 Addins or Replacine a Flat Field 2ivisc225cis sees ies E 208 95612 When to Update the Flat Field Data pase da dle 209 9 5 1 3 Displayine Existing PlatiBrelds La ina 209 DD 1 T la AAA O A ou lela eae Li 210 9 5 21 Applying Flat Field Correction During Image Capture occccccccnccncnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnininns 210 9 5 2 2 Applying Flat Field Correction after Image Capture ccccccccccccncnnncnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nn nn nnnnnnnnnnnananns 211 9 6 PERFORMING PHOTOBLEACH CORRECTIONS A 212 9 7 PERFORMING BACKGROUND SUBTRACTION venirse 213 9 8 CREATINGA PROJECTION IMAGE uo E 215 9 9 CREATING AN INTERPOLATED ISOTROPIC IMAGE cccccesseeeseeeeeeeeeeeseeseeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeeees 215 A A E RIA 216 9 10 1 Ger De ined CONVOIMIONK CFINCIS AS A A A AA A cubs Nat aulcutin ois 216 9 10 2 Gaussian Hot Pixel Mean and Median FUNCT S di 218 9 11 PERFORMING NO NEIGHBORS DECONVOLUTION ssssssseseeeceeccccceeeeeaaeesssssseeececceeceeeeeeaaaaaaaseseeeeeeeeeeees 219 9 11 1 Running No Neighbors DeconvolutiON oooocnnnnnnnnnnnnnnnnnnnnnnnnnnnannnannnnnnnnnnnnnn nana anna 219 9 11 2 Memory Requirements for DeconvolutiON cccccnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn nn 221 DAZ EXPORTIN
247. re and Import 6 5 Performing Auto White Balance for Color Capture If you are performing a color capture with an LCD filter you may first wish to perform a white balance procedure you must first define your filters as discussed on page 69 This will yield the truest color rendering To do so 1 In the focus window find a clear portion of your slide Alternatively use a piece of white lens paper 2 Make sure that the halogen lamp 1s set to 3200K and that you microscope 1s set for Kohler illumination 3 Select the green filter and check that your exposure time is at least 25ms this yields the best white balance results If you are saturating the camera at 25ms you should use neutral density filters 4 Once you have achieved an adequate exposure time go to Image gt Capture or select the image capture icon oi 5 Select the filter configuration checkbox corresponding to the color channel and then Test 6 Select the area to be imaged and any binning desired 7 Adjust the exposure time as described above 8 Select the area of the test view that you would like to use for performing white balance by clicking and dragging over the region 9 Select White Balance from the radio buttons and then click Update Extent Offset and Binning pixels C Image Bin Factor Width E Height lo i 1x1 F A X Offset CS Y Offset oo Full Chip 10 A white balance operation is performed and a dialog box similar to t
248. re are delays that may be reduced in order to increase the speed performance of the ASI Mad City piezoelectric stage and the Physik Instrumente PIFOC piezoelectric focusing collar The following adjustments allow the user to fine tune the amount of time the 139 SlideBook 5 0 User Manual software waits for a Z movement to complete The appropriate delay depends on system configuration and experimental conditions The following parameters must be set to give you the minimum possible delay to achieve maximum system performance Real world conditions may make 1t necessary to increase the delays to achieve scientifically acceptable results Should you experience issues with image quality we recommend you reset these parameters to the default value 7 8 3 1 ASI Mad City Piezoelectric Stage To increase the speed of this device perform the following steps 1 Select Edit gt Hardware Properties 2 Click on the sign to expand the section next to Advanced Z Stage Parameters Edit Hardware Properties E Advanced Capture Settings Property Information E Advanced stage Parameters Name StaticS eralMoveDelay Capture Direction Up Y StaticS erialMoveDelay Static TT LMoveDelay Maximum stagesizeMicrans e EN Enable Z Position Query Description The number of milliseconds to wait after a serial stage move during capture mz Categor Advanced Stage Parameters Analog Y Stage Control Properties Analog Z Stage Control Properties Ando
249. re four methods for viewing profiles 10 7 2 1 Method 1 Using Masks l 2 Select the regions that you would like to profile using masks see above If there are multiple objects in the mask for which you would like to plot intensity profiles generate objects by selecting Mask gt Define Objects in Mask see above for a discussion of objects Select Statistics gt Ratio Timelapse Data to generate a timelapse intensity graph 249 SlideBook 5 0 User Manual Ratio Timelapse Data Graph all regions 0 38 i 0 81 Graph all channels 0 75 Regions ratio Channels 0 19 0 00 16 00 32 00 48 00 64 00 30 00 96 00 112 00 a Time Point Intensity Ratio 7 Delta F F with baseline fo E E Espot Close 4 Use the radio buttons in the upper left hand corner to either Graph all regions or Graph all channels e Graph all regions displays all regions for a single channel If you have multiple fluors you may select the channel to display from the Channels list e Graph all Channels displays all channels for a single region If you have multiple regions defined you may select the region to display from the Regions list 5 To export the data click Export You will be prompted to enter a filename 6 Navigate to the desired file location enter a filename and choose Save The intensity profile data will be saved as a text file that can be opened in programs such as
250. register the changes that you have made 4 4 5 Removing a Filter 3 4 Select Edit gt Define Optics gt Filter Configurations The Filter Configurations Parameters dialog box will appear Select the appropriate filter using the Filter Configuration dropdown list Choose Remove then OK to save the changes Restart SlideBook to register the changes that you have made 4 5 Defining Magnification Changers A magnification changer or optovar 1s a component which your microscope may or may not have It acts to increase the effective magnification of an objective If you do not have a magnification changer you should use the predefined 1 0 x definition 4 5 1 Adding a New Magnification Changer l 2 Selecting Edit gt Define Optics gt Mag Changers The Mag Changer Parameters dialog box will appear Mag Changer Parameters Mag Chance GAIA gt Add Parameterz Remove ae oe MES Magnification y Cancel Cancel Turret Position f Unmounted Click on the Add button 73 SlideBook 5 0 User Manual 3 Enter a Name in the data entry field and the Magnification value 4 Click OK to save the definition 5 Restart SlideBook to register the changes that you have made 4 5 2 Removing or Modifying Magnification Changer Definitions You may remove and alter magnification changer definitions as you would for objectives and filter configurations Note that if you switch between several camera
251. rom the series of projection images 3 You may now generate a series movie as discussed above in steps 3 7 OR 1 Select an individual z plane for the movie by scrolling to the desired z plane and selecting the thumbnail button 2 You may now generate a series movie as discussed above in steps 3 7 8 6 Exporting Views Views may be exported as a variety of different file types These images will be 8 bit for each color and should not be used for intensity analysis However an exported view may be loaded into Adobe Photoshop or any other illustration program or inserted directly into a word processor document 196 Chapter 9 Preparing an Image for Analysis or Export NOTE If you would like to use the image in another analysis program for purposes of deriving intensity information you will need to export the image as described in Exporting Images on page 221 NOTE If you would like to show annotations such as scale bars time stamps etc you should export the image from the View menu 8 6 1 Exporting a Main View Three View Channel View or Tile View as a TIFF Open a Main View Three View Tile View or Channel View of the image and adjust the look of the data as desired This may include altering the color display and renormalization parameters or cropping the image Select View gt Export gt TIFF to export the view as a single TIFF tif file Enter the file name and select Save The TIFF image can now be used
252. rs This folder will contain files specific to individual users or the default user This is where saved capture settings point lists and analysis protocols are saved 74 Chapter 4 Configuring Your System 4 6 2 Restoring SlideBook Configuration 10 11 12 In order to restore your system preferences you must first open the backup configuration folder This file may be located at C Program Files Intelligent Imaging Innovations Inc SlideBook 5 0 ora user designated location as specified in Step 2 above Double click on the Global Preferences folder and select all of the files and folders Copy the data by right clicking and selecting Copy from the menu Now return to SlideBook and go to Edit gt Setup Guides gt Open System Preferences Folder Click inside of the Windows Explorer folder view that opens Right click and select Paste from the menu You will be informed that there is already a file in this location with the same name Select the option to overwrite all files or folders and click the box to apply to all files and continue You have now replaced the active system configuration files with the backup copies Now you will restore the user preferences Go back to the backup configuration from Step 1 Double click on the Users folder and select all of the files and folders Copy the data by right clicking and selecting Copy from the menu Now return to SlideBook and go to Edit gt Setup Guides gt
253. rs to be defined in up to five positions within the optic path For systems equipped with filter wheels or a rapid filter switcher such as the Sutter DG 4 or TILL Polychrome V use the Excitation Wheel Position and Emission Wheel Position fields If your system has a motorized filter cube turret you must specify the Internal Turret Position for the filter configuration s cube If your system does not have a motorized filter cube turret you should still enter the position of the cube Then when you select the filter from the Focus Controls SlideBook will prompt you to move the turret to the appropriate position Some examples of how to use filter positions are described in the section Adding a New Filter Configuration on page 68 NOTE If you have a manual filter turret SlideBook will prompt you to move the beam splitter when performing multi channel capture In cases where you do not want a prompt for a different beam splitter or in the motorized case you do not want the internal turret to move you can simply set Internal Turret Position to Unmounted If you believe you are seelng a prompt in error you may wish to review your filter configuration to make sure that your positions are defined correctly and also check your hardware configuration to ensure that any motorized elements are defined properly Finally if you have an LCD color filter as part of your system the LCD BF Filter Position field can be set to select positions for in
254. rt or re export the SOFTWARE PRODUCT any part thereof or any process or service that is the direct product of the SOFTWARE PRODUCT the foregoing collectively referred to as the Restricted Components to any country person or entity subject to U S export restrictions You specifically agree not to export or re export any of the Restricted Components i to any country to which the U S has embargoed or restricted the export of goods or services which currently include but are not necessarily limited to Cuba Iran Iraq Libya North Korea Sudan and Syria or to any national of any such country wherever located who intends to transmit or transport the Restricted Components back to such country ii to any person or entity who you know or have reason to know will utilize the Restricted Components in the design development or production of nuclear chemical or biological weapons or iii to any person or entity who has been prohibited from participating in U S export transactions by any federal agency of the U S government You warrant and represent that neither the BXA nor any other U S federal agency has suspended revoked or denied your export privileges 10 INDEMNIFICATION You hereby agree to indemnify 31 and hold 31 harmless from and against and shall defend against any and all claims and damages of every kind including but not limited to fines penalties compensatory damages consequential damages punitive damages attorneys fees
255. s 8 4 1 2 Making a 3D Selection in a Main Three and Channel View 1 Select the Marquee tool from the tool menu 2 Click and drag to designate a 2D selection and continue to hold the mouse button down 3 Hold the shift key down while continuing to hold the mouse button and move the mouse up or down to move the display along the invisible axis 4 Release the shift key and then the mouse button 8 4 1 3 Making a 3D Selection in any Data View 1 Select the Marquee tool from the tool menu 2 Click and drag to designate a 2D selection and release the mouse button 3 Extend the 2D selection across the third dimension by a Selecting View gt Select Rect in all Planes This will extend the selection to all planes of the third dimension OR 186 Chapter 9 Preparing an Image for Analysis or Export b Selecting View gt Define Selection Cube and entering the location range of the z or t planes that you would like to include in your selection You must click OK to complete the selection NOTE You may also use the View gt Define Selection Cube menu selection to enter x y and z or t dimensions The x and y dimensions are pixel locations while the z and t dimensions are plane numbers 8 4 1 4 Selecting an Entire Image If you would like to return your selection to the entire image simply select View gt Select All This will negate any previous selections 8 4 2 Scrolling through a Three View The point sele
256. s at conclusion of capture 7 1 2 Method 2 Use this method if you already know the distance above and below the plane of focus or other reference position that you would like to capture 1 Bring your sample into view and focus by following steps 1 2 as described above in Method 1 Setting Focus Window Parameters If desired you may set another position of interest by pressing the Set button next to Reference 2 Set capture preferences as described above in Method 1 Setting Capture Preferences 3 Open the Capture dialog box by selecting Image gt Capture or by selecting the capture icon in the SlideBook toolbar ro 4 Follow steps 2 through 5 as described above in Method 1 Setting Capture Window Parameters If you have set a reference position the dialog will appear as follows 109 SlideBook 5 0 User Manual Available 68 96 GB Required 41 42 MB Capture Use current IS ee position Range um lo m Use reference Planes 15 position Step Size um 0 27 _ Use top and bottom positions Offset um 0 i Range around reference 5 The edit fields that appear are described below e Range um Total distance traveled in microns e Number of Planes Total number of z planes acquired during capture e Step Size um Distance the stage upright or objective inverted or fixed stage must move between z planes A positive number typically corresponds to the stage or objective moving up durin
257. s described below e Current Image Operation will be performed on the selected image only 212 Chapter 9 Preparing an Image for Analysis or Export e All Images with Same Capture Type Operation will be performed on all images in the slide with the same capture type 2D 3D 4D etc as the selected image e All Images with Same Channels Operation will be performed on all images in the slide with the same channels as the selected image e All Images Operation will be performed on all images in the slide 3 Select the timepoints to modify and whether or not you would like to overwrite your data 4 Select OK to perform the operation WARNING Photobleach correction 1s an irreversible operation Please be sure to save a copy of your original data 9 7 Performing Background Subtraction You may also select a region of interest of arbitrary size and subtract the background mean intensities from your image using channel math Please see the example in Chapter 2 Creating Masks Manually Background Subtraction Example on page 41 You may also permanently subtract a background ROI 1 Make a copy of the data that you are working with 2 Use the marquee tool to select a background region in your image 3 Select Image gt Select Background 4 Select Image gt Advanced Operations gt Subtract Region Background The average values from your background ROI will be subtracted on a channel by channel basis
258. s described in the section Entering Image Information and Optical Parameters before Capture in Chapter 6 on page 93 Click on the 3D checkbox in the Capture Type section The radio buttons and edit fields in the 3D Capture section will become active Note that default value of the Planes field is one signifying a single plane capture Available 68 96 GB Required 11 04 MB Capture Use current A z E position Range um 1 e Planes 4 Step Size um 0 27 gt Use top and bottom positions Offset um lo Range around current Return to current position after capture Import the z parameters that you set in the focus window by selecting the Use top and bottom positions radio button The Planes field will display the number of planes to be captured see dialog box below The Spacing field determines how many microns the stage needs to move between planes The Offset field 1s not used with this method of setting 3D capture parameters 107 SlideBook 5 0 User Manual 7 If you wish for your z focus position to return to the center of the volume once capture 1s completed select the Return to center of volume after capture checkbox 8 Click OK to begin capture The Capture Controls window will appear and display each plane as it is captured You may choose to Pause Cancel or Stop Available 68 96 GB Required 11 04 MB 30 Capture Use current ee ie position Range um lo a Planes 15 A Step Size u
259. s discussed in Chapter 5 Controlling the Camera and Microscope Hardware Focus Window on page 77 3 To select a field to be visited during capture first click on the XY tab 4 Choose Set Point to add a field of view to be visited during capture The xy location coordinates will be added to the window of the XY tab NOTE If your system also has encoded z focusing you may specify xyz coordinates Scope Fi xY Camera Stream 0 0 0 0 839 2 Montage Extent 384 0 524 0 839 2 set Point al gt 962 0 948 0 850 6 Visit Point gt Update srz z 4 Update lt Reset All Clear Point Clear All Edit Description Home HI Once you have added a location to the list you may remove visit or add locations To perform these operations first highlight the location by clicking on the coordinates in the list then click on the appropriate button to perform the following actions e Visit Point moves the stage and z focus to the selected xyz location e New Point Z allows you to alter the z position of a given xyz location This allows you to update the focus position of a certain sample if it has drifted e Reset All Z resets the z location on all points to the current z location e Clear Point removes the selected location from the list e Clear All clears all locations from the list 128 Chapter 7 Advanced Capture 5 Once you are satisfied with your list of locati
260. s mean value A hot pixel in an image is generally caused by a bad pixel on a CCD chip To apply the Gaussian Gaussian Derivative Mean Median and Hot Pixel Filters E 218 Select the image that you would like to filter by clicking on 1t in the Slide View You may wish to copy the image before applying the filter as this is an irreversible operation see Copying an Image on page 159 You may also start from an open view of the image Select the appropriate filter from the Image gt Filter hierarchical menu A dialog box will appear that 1s similar to that shown below Gaussian Filter Filter Description Gaussian Blur Filter uses a gaussian kernel of a given radius to remove noise e Channel Parameters For the following channels Sigma 1 33 Destination C Replace each channel Create a new filtered channel Cancel Chapter 9 Preparing an Image for Analysis or Export 3 Select the batch channel and destination options for the filter as described in the above section User Defined Convolution Kernels on page 216 4 Each filter has different user definable parameters Enter the parameters for the filter based on the following descriptions e Gaussian filter A Gaussian kernel of a designated Radius will be used to determine a pixels intensity value e Gaussian Derivative A first derivative Gaussian kernel of a designated Radius will be used to determine a pixels intensity value e Mean or Media
261. s necessary if the pieza stage doesn t settle after a lange move default No Value e es C Ho Apply Change Default Value O a Revert To Default Cloze 9 Select Close and then restart SlideBook to register the change 143 SlideBook 5 0 User Manual You may find that you need to increase the delay above Oms in order to achieve scientifically acceptable results 7 9 Autofocus SlideBook offers the ability to perform an automatic focus routine during a 2D timelapse a 2D multipoint timelapse or a montage capture As with the other types of capture you will set parameters in the focus window and in the capture window The basic procedure for adding an auto focus routine to a multiplane capture is given below 7 9 1 Determining Auto Focus Parameters 144 Open the Focus Window by either selecting Window gt Focus Window or clicking on the focus window button in the SlideBook toolbar E Select your objective and filter configuration then bring your sample into view as discussed in Chapter 5 Controlling the Camera and Microscope Hardware Focus Window on page 77 NOTE It is critical that the Objective information is set correctly under Edit gt Optical Parameters gt Objectives SlideBook uses 1ts knowledge of the objective to compute the z spacing that it will use during auto focus If your microscope has an automated objective nosepiece SlideBook can read its position however if your microscope h
262. save and reuse your particle tracking parameters and apply them in a batch fashion Below we describe how to use the Particle Tracking Protocol Each step in the protocol can be accessed via a menu item which is also noted You may wish to practice particle tracking 232 Chapter 10 Using Masks for Image Analysis using the sample file ParticleTrackingDemo sld You may download this file at the SlideBook download site 1 Select the objects you wish to track by generating a mask using thresholding and or manual marking see Creating a Mask using Threshold Techniques on page 224 or Creating Masks Manually on page 226 You may need to separate objects that are touching using mask filters Mask gt Mask Filter gt Erode or an object splitting algorithm see Splitting Objects on page 231 For ParticleTrackingDemo sld create a mask using a threshold of 217 for the 1 485 channel 2 Begin by selecting Mask gt Particle Tracking gt Particle Tracking Protocol The following dialog will appear Particle Tracking Protocol Path Creation SaveLoad Settings Tracking Parameters Defaut xl Mask Name Mask 1 Remove Objects p m y smaller en 10 Path Creation 3 Path Statistics l Object Statistics Track with Center Of Area Apply to Batch Channel Maximum C microns Movement E 0 pixels Minimum Fath Length z E in timepoints E Track and Continue Track This operation tracks particles in ether 0 ti
263. set they can be accessed using the drop down menu Depending on the configuration of your microscope system clicking on a filter configuration tab will change one or more of the following excitation filter wheel position emission filter wheel position internal filter turret or slider position camera that is used for display when the system has multiple cameras The filter configuration that 1s currently selected will be highlighted and displayed in bold letters while the rest of the configurations will have a normal button appearance 5 1 1 4 XY Stage On systems equipped with a motorized xy stage you can use the directional buttons to move the stage in increments specified by the value in the adjacent edit field This value is always given in microns or fractions thereof You can change this increment distance at any time The defaults are 100 10 1 and FOV Field of View is an integer value defined by the pixel array of the CCD which can be selected from the drop down menu TIP If you double click on an object in the image display the xy stage will move so that the object 1s centered The current x and y positions read from the stage are also displayed in this section 79 SlideBook 5 0 User Manual 5 1 1 5 Z Stage On systems equipped with motorized z focusing you can move the focus up and down using the directional buttons There are three sets of buttons with predefined stage movement increments 10 1 and 0
264. sing manual editing or segmentation techniques 2 Goto Mask gt Advanced Operations gt Split Object Automatically The following dialog will appear 231 SlideBook 5 0 User Manual Watershed Separation 3 The slider bar controls how aggressive the splitting algorithm will be A value of 100 refers to no splitting while 0 refers to the most aggressive splitting algorithm Adjust this slider to a position you feel will give you clear object separation 4 Click Preview You will notice that the mask is updated on the image 5 If the splitting is adequate click OK If the splitting is inadequate repeat steps 3 and 4 until you are satisfied with the result then click OK 10 2 5 Removing Objects from Edge of Image In some instances you may wish to eliminate objects that touch the edges of your image To do so select Mask gt Advanced Operations gt Remove Edge Objects The following dialog box will appear Select the desired Image Range and select OK Image Range 6 Just the current image o Cancel 10 2 6 Tracking Objects Particle Tracking Once you have defined objects you may wish to track them over time SlideBook offers two methods for particle tracking automated and manual 10 2 6 1 Automated Particle Tracking Tutorial To perform particle tracking you may either use the Particle Tracking Protocol or individual dialog boxes The Particle Tracking Protocol has the advantage of being able to
265. single channel image in the Status tab The Live View will show a larger resizable version of the current capture which can combine multiple channels 119 SlideBook 5 0 User Manual Capture Controls Status FRAP Notes Live Mean 1969 Scale Display C Manual amp Auto 65535 Show Full Dynamic Range Next Capture 00 00 04 Time Remaining 00 16 34 Elapsed Time 00 00 06 Capturing channel TxRed timepoint 1 of 100 Graph Channels Regions e TxRed Select All Set Background sep rose 7 2 5 1 Creating Graphs to Monitor Regions of Interest If you have created ROIs you may choose to display intensity graphs To do so 1 Select the channel that you wish to graph from the drop down in Graph Channels section and click Show You may choose to display the graphs for multiple channels individually or by type 120 Chapter 7 Advanced Capture Open Data Table in Excel Save Graph as Tiff Save Data Table as txt Channel Menus Bh Grdph secs ad _Fura 2 340 mle banal LEN Dotted None 2580 00 ri 2304 00 1752 00 4 sr y 560 fe TL 2 f a 2 i 1280 00 et Y Erpand E 1024 00 Se Intensit Seja R Expand Time iii mange Pane 512 00 ri RANSE T Zoom In Out Zoom In Out Y axis a 0 2 4 La E 10 12 i4 16 18 20 22 24 26 28 3 u X axis Timepcint JL gt 2 Use the zoom in and zoom out tools to explore your data 3 Once your capture 1s finished
266. stage as described in Step 3 The stage will appear in the Not In Sequence frame You may use the gt or lt buttons to move events from the Stages frame to the Not In Sequence frame and vice versa To change the sequence of stages use the Up and Down buttons to move the stage in the list up or down one place 6 To remove a stage select the stage from the list by clicking on the stage name and then clicking Remove NOTE The overall time duration and the number of timepoints of all sequential stages 1s shown 7 After you have set up your sequence press OK If you have selected to perform an automated sequence the appropriate parameters will be loaded into the Capture dialog box 7 10 2 Setting Capture Dialog Box Parameters 1 Open the Capture dialog box by selecting Image gt Capture or by selecting the Capture icon in the tool bar i 2 Ifitis not already checked check the Timelapse checkbox in the Capture Type section of the Capture dialog box You now have the option to define a variety of timelapse parameters Enter the desired values in the following fields e of Timepoints The number of timepoints that will be captured e Duration The total length of time for the experiment Units of time in milliseconds ms seconds s minutes m or hours h can be selected from the dropdown menu e Interval The delay between the beginning of one timepoint and the beginning of the next timepoint The interval uni
267. t are the same size will overlay exactly 6 Click OK to insert the channel The new channel can now be selected from the channel menus in the info tool bar and displayed in any view The channel may also be selected when performing channel math see Using Channel Math on page 202 9 1 2 Removing a Channel You may also wish to remove a channel that was either added improperly or does not contain useful data To do so 1 Click on the image that contains the channel you would like to remove You may also start from an open view of the image 2 Select Image gt Channel Operations gt Remove Channel The following dialog box will appear 201 SlideBook 5 0 User Manual Remove Channel Image Range f Current Image m Pa m Selected Images Channel sd GFP Capture 5 Cancel The dialog box has the following options for Image Range e Current Image Operation will be performed on the selected image only e Current Capture Type Operation will be performed on all images in the slide with the same capture type 2D 3D 4D etc as the selected image e All Images with Same Channels Operation will be performed on all images in the slide with the same channels as the selected image 3 Select the channel that you would like to remove from the dropdown menu 4 Select the image extent for which you would like to remove channels You may select and deselect images using the checkboxes in the Selected
268. t can be selected from the dropdown menu If the capture sequence at one timepoint takes longer than the interval SlideBook will capture the next timepoint immediately following the preceding timepoint NOTE As you type in two of these values the third field will be calculated automatically Timelapse Capture of Time Points 124 min ms l Display Renormalize to t 0 NIN Interval 1000 151 SlideBook 5 0 User Manual 3 To begin a capture click OK The Capture Status Window will appear 4 Click on the Stages tab Capture Controls Status FRAP Notes Live Stages Mot in S quence Rapid capture Execute Stage Description Static Next Capture 00 00 02 Time Remaining 00 10 12 Elapsed Time 00 00 08 Capturing channel TxRed timepoint 2 of 124 Graph Channels Regions Select All Set Background Next Interval 00 00 52 foo Pe 2 Interval Timepoint This tab has two lists Stages and Not in Sequence 5 If you have opted to start a sequence at the beginning of capture the first stage in the top frame will be marked with a To manually execute a stage select the stage by clicking on it in the list and then click on the Execute Stage button The number of timepoints in the stage and the timelapse interval will be displayed at the bottom of the window 7 10 3 Examples for Variable Capture 7 10 3 1 Example 1 Suppose you have set up a stage with 22 timepoints and a 200
269. t lUnmounted positions Phase condenser lUnmounted turret position Field diaphram position Aperture diaphram postor Parfocal offset lens ens E Parcentric offset um 0 le E Top lens Polarizer DIC turret position 2 To add a new objective definition click on the Add button 3 Enter the objective name magnification numerical aperture working distance and um pixel You must give the lens a name and fill out all parameter fields except those in the Parfocality and Parcentricity and Transmitted Light Configuration groups NOTE For optimal results objective pixel size should be measured by traditional means using a stage micrometer If this is not possible a close estimate can be calculated by dividing the camera pixel size by the magnification of the objective Please note that this will not take into account any optics that alter magnification such as the Spherical Aberration Correction device by 31 4 Select the location of objectives from the Position drop down menu If you have a motorized objective turret Position should reflect the position of the objective 62 8 g Chapter 4 Configuring Your System on the microscope if you are using SlideBook offline or with a non motorized turret you should select Unmounted If you are using a non motorized turret but have selected Manual Turret in your hardware configuration select the appropriate turret position
270. t number in the table Notice that the corresponding object in the image is highlighted in pink 8 QuickTourWIN2 HeLa Cells 1 So le Ez 100 v 478 47 O 316 Cys 320 REI None None Notice that some of the objects are of very small size If you would like to exclude those regions when making objects you may gate the objects by size This can be done either by going back to the Define Objects dialog box or through the Statistics View itself 49 SlideBook 5 0 User Manual 50 12 13 14 15 16 To gate objects by size using the Define Objects window first close the statistics window Go to Mask gt Update Object Definitions Note the menu item is now named Update Object Definitions as opposed to Define Objects since objects already exist for this image The following dialog box will appear Define Objects Size Filter i Generate for all similarly named masks in curent slide Cancel For the purposes of this demonstration select the Gate Objects by Size checkbox and enter a minimum size gate of 100 voxels This will exclude any objects from the mask that are less than 100 voxels Select OK to update the object definitions Notice that SlideBook has generated fewer objects than when this operation was performed without a size gate Statistics can now be generated on these new objects as described above To gate objects by size using the Statistics windo
271. t running at appropriate speeds go to the Camera tab in the Focus Window and make sure that the drop down Speed menu is set to 0 Also you may choose to set the clear cycle on the camera so that charge 1s not cleared from the chip before each image 1s captured Charge accumulates between image captures and the default action of SlideBook is to clear the accumulated charge before capturing an image If you choose not to perform the clear action the camera will run at a faster rate However the accuracy of the intensities measured will be reduced To change the clear cycle setting do the following 1 Select Edit gt Hardware Properties 2 Click on the sign to expand the PVCAM section 3 Click on Clear Cycles and the right hand side of the dialog box will update 138 Edit Hardware Properties FRAP Parameters Hamamatsu Properties Hardware Properties Version Info Leica CTR Leica DMs000 Lifetime Ludi Info Marzhauser Corvus Memory Cache HAI Fiber Switcher Mikon TE 2000 Chapter 7 Advanced Capture Property Information Name Clearlucles Category PICA Description How many clears should be performed before each exposure A clear cepele removes any charge build up From the COD If you do not perform any clears pour data may not be quantitatively correct but you will capture Ocular Lightpath at a faster rate Olympus OP Camera Olympus OSU Settings Olympus MAL Olympus TTL TIRE S
272. tensity average marked voxel intensity value for each channel Sum intensity sum of all marked voxel intensity values for each channel Note that this value is equivalent to the product of the mean intensity and the volume or area Minimum Intensity the minimum voxel value in the object Chapter 10 Using Masks for Image Analysis e Maximum Intensity the maximum voxel value in the object e Median Intensity the median voxel value in the object e Variance the variance of intensities of the marked voxels e Standard Deviation the standard deviation of intensities of the marked voxels e Background Intensity the average background intensity in the channel Cross Channel e Pearson s Correlation the correlation of intensity in one channel with another a O value signifies no correlation while a value of 1 0 signifies perfect correlation A negative value signifies anti correlation This statistic can be used for measuring colocalization The equation used is shown below Ri is the intensity in channel 1 for pixel I and Giis the intensity in channel 2 for the same pixel Rav and Gav are the average mean intensity values over all pixels Ri Ray Gi Gay r UR Rav i 2 Gi Gay E i i e Mander s Coefficients these coefficients indicate the percent of protein 1 colocalized with protein 2 M1 and the percent of protein 2 colocalized with protein 1 M2 as g
273. th filter wheels will need different beam splitters to be in position for different sets of fluorophores For instance a system may have excitation and emission filter wheels loaded with filters for DAPI CFP FITC YFP CY3 and CY5 The DAPI FITC CY3 and CY5 filters require a quad pass beam splitter designed for that set of fluorophores while CFP and YFP require a dual pass beam splitter which is designed specifically for those two fluorophores In a system with filter wheels and a motorized turret Internal Turret Position should be set to the appropriate beam splitter or dichroic position for each filter configuration Ina system with filter wheels but no motorized turret the Internal Turret Position field should be set differently for each set of fluors that require a different beam splitter if you want SlideBook to prompt you to move to the necessary beam splitter Given the example above even if the system does not have a motorized turret the filter configurations for DAPI FITC CY3 and CY5 should have Internal Filter Position set to say 1 while CFP and YFP have Internal Filter Position set to 2 4 4 3 2 Configuring Channels for Color Cameras or Color LCD sliders If you have a color camera you will need one filter definition that appears as follows you must enter the appropriate location of an empty turret and emission wheel position if applicable E Filter Configuration Parameters Filter Configuration New Fluor
274. the ability to move the z focus of the microscope to any position along the specified range This is useful for confirming that the specified region covers all of and not significantly more than the volume you wish to capture and provides a means of returning the z focus to the brightest part of the sample without altering the 3D capture range When you later wish to find optimal exposure times that won t saturate the camera 1t is best to be focused on the z position with the highest peak intensities 5 1 3 3 Capture Information Choosing top and bottom positions in the Focus Window to set the extent of z travel is only one part of setting up a 3D capture You also need to specify how many planes discrete z focus positions should be collected Typically the most important consideration 1s the step size as this governs the ultimate z axis resolution of the acquired image The step size in microns is specified in the corresponding edit box SlideBook suggests a z step size based on the theoretical resolution of the lens The theoretical resolution is a function of wavelength of the chosen filter and numerical aperture You may adjust this step size by entering a value in the Step Size edit field Once top and bottom positions are defined SlideBook will use the step size to compute how many planes must be acquired to cover the specified range At any point a different step size can be entered and the computed number of planes will be updated Note th
275. the camera field at the current magnification to compute the number of images needed to span the montage extent If your microscope has an automated objective nosepiece SlideBook can read its position and the Objective field should already be set correctly If it has an automated magnification changer turret or only a single magnification changer setting then the Mag Changer field should also be set correctly However if your microscope has manual selection of objectives or more than one manually selected magnification changer you must make sure the Objective and Mag Changer fields are correct before proceeding Click on the Entire List Montage radio button in the Multiple XY Location Capture section of the Capture dialog The image extent is adjusted to reflect the montage extent in pixels Multiple XY Location Capture C CurrentLocation Entire List Montage NOTE If the Entire List Montage button is not enabled a montage extent was not properly defined in the Focus Window Click on the OK button to start the capture SlideBook will return the xy stage to the upper left corner and begin its acquisition sequence The Capture Status Window will appear and display each plane as it is captured You may choose to pause cancel or stop your capture any time after it has started Once the capture is complete the current slide will contain images of all of the fields that contributed to the montage To generate a montage image please see pa
276. the displayed portion of an image that is in a window smaller than the extent of the displayed axes 1 Zoom in on the Main View image until it is larger than the confines of the window as illustrated on the next page SlideBook 5 0 User Manual A QuickTourWIN1 Metaphase B Cell 2 lo E aa 200 44 52 0 r40 Cra 306 FITC Ara 596 DAPI H am o 41 m a Lee E o Noe 2 Select the hand tool button from the tool menu 3 Click and drag the hand tool in the window moving the portion of the image that is displayed Notice that the hand tool performs the same function as the scroll bars on the right and bottom sides of the Main View 4 Return to a 100 zoom factor by any of the methods discussed previously 2 3 4 Axis Menu SlideBook is capable of visualizing data along all three axes 1 Click on the axis menu on the tool bar and select y Enlarge the image by using the zoom tool 2 Use the up and down arrows to move through the planes 8 QuickTourWIN1 Metaphase B Cell 2 lo E aaa 200 33 04 3 5495 Cra 378 FITC Bod DAPI Mone 30 Chapter 2 Quick Tour The y axis 1s now the invisible axis You are observing the xz plane and scrolling through the y axis You may view all three Cartesian planes simultaneously using the Three View which will be discussed later in the tour The axis menu options are unavailable in the Three View because all three planes are a
277. the following descriptions e Subtraction Constant Adjusts the amount of intensity subtraction used in the deconvolution algorithm Increasing the subtraction constant will increase the amount of deblurring but can also result in a loss of image data 220 Chapter 9 Preparing an Image for Analysis or Export e Theoretical Spacing Adjusts the distance between theoretical image planes used for de blurring The closer the theoretical planes the greater the amount of de blurring Make sure to enter a value greater than zero for this parameter e Edge Padding creates a space around the border of the image in order to resolve fluorescent point sources that exist outside of the image 7 Once the parameters are set select OK to begin the deconvolution In addition to the user definable parameters the No Neighbors Deconvolution dialog box also displays the following information e Microscope Components Displays the optical components that were used to take the image e Optical Parameters Displays the optical parameters associated with the microscope components used for image capture To change these parameters refer to Chapter 4 Configuring Your System on page 54 9 11 2 Memory Requirements for Deconvolution Deconvolution 1s a memory intensive process and is limited by the size of the image that is being deconvolved On the PC any given program can use up to 2 GB of memory This results in an 800MB size restricti
278. the objected weighted by intensity values that are above the mean intensity This is a useful feature to track if you have delineated your objects by hand By using values that fall above the mean any imprecision in your hand delineation does not impact the center of intensity calculation If you select this feature you will also need to select the channel that is used for weighting Maximum Movement enter the maximum movement that you expect from any given object from one timepoint to the next This will help the particle tracking algorithm distinguish between objects that may have similar centroid values Minimum Path Length enter the minimum number of timepoints that must be tracked in order to determine a path For instance if you enter 8 any object that cannot be followed for at least three timepoints will be removed for the mask NOTE This dialog box may also be found via the Mask gt Particle Tracking gt Basic Particle Tracking menu 4 For the tutorial select the parameters as shown in the dialog below Chapter 10 Using Masks for Image Analysis Particle Tracking Protocol Path Creation SaveLoad Settings Tracking Parameters Default Wask Mame Mask 1 r Remove Objects C microns smaller than 10 Path Creation pixels Path Statistics Object Statistics Track with Center OF Intensity Apply to Batch Channel l 4865 kasi Co microns Movement 25 f pixels Minimun Path Len
279. the optimal auto focus plane during the image capture series Thus after an auto focus routine has determined an optimal focal plane all subsequent images will be captured at this z position until another auto focus routine 1s initiated The z position of this new auto focus routine will be determined by the results of the previous auto focus 4 Select the channel that will be used for auto focus This should be the same channel that was used to test the auto focus parameters in the focus window 5 The Total Search Range Post Focus Offset and Peak Delta Threshold values will be automatically entered based on the auto focus parameters determined in the focus window If needed these values can be further modified 147 SlideBook 5 0 User Manual 148 If the autofocus channel will be a captured channel during the image series click the OK button and proceed with setting up the timelapse image capture parameters as described on page 112 Make sure to expose the autofocus channel in the image capture window and set an appropriate exposure time SlideBook will use these capture parameters image extent bin factor and exposure time when acquiring the image stack for auto focus computation If you do not want to capture the autofocus channel or you want to use a different image extent bin factor or exposure time for auto focusing select the Expose uncaptured auto focus channel checkbox Uncaptured autofocus channel ie el 1100 ms
280. tomatically compute the number of planes to image based on the total search range and the calculated depth of field of the microscope objective This information is displayed below the Total Search Range edit box For instance in the above example in order to span the 8 micron search distance with an objective that has a focal distance FD of 0 27 microns a total of 29 planes will be imaged e Post Focus Offset A positive or negative offset distance that the z stage will be moved from the optimal focus position This feature is useful for always focusing a constant distance above or below the automatically determined focal plane e Peak Delta Threshold Used to determine when no optimal plane of focus should be computed from a particular image position When performing a montage capture for example some fields might not contain tissue and should not be used to change the current z position This value can range between 0 0 and 1 0 but should not be decreased below 0 1 in 145 SlideBook 5 0 User Manual most cases For captures where you know that each image in the sequence will have sufficient detail to focus this threshold can be raised If the value is set to 1 0 then the threshold will be ignored e X Y Threshold Not currently used e Exposure The exposure time used for image capture during the auto focus routine This edit box will have the same value as the exposure time used in the focus window e Save TIFFs
281. tons or by adjusting the fine focus of the microscope itself After you are finished click on the Set Top button A number will appear above the Set Top button indicating that the top z position has been set Next focus in the opposite direction until the focal plane is at the bottom of the volume of interest Click on the Set Bottom button Again a number will appear below the Set Bottom button indicating that the bottom z position has been set Once you have set a top and bottom position you may drag the z position slider to return to either of those positions In addition SlideBook will calculate the Center position based on your top and bottom positions You may select Go to return to the center position You may also set a reference or home position This reference position can be the point of best focus or some other position that is of importance for your experiment Once you set this reference position it may be used for 3D capture To set the reference position press Set This position will be saved until you reset the position or close SlideBook NOTE If you do not wish to continuously expose your sample while performing z focusing you may set these ranges using single images generated in the Capture dialog box Please see 8D Capture on page 105 82 Chapter 5 Controlling the Camera and Microscope Hardware 5 1 3 2 Slider Once top and bottom z positions have been selected the slider to the left of the buttons provides
282. ts Capture Date Capture Type Channels 2 Double click anywhere on the thumbnail or within the shaded region that surrounds the thumbnail A new window will pop up that contains the image This window is a Main View of the image an QuickTourWIN1 Metaphase B Cell 2 200 186 36 0 Alternatively you may open a Main View using the View menu To do this 1 Click once anywhere on the thumbnail or within the shaded region that surrounds the thumbnail to highlight the image 2 Choose View gt New Main View to display a new Main View When working with 2D Timelapse or 4D Capture types the Main View window will have controls for time as shown below 161 SlideBook 5 0 User Manual 3 ratiosample Untitled 1 Sfax gt 306 74 11 Ratio 1 576 Fura 2 3801 PEC CI 1952 ODER FPS 10 0 For 4D capture types the Main View operates similarly to Main Views for 3D and 2D data Additionally there is a slider that you may click and drag to view individual timepoints of the 4D capture You may scroll through the z axis by using the up and down arrow bars in the tool bar see Scrolling through the Invisible Axis in a 3D or Timelapse Image on page 180 8 2 2 Displaying a Three View The Three View is equivalent to three Main Views put together one for each axis It is useful for exploring 3D 4D and 2D Timelapse data
283. ture 117 SlideBook 5 0 User Manual Capture Controls rmm Status FRAP Notes live stages Note 1 add Note 2 add chelator OO ms Next Capture 00 00 07 Time Remaining 00 16 27 Elapsed Time 00 00 37 Capturing channel 340 timepoint 2 of 100 Graph Channels Regions o 340 a Select All Set Background Cancel 3 P Fause NOTE You can make multiple clicks during capture even of the same event but currently only one event can be recorded with any single timepoint After capture elapsed time and timelapse annotations can be displayed as a text overlay in the image by selecting Annotations gt Timestamps In addition these events are displayed next to the annotated timepoint when data is exported using Statistics gt Ratio Timelapse Data 7 2 4 Creating ROIs and Graphs to Monitor Regions of Interest Before beginning your capture you may wish to create regions of interest in order to monitor intensities during You may also do this during capture To draw regions before capture perform the following 1 Make sure that the Live View 1s displaying the specimen You can do this by pressing Test in the Capture dialog box 2 Choose one of the ROI tools which are located on the SlideBook toolbar at the top of the application window fa Rectangle Tool click and drag to draw a rectangle ll Ellipse Tool click and drag to draw an ellipse 118 Chapter 7 Advanced Capture
284. ture New Image or by clicking on the capture button in the toolbar Al You may also import images that were captured using other programs by selecting Image gt Import 1 9 Chapter 7 Advanced Capture In this chapter you will learn to perform more advanced capture sequences such as timelapse 3D 4D and montage capture XV SlideBook 5 0 User Manual 1 10 Chapter 8 Image Display and Manipulation Once you have captured an image set it may be displayed in various modes using the View menu Interactive data views allow you to display any combination of different channels and masks ROIs as well as change lookup table parameters You may also perform operations such as volume rendering Note that operations from this menu do not alter the underlying intensity data contained in an image 1 11 Chapter 9 Preparing an Image for Analysis or Export Before performing analysis on an image you may want to perform additional operations on an image For example you may wish to eliminate any constant artifacts from the image by performing flat field correction It may also be useful to deblur an image by performing deconvolution Other operations that may be necessary or useful prior to analysis are cropping aligning channels or applying filters Operations such as these are found under the Image menu 1 12 Chapter 10 Image Analysis Mathematical analysis can be performed on regions of the image that you select You may select thes
285. ture and Import fa TxRed 100 ms Fite JDAPI OFEN 5 Enter an exposure time in the Expose field if a different one is desired and press Test Pressing the Test button for a particular filter configuration will give you a sample single plane image at the specified exposure time for that channel You may resize the image by dragging and dropping the corners or edges of the Live View An intensity histogram and the minimum and maximum data values will be displayed in the Capture dialog in order to show you where in the dynamic range of the CCD your exposure lies Making a test image for each channel gives you the opportunity to choose the best exposure times and of course make sure that the light is directed towards the CCD camera and not the eyepieces 6 Press the Once button in the Adjust Exposure section of the dialog and notice that the exposure time has been adjusted to take advantage of the CCD camera s full dynamic range 7 Press the Test button again to update the image in the test view 8 Repeat the test and adjust exposure process until the histogram shows an adequate intensity distribution see note below For multi channel capture simply repeat this process for each channel that you wish to expose For instance a two channel capture using both the DAPI and the FITC filter configurations will appear as follows Filter Set Widefield y e FItC 100 ms DAPI 35 ms OPEN NOTE The m
286. uent channels represents the underlying data Operations that can alter the underlying data such as deconvolution flat field correction and cropping are found under the Image menu 3 2 2 Views Unlike images views represent a visual interpretation of the image s data SlideBook currently has two classes of views data views and display views It is within a view that data can be renormalized altering the mapping from image intensity levels to red green or blue intensities on the display and colors can be assigned to different channels You can have multiple views open on a given image at one time Commands used to create views are found under the View menu 3 2 3 Masks and Statistics A mask is a two three or four dimensional set of binary values that has the same extent as the image to which it 1s assigned Masks are often termed regions of interest ROIs in other imaging programs Masks are used for performing advanced selection and analysis of image data and can either be created and edited manually or generated automatically through threshold segmentation and other techniques A single image can contain several different masks Commands used to create and manipulate masks are found under the Mask menu Statistics can be viewed and exported for both masks and image tools from the Statistics menu 3 2 4 Slides Images in SlideBook are organized into slides Typically a slide corresponds to a number of images captured from a si
287. uld like to view images as they are captured select the Live Picture during Capture checkbox 6 Click Start to begin capture 7 6 7 Exporting Point List To capture 3D images or to take advantage of other advanced capture options you must use the Export Points button to transfer the list of positions for each selected well to the XY tab of the Focus Controls window Once transferred you can perform a multipoint capture using the exported points see Multipoint Capture on page 127 The well name and position index within the well will be saved as the image name and spool file name for each image captured 7 7 Simultaneous Capture SlideBook allows for simultaneous dual camera or image splitter operation for systems so equipped see Configuring an Image Splitter on page 60 and Defining Filters when using an Image Splitter on page 72 Simply set up for the type of capture that you would like to perform as described in the sections above Before initiating capture make sure that the Simultaneous Capture checkbox is selected in the Multiple XY Location Capture section of the Capture dialog box 7 8 Configuring Your Hardware for Speed The following sections describe how to get the best speed performance when performing timelapse imaging In addition to get best speed performance you may wish to change the frequency of image and histogram updates during capture see page 114 7 8 1 Photometrics Cameras If you feel that your camera 1s no
288. uration of color in the display Thus any data whose value is lower than the low bar will not appear in the view Any data whose value is above the high bar will show as fully saturated color The display s dynamic range then is a linear interpolation of the values in between 4 You may move the red and green bars and alter the display two ways a Click and drag the red and green bars in the histogram window to the left or right The change will automatically register once you release the mouse button OR b Enter the desired minimum and maximum intensities in the Low and High edit fields and click on Apply to register the change and update the image This gives you the opportunity to see the effect of the values and make Chapter 9 Preparing an Image for Analysis or Export changes without closing and reopening the dialog Choosing Close after entering intensities manually will close the dialog box without registering the change Again the underlying intensity values are not affected by the renormalization process 5 Follow steps 3 and 4 for any other channels that you wish to renormalize 6 Choose OK to exit the Renormalize Image dialog box If you would like subsequent views of the image to contain the same renormalization parameters click on the thumbnail button NOTE You can reset the low and high values for a single channel to correspond to the minimum and maximum intensities for that channel by pressing the R
289. ure time that you have selected in the Focus Window If desired select the Number of frames to average from the drop down list 4 Name the file in the Image Name field 5 Press Record to save the captures to disk Once you have pressed record images will be saved to disk and the Record button will convert to Pause To end the recording press Stop This will also stop the streaming update To resume the streaming update press Start You will not be able to change camera settings while the camera 1s streaming You must press Stop and make changes to camera settings then press Start again CAUTION Recording at this speed will rapidly use computer disk space Spool files will be opened automatically once you press Stop They will also be placed in the open slide with the name designated in Step 4 Chapter 6 Image Capture and Import 6 Image Capture and Import Image capture parameters are set in SlideBook s Capture window In order to capture an image you must first set your optics and bring your sample into view and focus using the Focus Window please see Chapter 5 Controlling the Camera and Microscope Hardware Focus Window on page 77 Next you must set camera parameters such as binning exposure time and image extent as described in this chapter Once all of these parameters have been set you may initiate capture You may also choose to import an image created using another program This chapter covers the following topics
290. urrent Image Operation will be performed on the selected image only e Current Capture Type Operation will be performed on all images in the slide with the same capture type 2D 3D 4D etc as the selected image e All Images with Same Channels Operation will be performed on all images in the slide with the same channels as the selected image 3 Press OK and to export a single multi plane image for each channel in the selected image s 222 Chapter 10 Using Masks for Image Analysis 10 Using Masks for Image Analysis Masks are the principal means to perform analysis in SlideBook A mask is a binary overlay on image that can be used to select an arbitrary region or set of regions of the image at the same resolution as the image itself Masks are often referred to as Regions of Interest ROIs in other imaging programs Once you have selected a region of interest s using masks you may generate a variety of morphometric measurements and intensity statistics on those regions including e Morphometrics Area and Volume Center of Mass Estimated perimeter or surface area Length along major axis Center of area VVVV WV e Intensity Statistics Mean Minimum and maximum Integrated intensity Center of intensity Correlation Colocalization VVVVV Plus many others In this chapter you will learn how to Display an Image Histogram and Mean Intensity Create Masks and Objects Display or Delete M
291. used make sure that its definition exists by selecting Edit gt Define Optics gt Mag Changers and looking at the available magnification changers in the dropdown list If the magnification changer is not available add the definition as described on page 73 4 Make sure that the filter definitions exist by selecting Edit gt Define Optics gt Filter Configurations and looking at the available filters in the dropdown list If the filters are not available add the filter definitions as described on page 68 5 Open a Main View of the imported image by either double clicking on the thumbnail in the slide file or by clicking once on the thumbnail and then selecting View gt New Main View 6 Follow steps 2 7 above for getting and editing info for images that were captured in SlideBook You should also select the Capture Type for your imported image and make sure that the image information is accurate 104 Chapter 7 Advanced Capture 7 Advanced Capture SlideBook can automate many different types of capture This chapter covers the following topics 3D Capture Timelapse Capture 4D Capture Multipoint Capture Montage Capture Multiwell Capture Simultaneous Capture Configuring Your Hardware for Speed Auto Focus Varying Capture Rates for Timelapse Capture Sequence Feature Saving Images to Disk Spooled Capture Saving Capture Parameters 7 1 3D Capture Similarly to 2D capture 3D capture is accomplished by sett
292. uttons which are defined as follows e Inthe current image Creates mask for the current image only e In all images with the current capture type Creates mask for the current capture type only For instance if you have a 2D timelapse image open in a slide with at least one other timelapse image as well as other capture types only the 2D timelapse images would be segmented e In all images with same channels Creates mask for images that have the exact same number and type of channels For instance if you wish to segment a FITC channel and one image contains a FITC channel while another contains both DAPI and FITC both images will NOT have a newly created mask A mask will only be created for the active image 226 Chapter 10 Using Masks for Image Analysis 5 Click OK to create a new mask for the current image 10 2 2 2 Editing the Mask Manually Once you have created an empty mask you can use a variety of tools to construct a useful mask There are two means of editing the mask using drawing tools from the tool menu and performing fine adjustments using tools from the mask menu on the info tool bar i Make sure that your mask is the currently selected mask To do this click on the mask menu and select the desired mask from the list Select a tool from the drop down tool menu in the info tool bar see Using the Tool Menu on page 184 e Marquee tool click and drag to draw a box outline then select
293. w is designed for use with 3D Capture data This view will display each z plane in a montage From an open slide 1 Click once anywhere on the thumbnail or within the shaded region that surrounds the thumbnail to highlight the image 163 SlideBook 5 0 User Manual 2 Choose View gt 3D Tile View to display a new 3D Tile View A QuickTourWIN1 Metaphase B Cell 4 Shox 80 147 10 8 2 3 2 Displaying a Timelapse Tile The 3D Tile View is designed for use with 2D Timelapse or 4D Capture data This view will display each time point in a montage The arrows for z movement will be available for 4D Capture data From an open slide 3 Click once anywhere on the thumbnail or within the shaded region that surrounds the thumbnail to highlight the image 4 Choose View gt Timelapse Tile View to display a new Timelapse Tile View 164 Chapter 9 Preparing an Image for Analysis or Export DU mm E y 3 ratiosample Untitled 1 kol e E 100 v 135 1 6 range Batio1 f 302 _Fura 2 3801 00 02 06 00 02 22 00 02 37 00 03 09 00 03 25 00 03 41 8 2 3 3 Displaying a 3D or Timelapse Tile View of a Portion of the Image The Tile View is slightly different than the Main and Three Views in that it is tailored to display a portion of the image rather than the entire image Since the Tile View creates a pane for every plane that it displays a Tile View of the entire image will often be f
294. w select Statistics gt Mask Statistics The following dialog box will appear E Mask Statistics Image Scope Features G m Current 2D 3D Image Compus o E Date Of Capture none C C All Images in Slide with the Same Mask e O Morphomety none A co Name Export Only C Intensity none Description C Cross Channel none C Under Development none Mask Scope Display C Entire Mask Object Export Primary Mask Mask 1 Cancel V Remove objects smaller than 100 C Microns Pixels Some advanced features require a secondary mask Secondary Mask None sa 4 y Remove objects smaller than 10 e e Clear Now click the Remove objects smaller than check box located under the Primary Mask drop down menu and enter a minimum size gate of 100 in the active edit box Chapter 2 Quick Tour 17 Select the statistics you would like to have computed and click Display SlideBook will automatically remove objects smaller than the minimum size gate during statistics computation 18 The statistics table may be exported for use in other programs by clicking the Export button A dialog box will appear that will allow you to choose the file destination The file will be saved as a tab delineated text file txt which can be opened by various programs such as Excel 19 Close the slide by clicking on the in the upper right corner of the slide view or by selecting File gt Close
295. ximum pixel intensity values for the first timepoint in a timelapse capture series will be used to determine the renormalization values of all subsequent timepoints This option only affects the image display during capture and will not affect the captured image 7 2 3 Creating Notes SlideBook allows you to record the exact time of experimentally relevant actions To do this you may elther create notes prior to capture or during capture These notes will then appear as buttons in the Capture Status Window during capture If you do not wish to create notes proceed to the next section Initiating and Monitoring Timelapse Capture 116 Chapter 7 Advanced Capture 1 To create annotations first bring up the Capture Preferences dialog from the Capture dialog by clicking on the Advanced button 2 Select the Notes tab The following dialog box will appear Capture Preferences Focus Sequence Note 1 You may specify up to eight notes which will appear in Capture Controls and the exported data 3 Click OK once you have entered the desired annotations Once capture is initialized by clicking OK in the Capture dialog box the notes will appear in the Capture Status Window see below 4 To record an event simply click on the desired annotation button during capture You can access notes from the Notes tab of the Capture Controls You may also add new notes at this point You need not specify the notes before cap
296. y an image before performing manipulations that affect the underlying data 1 Make the slide that contains the image that you would like to copy the active slide by clicking on 1t 2 Make the image that you would like to move the selected image by clicking on 1t as discussed above 3 Select Edit gt Copy Image or click on the copy image icon Ha 4 Click on the slide that will hold the copy of the image This may be the current slide or another slide 5 Select Edit gt Paste Image or click on the paste image icon E l 8 1 2 4 Deleting an Image To permanently remove an image 1 Make the slide that contains the image that you would like to delete the active slide by clicking on it 2 Make the image that you would like to delete the selected image by clicking on 1t as discussed above 3 Select Edit gt Delete Image CAUTION This is a permanent removal that cannot be undone 8 1 3 Image Display SlideBook contains four different kinds of data views the Main View the Three View the Tile View and the Channel View Data views may be used not only to look at data but to generate statistics Display views are strictly used for viewing data The following display views are available Volume View Surface View and Timelapse Intensity Plot The displays that are most appropriate for different modes of imaging or capture types are shown in the table below Capture type is designated by the icon for any image as well as ident
297. y prior version for the same operating system on a single computer workstation terminal or other digital electronic device COMPUTER e Storage Network Use You may also store or install a copy of the SOFTWARE PRODUCT on a storage device such as a network server used only to store the SOFTWARE PRODUCT A license for the SOFTWARE PRODUCT may not be shared or used concurrently on different COMPUTERS e Reservation of Rights The SOFTWARE PRODUCT is licensed to You on a non exclusive basis All rights not expressly granted are reserved by 31 2 DESCRIPTION OF OTHER RIGHTS AND LIMITATIONS e Not for Resale Software If the SOFTWARE PRODUCT is labeled Not For Resale or NFR then notwithstanding other sections of this EULA your use of the SOFTWARE PRODUCT is limited to use for demonstration test or evaluation purposes and you may not resell or otherwise transfer for value the SOFTWARE PRODUCT e Limitations on Reverse Engineering Decompilation and Disassembly You may not reverse engineer decompile modify or disassemble the SOFTWARE PRODUCT except and only to the extent that such activity is expressly permitted by applicable law notwithstanding this limitation e Separation of Components The SOFTWARE PRODUCT is licensed as a single product Its component parts may not be separated for use on more than one COMPUTER e Trademarks This EULA does not grant you any rights in connection with any trademarks or service marks of
298. y selecting Mask gt Particle Tracking gt Path Statistics Select the statistics you wish to calculate using the checkbox Select Calculate and Continue if you wish to progress to the next step or simply select Calculate if you wish to view your statistics without going to the next step then select Next when you are ready to go to the next step The following dialog will appear Particle Tracking Protocol 1 x Capture Date pear month da SavevLoad Settings Defaut Elapsed Time ms Done Req d Operation Name Elapsed Time hh mm ss ms x Es Path Creation Area pixels A Path Statistics j rea Object Statistics Apply to Batch These statistics report information pertinent to objects in the mask such as volume or center of intensity Permeter Center OF Area Major Asie Coordinates Major Axis Length Minor Axis Length Longest Chord Length FT anuanu H ill Aras Select All Clear All Calculate and Continue Stop Menu Mask gt Statistics Hest gt Close NOTE Object statistics can also be calculated via the Mask Statistics dialog Mask gt Statistics Statistic definitions are given in the section Generating and Exporting Mask and Object Statistics on page 243 Select the statistics that you wish to calculate and either select Calculate and Continue or Calculate and then Next The following dialog will appear 237 SlideBook 5 0 User Manual
299. you have specified a timelapse interval that is shorter than the time required to capture a single 3D stack SlideBook will default to capturing 3D stacks as quickly as possible 124 Chapter 7 Advanced Capture In addition to all of the options available for 2D timelapse imaging SlideBook also allows you to perform mid volume capture and sequential multi channel z series as discussed below 7 3 1 Shifting the Volume During 4D Imaging You may find that your sample exhibits focus drift during image acquisition You may correct for this by selecting the Live tab during capture To move your volume 1 Click on the Live tab and press Pause to pause the capture 2 Click Start to go into live mode in the Live View Refocus your sample either manually or by using the Z Stage controls and select Recenter Z You may also shift the whole 3D volume by checking the Shift 3D Volume box Capture Controls status FRAP Notes Live stages Z Stage XYZ Position 3443 jA 10 1 1 02 Updating the Z position is not possible during a 6D capture um soto xyz Update z update xr Filter TxRed Go Captured Images 5 ow Sni Open Fluor E Open Bright _ Start HOUGH Next Capture 00 00 07 Time Remaining 00 01 27 Elapsed Time 00 00 13 Capturing channel DAPI timepoint 2 of 10 plane 2 of 5 Graph Channels Regions e TxRed o Select All Set Background Cancel Stop 7 3 2 Mid volume C

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