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133026_LI 4.2 User Manual.book

1. Close Preview Label Set All v Add to List Browse View Default y Configure Load as Group Load Remove J Close Location C Share Caliper LS Caliper Data Sample Data IVIS200 data TraserBeadsPC TLT20050624145507_SEQ SequencelInfo txt Snapshots of an image sequence A list of image types available for display for example overlay For more details on the different types of image displays see Table 2 2 page 8 Note If the acquisition included more than two imaging modes for example luminescent x ray and photograph additional drop down lists appear so you can choose any two images to overlay Display Luminescent oon BY Photograph To change the foreground or background click the button or select from the drop down list Options single image Enables you to select the type of information to include in the image label LVS ee E TLT20050624145507_005 Units Counts v Display Overlay v Options v aano wi Image TLT20050624145507_005 Series Male Nn nu Fri Jun 24 2005 07 56 46 E ment DOB 03 21 05 Em Filter 640 Bin M 8 FOV 12 6 f2 1s eR ii SS au Ima ge label Camera IVIS 200 Beta II SI620EEV Comment dorsal Luminescence 10000 8000 6000 4000 2000 Color Scale Min 586 Max 10348 92 Living Image Software User s Manual Table 7 2 Image window continued ltem Description Color Sc
2. frame Accumulate hiss e Put the mouse pointer over the J S graph to view a tooltip that J _ Aa shows the frame number and Maximan Vi Time Minimum time jin f Maximum e Right click the graph to view the 13487 available shortcut menu of File Size 110 9 MB 3 Time secs Thee Gece 17 05 Table 6 1 Kinetic acquisition settings ltem Acquisition Settings Luminescent v Overlay Dynamic Range 14 bit v Exposure Time msecs 35 4 Binning 4 v F Stop 1 v EM Gain 100 v Excitation Filter Block Emission Filter Open v FL Lamp Level High Photograph Light Level 40 7 a Color Scale Auto Minimum 125 Maximum 688 File Size 110 9 MB Description Select the type of data to acquire luminescent or fluorescent from this drop down list Choose the Overlay option to also acquire photographs Overlay Choose this option to acquire photographs 72 Living Image Software User s Manual Table 6 1 Kinetic acquisition settings continued Item Description Dynamic Range 14 bit If this option is chosen the signal intensities range from O to 16383 counts per pixel 16 bit If this option is chosen the signal intensities range from O to 65535 counts per pixel Note The14 bit dynamic range enables faster imaging Exposure Time The exposure time for the luminescent image
3. Description Living Image 4 2 Jan 24 2011 14 10 41 Version 4 2 0 12623 IVIS configuration file found and loaded gt gt KSA Logged IN lt lt Activity window Switching Fluorescent Lamp OFF Switching On Motion safety The software tracks user time on the system hr min sec per user ID from logon until switching users or system shut down The software creates a separate record for each month for example LI_USAGE_ lt MONTH gt _2009 csv located at C Program Files Caliper Life Sciences Living Image Usage Software Help There are several ways to obtain help on the software features e To view a tooltip about a button function put the mouse cursor over the button e To view a brief description about an item in the user interface click the k toolbar button then click the item Press F1 or select Help User Guide on the menu bar to display the Living Image Software User s Manual pdf Y Caliper 4 Optical Imaging Bioluminescent Optical Imaging 2 ee eee ee ee 21 Fluorescent Optical Imaging With Epi lllumination 25 Fluorescent Optical Imaging With Transillumination 28 Acquire a Sequence Using the Imaging Wizard 0 Ja Acquire Multiple Sequences in Batch Mode aoaaa a a a a a 39 Manually Set Up an Image Sequence a a ee eee ees 41 High Resolution Imaging With the Optical Zoom Lens Attachment aaa 45
4. 204 Living Image Software User s Manual 14 3 3D Reconstruction Results The Results tab displays information about the photon density voxels and algorithm parameters DLIT or FLIT Results NOTE For more details on DLIT see Appendix H page 317 Sometimes adjusting the DLIT algorithm parameters improves the fit of the simulated photon density to the measured photon density data Figure 14 13 Example DLIT 3D reconstruction results Tool Palette Value Final voxel size mm 1 25 Number of voxels 115 Reduced Chi2 9 73e 05 Index Of Refraction 1 40 Angle limit deg 70 Damping reduce 100 of samples 200 Show samples Starting voxel size 5 Sampling method Uniform Depart Rests Save Results Name DLIT_45507 v _ 3D Multi Modality Tools gt Spectral Unmixing Table 14 5 DLIT or FLIT 3D reconstruction results Item Final voxel size mm Description The voxel size length of a side mm that produces the optimum solution to the DLIT or FLIT analysis Number of voxels The number of voxels that describe the light source s Reduced Chi2 A measure of the difference between the computed and measured photon density maps at the optimum solution A smaller value indicates a better quality of fit Starting voxel size The voxel size at the start of the analysis The length of the side of the voxel cube in mm units for the coarsest initial gri
5. c Enter the syringe diameter in the infusion pump control panel NOTE Custom syringe information that is entered in the infusion pump control panel is not saved to the system 4 To automatically start the infusion pump after data acquisition begins choose Auto Start After and enter the amount of seconds For example enter 10 to start infusion 10 seconds after acquisition begins To manually start infusion click Start Now Nn To automatically stop infusion choose Auto Stop After Acquisition To manually stop infusion at any time click Stop Now If the auto stop option is not chosen and you do not manually stop the pump the pump continues to run after acquisition ends until the specified infusion volume is reached NOTE The information in the infusion pump control panel is saved in the click info file During acquisition if you start infusion then manually stop and restart the infusion only the last actual start and stop is saved to the click info file not the start stop settings in the panel YY Caliper _ Appendix IVIS Syringe Injection System 325 1 2 Tracking Infusion in the Maximum vs Time Graph During kinetic acquisition the blue shaded region in the Max vs Time graph indicates the infusion period During acquisition if you start infusion then manually stop and restart infusion only the last actual start and stop is recorded in the Maximum vs Time graph The graph stops recording
6. a a mA gla E Photo Adjustment Brightness E 100 Contrast 15 Opacity 100 Color Scale Epi fluorescence f 4 51e8 LH 4 69e9 Color Scale Limits Auto C Full D Manual Color Table ot a Reverse Logarithmic Scale Current image Corrections Filtering Image Information a A Wo Q i f E Unts cm Image Binning 4 Width 12 5 cm Height 12 5 cm Image X Y 1 033 12 379 an Radiance Image Data 2 48128 photons Sec on2 sr pisec cm2 sr Crop Distance aessesens z r A i 0 00 0 00 B 0 00 0 00 Borasassssos Color Scale Min 4 51e8 Max 4 69e9 f i 0 00 0 00 Distance 0 00 gt i Accumulate i82 pf Extract ut ROI Tools Current image number top slider position To select a particular image enter a new number or move the top slider Pi 128 e P m BA E Accumulate 51 j i2 E pj Extract Start frame image in Use the bottom sliders to select a ehd frame image in the selected data range range of data for viewing or export the selected data range left slider position right slider position 80 Living Image Software User s Manual Figure 6 9 Image window selecting kinetic data for export Put the mouse arrow over a bottom slider to view a tooltip that shows the number of selected images frames E vazoenione2612 a units Radence Poke ospiey Overes Op
7. Shading styles for the surface Reflect smoot surface face Smooth surface gal Surface face face y Reflect surface face m Click to open the color palette from which you can select a display color for the surface and the cross section views Opacity Adjusts the surface opacity Display Photon Density Map Choose this option to display the photon density on the surface Apply Choose measured or simulated photon density maps for display Wavelengths DLIT Images FLIT Choose the data to display in the photon density map Intensity Set the maximum intensity of the photon density map using the slider or by entering a value Color Table Color scheme for the photon density map 222 Living Image Software User s Manual Table 14 10 Surface tools continued Item Description Reverse Choose this option to apply the colors of the selected color table in reverse order For example the Red color table represents the source intensity photons sec from low to high using a color scale from transparent to red If Reverse is chosen the source intensity photons sec from low to high is represented using the color scale from red to transparent Log Scale Choose this option to apply a logarithmic scale to the photon density scale 14 11 3D Tools Source Use the source tools to e Adjust the appearance of sources in DLIT or FLIT reconstructions e Make
8. NOTE The software automatically includes the user ID and a date and time stamp with the data 4 9 Exporting Image Data You can save the active image view in different file formats for example bmp dcm 1 Open an image or image sequence 2 Click the Export Graphics button ag Figure 4 30 Exporting an image to a graphic file E 11120050624145507_006 urs or Batmap image bmp JPEG Image jpg Postscnpt Enhanced Metafile eps DICOM dem 3 In the dialog box that appears select a directory choose a file type and enter a file name 4 Click Save NOTE To export a sequence to DICOM dcm format select Export gt Image Sequence as DICOM on the menu bar This creates a directory that contains the dcm files and a Sequencelnfo txt 48 Living Image Software User s Manual This page intentionally blank Y Ca i a 5 X Ray Imaging Acquire an X Ray Image 1 a a a 49 Acquire an Image Sequence That Includes X Ray Images 54 Acquire Multiple Sequences in Batch Mode aoaaa aa aa a a 082 88 61 High Resolution Imaging With the Optical Zoom Lens Attachment aaa 63 Measuring Relative Density aoaaa a a a ee ee ee 65 X ray images can be acquired on the Lumina XR Imaging System An X ray image luminescent or fluorescent image and a photograph can be acquired at the same time You can choose two images to create an overlay Figure 5 1 F
9. 258 Living Image Software User s Manual To load results 1 Select the results from the Name drop down list 2 Click Load To delete results 1 Select the results from the Name drop down list 2 Click Delete 3 Click Yes in the confirmation message that appears 15 8 Volume Data Viewer The Living Image software provides a viewer for volumetric data The 3D Multi Modality tools are not required to view DICOM or TIFF data 1 Select View Volume Data Viewer on the menu bar The Volume Data Viewer appears 2 Select volume data by doing either of the following e Drag the data file DICOM TIFF from Windows Explorer to the Volume Data Viewer window or e In the Volume Data Viewer click the Open button Ga and in the dialog box that appears select a DICOM or TIFF file and click Open 3 To clear the Volume Data Viewer click the El button Figure 15 24 Drag volume data from Windows Explorer to the Volume Data Viewer GU Je TIFF gt mouse 3 3 20100528 140051 FOVEOm 5 Search mouse 3 3 20100528 140051 F Organize H Open Share with Slide show Print E mail Bun Fil i AL20110120171831_SEQ Z mouse 3_3_20100528_140051 gt d AutoFluorBkgCorr Ar mae bd BkgSub do dem 20110306103654 b gt J DLT Datasets bi 1 20110114162356_SEQ di Katherine Quantum do Kidney gt E K5A20110305104918_5EQ LognitudinalData tal Lung do MAT20090302190433 p Ji Multi Modality
10. Remove Comment Remove All Tags Remove All Comments Color Scale Min 586 Max 10348 Copy All ROIs Paste ROI Hide ROI Tags Delete All ROIs Sort ROIs Zoom Area Zoom In Zoom Out Reset Zoom Co TLT20050624145507_005 E TLT20050624145507_005 Units Counts v Display Overlay v Options vY Info i8 Luminescence 10000 8000 6000 4000 2000 Counts Color Scale Min 586 Max 10348 Y Y Caliper Chapter 7 Working With Optical Image Data 97 To move a tag 1 Position the mouse pointer over the tag 2 When the hand tool appears use a click and drag operation to move the tag then click the mouse to set the tag location A line between the pixel and the tag identifies the location associated with the tag 7 5 Adjusting Image Appearance Use the image adjust tools to adjust the appearance of an image Figure 7 14 NOTE Not all tools are available for all image display modes Figure 7 14 Tool palette Image Adjust tools Image TLT20050624145507_002 Fri Jun 24 2005 07 56 11 Camera IVIS 200 Beta II SI620EEV Units Counts Display Em Filter 580 Bin M 8 FOV 12 6 f1 1s 7 Image adjust OOOO Series Male Nn nu a a maA E E65 Experiment DOB 03 21 05 Photo Adjustment Label kidney Brightness 100 Comment dorsal Contrast P 1 5 Image Opacity Jw 6 Adjust tools Color
11. Pi HX 20070420121444 SEO gt Image Adjust gt gt ROI Tools bi Units Counts _ Use Saved Colors gt Spectral Unmixing Vel He ee ELE LE ie ce oe 7 i i an ae a a tr es ij Sequence View 2 Select Tools gt Colorize on the menu bar The software renders each luminescent or fluorescent image in color and combines them into a single image Figure 7 29 Figure 7 29 Colorize view 7 mannna ele Ci Sequence ew Sperra Cokripe Wam E al Tokona MIR Color Range os Alber Range Log Scale P Reel Coy ih i amp 114 Living Image Software User s Manual Table 7 12 Colorize tools Item Description Colorize View Color Map NIR A special camera setup that extends the color response into the near infrared range Near infrared fluorophores appear red to purple using the NIR camera setup VIS Regular camera setup that mainly renders color in the visible range It is similar to the color response of a commercial digital camera NIR fluorophores appear dark red to invisible using the VIS camera setup Color Range The color map indicates the color range of the selected camera setup from short to long wavelength The two sliders determine the lower and upper limits of the color range that is used to render color The parts of the color map outside the selected range are not used in the color rendering process By default the entire color range is selected Filter Range
12. Units Counts v Display Options v Info a Luminescence 10000 Counts BKG 1 2 2 0e 03 8000 6000 ROI 50 J 3 349e 05 4000 ROT 8 50 J 1 763e 05 2000 Color Scale Min 586 Max 10348 falas C ROI Properties ro Ea ROI Label BKG 1l Shape Cirde Type Manual Background ROI Subject ROI T Use as BKG for future ROIs in C Entire sequence TLT20050624145507_005 only T Lock Position Xc pix 116 73277 Yc pix 94 18487 Angle deg 0 0000 E Lock Size Width pix 19 60336 Height pix 19 60336 Line Size 2 Label Shape Type ROI 8 Contour Auto 50 Image Number TLT20050624145507_005 ROI _none_ ID Label EF ROI Properties 33 Beckground Ror SubjectRor une cor TT C v Lock Position Xc pix 106 81818 4927 EAn Yc pix Angle deg 0 0000 Lock Size Width pix 11 00000 Line Size 2 a ure coor M C ROI selected in the image Label of the ROI selected in the image Double click to edit ___ Selected image Y Ca i a Chapter 8 ROI Tools 137 Figure 8 20 ROI properties Subject tab The items in the ROI Properties box depend on the type of ROI selected in the image For more details see Table 8 3 page 137 Background ROT E Lock Position E Lock Size Subj ROI tab
13. 2 Click an ROI shape button Circle OJ Square Gj or Contour and select Auto All from the drop down list The ROIs appear on the image or sequence thumbnails The ROI label includes the ROI intensity threshold Threshold and intensity measurement NOTE Auto ROIs are created and numbered in order from highest to lowest maximum signal within the ROI ROI 1 contains the highest maximum signal You may want to arrange the ROIs in a known order for easier comparison between images To renumber the ROIs ascending order from right to left right click the image and select Sort ROIs on the shortcut menu If the Apply to Sequence option is selected in the ROI tools choose Sort ROIs in Sequence to sort all of the ROIs in the sequence The sort options are only available if the ROIs have not been sorted TeolPalete e _ j ROI Tools O al z Y Meare ROls Type Meseuremer Save ROS Name Auto RO Parameters Threshold d 2 i Use Sig Offset Regtace ROIs seve Load Planar Spectral Imaging Figure 8 8 Automatically drawing measurement ROIs detected by the software 7 Surface Topography l i _ Spectral Unmiang S cl 7 s0 3 349 OF SSO e 5 675605 TEDI 5 ROL SSO 62le 06 eA or i ule l Ge 4 SPI 3 Click the Measure button W R05 in the ROI tools to show the ROI Measurements table 128 Living Image Software User s Manual Figure 8 9 ROI Mea
14. Luminescence 10000 A ROI Measurements ESE 2 8000 ROI Measurements I Refresh Image Number ROI Image Layer Total Counts Avg Counts Stdev Counts Min Counts Max Counts 6000 TLT20050624145507_005 ROI1 Overlay 6 504e 05 5 559e 03 2 354e 03 2 929e 03 1171e 04 TLT20050624145507_005 ROI2 Overlay 3 087e 05 1 319e 03 5 613e 02 6 653e 02 2 655e 03 4000 ROI 21 25 3 087 e 05 ail Customized Selections 2000 Measurements Types ae Arbus ROL Dimensions wee centaure Cert aa i Counts Color Scale Min 586 Max 10348 In counts mode the ROI measurements table includes Total Counts Sum of all counts for all pixels inside the ROI Average Counts Total Counts Number of pixels or superpixels Quantity ROI Pixels Number of binned pixels inside the ROI Area CCD pixels Number of unbinned CCD pixels inside the ROI Radiance When image data is displayed in radiance units photons for short the photon emission from the subject is displayed in photons sec cm2 sr Counts are a relative measure of the photons incident on the CCD camera and radiance is in absolute physical units that measure the photon emission from the subject The radiance unit of photons sec cm2 sr is the number of photons per second that leave a square centimeter of tissue and radiate into a solid angle of one steradian sr Figure D 3 Figure D 3 Isotropic radiation Isotropic radiation from a cell is
15. NOTE The Normalization option is selected by default so that NTF Efficiency images can be produced Figure 4 10 Acquisition Control Panel Field of view System Status Acquire E Mouse Imaging Shuttle Es Oo Subject height 1 50 E I Sequence setup Focus Se j Temperature A Locke SSS 30 Living Image Software User s Manual 2 Select an excitation and emission filter from the drop down lists For more information about the standard filter sets see Table F 1 page 296 3 Click Setup If you are prompted to acquire a subject photograph click Yes 4 Inthe Transillumination Setup box that appears Figure 4 11 choose the location for transillumination and image acquisition click a W square Figure 4 11 Opening the Transillumination Setup dialog box P MS Acquisition Control Panel i Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter a 1 00 sec Medium 2 j Blok Open m T al Field of View c A System Status Mouse Imaging Shuttle Idle j Transillumination Setup Single Location Mode C Move Motors To Selected Spot C Mask Grid Points To Subject Grid Type 15x23 J F Raster Scan Hem Service 13 4 cm Subject height 1 50 gt cm Focus use subject height v Temperature I Locked Single location mode acquires one Image
16. NOTE When the Zoom Lens attachment is installed single images or sequential images can only be acquired at the Z field of view setting During sequence setup if you attempt to change away from the Z setting the sequence table will be cleared If you attempt to change from another field of view setting to the Z setting the sequence table is cleared of all previous settings before the camera settings for the Z position are added Removing the Zoom Len Attachment The imaging system is set to the Z field of view until the Zoom Lens attachment is removed 1 Remove the check mark next to Zoom in the control panel Figure 5 17 The stage moves to position C then you are prompted to remove the lens attachment 2 After you remove the Zoom Lens attachment click OK in the prompt Always store the lens wrapped in its protective container Figure 5 17 Control panel remove the check mark next to Zoom J Exposure Time Binning F Stop Excitation Filter auto Ea medun 2 gt magra wasa M a Please remowe the zoom lens by pulling down firmly from its magnetic attachment Click OK when done YY Caliper A per Chapter 5 X Ray Imaging 65 5 5 Measuring Relative Density As density increases tissue absorbs more X ray energy and appears darker in a raw Lumina XR X ray image Figure 5 18 To enable measurements of this X ray absorption the raw X ray image is mathematically transformed resul
17. Pixel Spacing Row Column 0 1000 0 0016 mm Slice Spacing 0 0016 mm Subject Orientation Invert X Axis Invert Y Axis Invert Z Axis ox _ cance 256 Living Image Software User s Manual 15 6 Viewing a Slice Slice Tab In the 3D Multi Modality tools the Slice tab contains the color table options for the slice views Figure 15 22 Perspective view and slice views displayed using different color tables E ELADIOTILIIA AR SEN Toal Palette ROT Tools E Sequence View 30 View Spectra Planar Spectral imaging E _ B Surfoce Topearephy k E 4 e m ee l DLT JU Reconstrcton 3D Multi Modality Toots Volumes Coder Table J 1 To select a color table for the slice views choose the Slice Color Table option then make a selection from the Color Table drop down list Figure 15 22 To apply the reverse of the selected color table choose the Reverse option Black areas that appear around the optical sources in the overlay with the CT slices are due to the black color level at the low end of the color palette To correct this go to Sources tab in the 3D Tools and move the colorbar low end slider up from the black level 2 To apply the same color table as the color opacity map choose the Volume Color Table option The volume color table is selected in the Volume tab ty Caliper p Chapter 15 3D Multi Modality
18. ROI Properties ROI Subject 1 Label Subject 1 Shape Square Type Manual Subject ROI Kel pix 122 21444 e pix 127 40694 Width pix 78 65097 Height pix 197 84919 Line Size 2 gt Line Color NE Done Drop down list of subject ROIs in the image ROI label name Edit the label here Enter information about the selected ROI optional Table 8 3 ROI Properties ltem ROI Description A drop down list of ROIs in the active image or image sequence To select an ROI double click the ROI in the image or make a selection from the drop down list Shape The shape of the ROI circle square grid or contour selected in the image Type Indicates the method that was used to draw the selected ROI automatic manual or free draw ROI Label Click to edit the selected ROI label name Image Number A drop down list of open images Background ROI tab The Background ROI tab shows a drop down list shows all average background ROIs in active image that can be linked to a user specified measurement ROI or subject ROI selected from the drop down list at the top of the dialog box 138 Living Image Software User s Manual Moving an ROI Table 8 3 ROI Properties ltem Description Subj ROI The Subject ROI tab shows a drop down list of all subject ROIs in the image number selected above that can be linked to a user specified measurement ROI or average b
19. 7 Well Plate Quantification Window For Sequence EL20090414101005 SEQ Click EL20090414101005_001 Fluorophore Type Dye molecules Cells These options only available for fluorescence data Set gt For Click EL20090414101005 001 4 Select the well plate dimensions from the Well Plate Type drop down list The first image in the sequence opens and a grid ROI appears on the image Figure 13 3 Well plate image with grid ROI p IE EL20090414101005_001 AEA Units Efficiency X Display Overlay X Options Info ie Ter HF 5 Adjust the grid ROI to closely fit the plate wells VW Caliper Chapter 13 3D Quantification Database 187 feSciences 6 In the well plate table select the sample cells and click Set Figure 13 4 Clicking a row or column header selects the entire row or column 7 Toremove the sample designations from table cells select the cells and click the button 8 To apply a color to table cells a Select the table cells and click the e button Alternatively right click the selected table cells and choose Background Color on the shortcut menu b Choose a color from the color palette that appears Figure 13 4 Select the sample wells and enter the number of cells or molecules 7 Well Plate Quantification Window mm rn L Wel Plate Quantification Window ace oll For Sequence EL20090414101005_SEQ Click EL2009
20. Alternatively put the pencil over the image and click the mouse key or press and hold the mouse key while moving the pencil over an area of the image 200 Living Image Software User s Manual NOTE If the pencil tool markings are applied to the image only the marked pixels are included in the analysis Figure 14 8 Selecting regions to include in reconstruction j DataAdjustment Threshold Tools Adjustment i 0 5 S Region Selection Tools Draw Erase Painting size Large Segment Wre v Opacity i Use these tools to select particular image data to include in the analysis Table 14 4 Region Selection Tools Item Description Draw Choose this option to display the pencil tool Use this tool to apply markings that select regions to include in the reconstruction Erase Choose this option to display the eraser tool Use the eraser to remove pencil tool markings exclude pixels from the image Painting size Adjusts the width of the pencil tool mark or the eraser tool Segment Colors available for the pencil tool Opacity Adjusts the opacity of the pencil tool markings Reset Removes all pencil tool markings v ie Y Caliper Chapter 14 3D Reconstruction of Sources 201 14 2 Reconstructing Fluorescent Sources Image Sequence Requirements Use the Imaging Wizard to setup the image sequence required for FLIT analysis For more details on t
21. CSV The data can be opened in a spreadsheet application such as Microsoft Excel 162 Living Image Software User s Manual 10 4 Managing Planar Spectral Imaging Results Go to the Results tab to select results that you want to view or manage Figure 10 6 Planar spectral imaging tools Results tab Tool Palette Plot Linear Fit Plot Intensity Save Results Name Splm_1 Delete To save results 1 Select results Splm_ lt name gt from the Name drop down list 2 Click Save The planar spectral imaging results are saved with the image To view results 1 Select results from the Name drop down list 2 Click Load To delete results 1 Select the results that you want to delete from the Name drop down list 2 Click Delete To copy selected results 1 Right click the results row and select Copy from the shortcut menu that appears The selected results are copied to the system clipboard To copy all results 1 In the Results tab right click the results table and choose Select All from the shortcut menu that appears All of the results are copied to the system clipboard Tier x Y Caliper Chapter 10 Planar Spectral Image Analysis 163 To export results 1 Right click the results table and select Export Results from the shortcut menu that appears In the dialog box that appears choose a folder for the results enter a file name txt and click Save 164 Living
22. Close Location C Share Caliper LS Caliper Data Sample Data IVIS200 data TraserBeadsPC TLT20050624145507_SEQ Sequencelnfo txt Snapshot Color scale Provides a reference for the pixel intensities in a luminescent or fluorescent image Pixels less than the color scale minimum do not appear in the image Pixels greater than the color scale maximum are displayed in the maximum color Image label Information about the image that the software automatically records and user specified information entered in the Edit Image Label dialog box 5 2 Acquire an Image Sequence That Includes X Ray Images To acquire an image sequence first specify the acquisition parameters for each image in the sequence table Figure 5 7 The Imaging Wizard provides a convenient way to do this for some imaging applications The wizard guides you through a series of steps prompting you for the information that the software needs to set up the sequence in the sequence table If you don t use the wizard you can set up a sequence manually for more details see page 41 Sequence Setup Using the Imaging Wizard l Click Sequence Setup in the control panel Figure 5 7 The sequence table appears table If necessary click the Remove button x Remow and select All to clear the sequence Click the Imaging Wizard button Imaging Wizard YP Cali er REF nce Chapter 5 I X Ray Imaging 55 Figure 5 7 Opening
23. Description Spectral Unmixing Results Number of The number of iterations that the algorithm used Iterations Number of The number of components unmixed Components Number of The number of wavelength pairs used in the analysis Wavelengths Number of Samples The number of pixel samples used in the analysis Lack of Fit PCA The fitting residue compared to the data filtered by principal component analysis Lack of Fit EXP The fitting residue compared to the experimental data Divergence Counter The number of divergences that occurred Maximum Iterations The maximum number of iterations allowed Denoise PCA Indicates how much of the data was filtered by principal component analysis Normalization The normalization method used in the analysis Non negativity Method The non negativity method used in the analysis Weighting Mode The weighting method applied to the data Column Weighting Mode Indicates if column wise weighting was used Row Weighting Mode Save Results Indicates if row wise weighting was used 174 Living Image Software User s Manual Table 11 5 Spectral unmixing tools Results tab continued Item Description Name The name of the selected spectral unmixing results Delete Removes the selected spectral unmixing results from the system Save Saves the spectral unmixing results 11 5 Spectral Unmixing Optio
24. Figure 7 19 View a histogram of pixel intensities Tool Palette gt DAPA cir E us Image Binning amp Width 12 6 cm Height 12 6 cm Histogram Wir Ful min gin sse S Maxein 10348 Bins 512 TP aeeneenen TLT20050624145507_005 Overlay i 0 00 0 00 15 NOTE By default the Auto min max range of the image data determines the histogram range and bins the software sets the min and max values to optimize image display and suppress background noise To display the histogram using the full intensity range of the image click Full in the Histogram window 2 To edit the minimum or maximum bin intensity enter a new value in the Min Bin or Max Bin box or click the arrows 3 To edit the number of bins enter a new value in the Bins box or click the arrows NOTE In the Overlay display mode the histogram plots the luminescent data To obtain a histogram of the photograph select Photograph from the Display drop down list Table 7 6 Histogram window Item Description Full Displays the histogram using the full intensity range of the image Min Bin The lowest intensity bin Max Bin The highest intensity bin Bins The total number of bins pE Opens a dialog box that enables you to export the histogram csv 104 Living Image Software User s Manual Table 7 6 Histogram window continued Item Description Copies the
25. For an optimum fit when there is a large difference between the orientation or size of the atlas organs and surface first use the transformation tool to manually register the surface and atlas organs then click a registration tool to automatically fit the organs For more details on manual registration see below 4 If necessary adjust the opacity of the organs using the slider or enter a number in the box The organs are easier to view if you uncheck Skin in the Organs list YY Caliper Nn a Chapter 141 3D Reconstruction of Sources 227 To clear all organs from the surface click the Deselect All button ma To hide a particular organ remove the check mark next to the organ name To display a specific organ s choose the organ name To display all organs on the surface click the Select All button NOTE After fitting organs to the surface using the i or pa tool if necessary you can click Reset button to restore the default fit j To manually adjust the scale or location of organs 1 SY Load reconstruction results and confirm that the surface is in the perspective view click the toolbar button in the 3D View window or press the R key In the 3D registration tools choose the Display Organs option and select an organ atlas The organs in the selected atlas appear on the surface In Figure 14 32 only Skin is selected Click the Transform tool button amp The transform to
26. m Play Frames Per Second Total Duration secs 5 Record i i i im Save 6 To capture the first key frame click the f button The first key frame is added to the key frame box 7 Adjust the position of the reconstruction in the 3D View using an image tool for example 4 or For more details on the image tools see page 197 8 Click the f button The second key frame is added to the key frame box YW Caliper Chapter 141 3D Reconstruction of Sources 235 feSciences Figure 14 40 Example key frames for a custom animation Frame Factor Animation Setup Time Scale la Key Frame 1 Key Frame 2 Key Frame 3 Key Frame 1 Key Frame 2 Frames Per Second 10 Total Duration secs 5 photons sec jatans sec Source Intensity Source Intensity Perspective Perspective Key Frame 3 Key Frame 4 Load 9 Repeat step 7 to step 8 until all of the key frames are captured For details on how to edit the key frame sequence see page 236 Click a key frame to display the associated 3D view and the time stamp position in the time scale 0 100 at which the frame occurs in the animated sequence 10 Confirm the defaults for FPS frames per second and Total Duration length of animation or enter new values FPS x Total Duration No of frames generated to create the animation The number of generated frames should be to the number of key fram
27. 252 Living Image Software User s Manual Figure 15 17 Example intensity histograms Color Opacity Map Color Opacity Map Grays Grays Y 1 0 Opacity F Reverse 1 0 Opacity E Reverse 0 00 Intensity 5 643 an inienoy ia E Logarithmic Histogram Logarithmic Histogram E Maximum Intensity Projection MIP E Gradient Illumination E Maximum Intensity Projection MIP E Gradient Illumination Linear intensity scale Logarithmic intensity scale Managing Control Points 1 Place a control point on the histogram by clicking anywhere on the histogram between the point represents the lowest intensity in the volume and point represents the highest intensity in the volume During volume rendering the color opacity maps are used to map color and opacity to the corresponding intensity value as well as interpolate color and opacity for all data between adjacent control points 2 Drag any control point up or down to set the opacity level that is associated with the intensity value represented by the point Drag a user added control point left or right to change the intensity associated with the opacity specified by the point When you add delete or modify a control point the color opacity map and the rendering of the volume data are updated in real time NOTE The minimum and maximum intensity levels associated with the and O control points cannot be changed The opacity level ass
28. Acquisition menu ae 5 To enable autosave click Yes in the prompt and choose a folder in the dialog box that appears If autosave is enabled all images acquired during the session are automatically saved to a user selected folder You can choose a different folder at any time select Acquisition Auto Save on the menu bar Image acquisition proceeds The Sequence View window appears and displays the images as they are acquired The Tool Palette appears when acquisition is completed Figure 4 20 To stop acquisition click the Stop in the control panel To pause acquisition click Pause in the control panel YP Cali Der nce er Chapter 41 Optical Imaging 37 Figure 4 20 Sequence View window amp Tool Palette Double click an image in the sequence to open it in a separate image window File fee View losis Aequistiem Window Hep ea mS D g E mjia 0 ir tn al n e E a haquetce ew cotcra is Counts mn Sequence View window Check the image min and max in the color scale to determine whether the signal of interest is above the noise level and below CCD saturation NOTE The Spectra window is available if the acquisition included multiple wavelengths The Spectra window provides a convenient way to view probe spectra from the factory installed library and ROIs For more details see page 171 Table 4 4 Sequence View window Item Description
29. ROI Measurements o e ls Auto ROI Param ROIMeasurements Grid ROI Measurements EY Refresh Image Number ROI Image Laye Total Coun Avg Count Stdev Cour Min Count Max Count ROI Pixels Area Xc Yc Widt ccd Pixels pixels pixels pixe HX20070420121444 001 ROI1 Overlay 1 425e 06 4 167e 02 6 726e 02 8 462e 00 2 545e 03 3420 2 189e 05 4 141e 02 1 020e 03 2 87 HX20070420121444 001 ROI2 Overlay 1 489e 06 3 309e 02 6 061le 02 8 642e 00 2 545e 03 4500 2 880e 05 4 168e 02 9 001e 02 2 822 HX20070420121444 001 ROI3 Overlay 1 676e 06 3 637e 02 5 988e 02 1 056e 01 2 545e 03 4608 2 949e 05 4 168e 02 1 152e 03 2 846 HX20070420121444 002 ROI1 Overlay 7 261e 06 2 123e 03 3 445e 03 2 181e 01 1 031e 04 3420 2 189e 05 4 141e 02 1 020e 03 2 87 HX20070420121444 002 ROI2 Overlay 7 572e 06 1 683e 03 3 107e 03 2 181e 01 1 031e 04 4500 2 880e 05 4 168e 02 9 001le 02 2 82 _ 4 m Customized Selections Measurements Types Image Attributes ROI Dimensions Counts x All Possible Values Pixels X pe ROI Measurements Lo S ss ROI Measurements Grid ROI Measurements Click x20070420121444 001 ROT Measurement Total Counts amp Refresh 1 2 3 5 6 1 6 204e 04 5 459e 04 3 597e 05 1 856e 05 5 045e 05 2 685e 05 2 4 374e 05 1 613e 05 1 390e 05 6 498e 04 1 490e 05 9 588e 04 Grid ROIs 3 6 531e 05 2 505e 05 3 515e 05 1 708e 05 5 025e 04 5 172e 04 4 1 965e 05 1 0
30. allowing the use of narrow band gaps of 25 nm The slope is less steep at infrared wavelengths 800 nm so a wider gap of up to 50 nm is necessary to avoid cross talk 296 Living Image Software User s Manual Fluorescent Filters and Imaging Wavelengths The IVIS Spectrum excitation and emission filters enable spectral scanning over the blue to NIR wavelength region and include e 10 narrow band excitation filters 415 nm 760 nm 30 nm bandwidth e 18 narrow band emission filters 490 nm 850 nm 20 nm bandwidth Figure F 8 IVIS Spectrum excitation and emission filters rans mission Vo o a ee ee ae a IPAS TESS tO a Excitation Filters p Ei SI 24 ABU 520 SEU BUU Ba0 GeO Teo Tao BUU ga Wavelength nim Emission Filters Wavelength men Eight excitation and four emission filters come standard with a fluorescence equipped IVIS Imaging System Table F 1 Custom filter sets are also available Fluorescent imaging on the IVIS Imaging System uses a wavelength range from 400 950 nm enabling a wide range of fluorescent dyes and proteins for fluorescent applications For in vivo applications it is important to note that wavelengths greater than 600 nm are preferred At wavelengths less than 600 nm animal tissue absorbs significant amounts of light This limits the depth to which light can penetrate For example fluorophores located deeper than a few millimeters are not excited T
31. c Step through the rest of the wizard Each page of the wizard guides you with step by step instructions and descriptions When you complete the wizard the sequence information appears in the sequence table Figure 4 15 3 To clear the sequence click the Remove button and select All Figure 4 15 Opening the sequence table and Imaging Wizard F Stop Excitation Filter Emission Filter wees Be oas amaiiecia Terveen I ci If this screen does not appear Double click here to set when the wizard starts click oF a bioluminescence Seam the option fee maging bokimmescant of Restart Wizard on Image sequence ipa umay the wizard screen to restart Double click here to EE A EE EE N ae the wizard set up a fluorescence image sequence When the wizard is finished the sequence is displayed Fstop Excitation Emision Strechare PUW Height 1 Block 550 Ves C 150 1 Blerk 580 Ne 1 50 l Blerck 62 No 1 Blerk 60 Ne c 13 une subject height Temperate cocked i Inebakire E Humbe of Segments 1 E Delay i0 F min Agpaty to A E Remove 2 update arose Ada Y Caliper per Chapter 4 Optical Imaging 35 Figure 4 16 Choose the type of image sequence f E Imaging Wizard Bioluminescence DE 7 Imaging Wizard Fluorescence EJ es Bioluminescence Open Filter Fluorescence Filter Pair Imaging Type Epi lllumination
32. mp4 or avi Use the animation tools to e View a preset animation generated from a factory loaded animation setup page 233 e Create a custom animation created from your custom animation setup page 234 e Save an animation setup page 235 e Record an animation to a movie file page 235 e Edit an animation setup page 236 Figure 14 37 Individual 3D views key frames in the preset animation Spin CW on Y Axis r 3DAnimation Preset Animations Presets Frame Factor 1 5 Animation Setup Time Scale _ 0o 6 iKey Framel Key Frame 2 Key Frame 3 Key Frame 4 Key Frame 5 Perspective Hek Perspective Perspective Xv Keyframe 1 Keyframe 2 Keyframe 3 Keyframe 4 Keyframe 5 The L box shows the key frames in the current animation setup Click a key z frame in this box to display the associated 3D view and time stamp position in the time scale 0 100 at which the frame occurs in the animation Frames Per Second 10 F Click to view the animation composed of the key frames Total Duration secs 5 i 232 Living Image Software User s Manual Table 14 13 3D animation tools ltem Time Scale Description The time stamp of a key frame in the animation on atime scale of 0 100 For example if the animation is 10 sec long and includes five key frames Key frame 1 Time stamp 0 first frame of the animation Key frame 2 Time stamp 25 frame occurs 2 5 seconds after
33. reader fet o E bytes qlee Enter the Pixel Spacing Row Column 0 1000 0 1000 mm e Width height and number of slices Slice Spacing 0 1000 mm ORS e Slice row column pixel spacing and Low Down samples volume data It conserves memory and improves performance the slice spacing In millimeters Full Loads and renders volume data using the original resolution Low Y Full Memory Requirement Status Good 3 If loading the data will cause low memory you are prompted to down sample the data Figure 15 7 Decrease the slice resolution by moving the Slice Resolution slider to the left until the Memory Requirement Status is Good Figure 15 7 Down sample 3D volumetric data to improve memory and performance Slice Information Pixel Spacing Row Column o 1000 4 0 1000 5 mm Slice Spacing 0 1000 4mm Slice Resolution Low Down samples volume data It co Full Loads and renders volume data using the original resolution Low I I I I I J serves memory and improves performance y Requirement Status Low Changing the Orientation of RAW Volumetric Data Occasionally RAW files raw or vox may be loaded with the orientation flipped or reversed along the x y or z axis As a result the slice views transaxial coronal sagittal may be flipped or rotated so that the actual view that is displayed does not match the 3D View windowpane name for example the
34. A drop down list of fluorescence excitation filters For fluorescent imaging choose the appropriate filter for your application For luminescent imaging Block is selected by default If you select Open no filter is present For systems equipped with spectral imaging capability choose the appropriate emission filter Tor your application Note On some models with standard filter sets the excitation filter selection automatically sets the emission filter Emission Filter A drop down list of fluorescence emission filters located in front of the CCD lens The emission filter wheel is equipped with filters for fluorescence or spectral imaging applications The number of filter positions 6 to 24 depends on the system For luminescent imaging the Open position no filter is automatically selected by default Photograph Choose this option to automatically acquire a photograph The illumination lights at the top of the imaging chamber are on during a photographic image so that the system can acquire a black and white photograph of the sample s Note You can adjust the appearance of the photographic image using the Bright and Contrast controls see Adjusting Image Appearance page 97 X ray Choose this option to acquire an X ray image Y Ca i cies Appendix A IVIS Acquisition Control Panel 261 Table A 1 IVIS acquisition control panel continued Item Structure Description Choose this option to take
35. Clear polypropylene pipecdem 2er Clear polystyrene Black polystyrene Two types of autofluorescent effects may occur Overall glow of the material Usually indicates the presence of autofluorescence Hot spots Indicates a specular reflection of the illumination source Figure F 13 The specular reflection is an optical illumination autofluorescence signal reflecting from the microplate surface and is not dependent on the microplate material Figure F 13 Specular reflection The four symmetric hot spots on this black polystyrene well plate illustrate the specular reflection of the illumination source Imaging parameters GFP filter set Fluorescence level Low Binning 8 FOV 15 f 1 Exp 4sec Specular Reflection Specular Reflection Specular Reflection E VY YO YO YC YO YC YC we WV 001000 N NED atata QG Ox Nf ne VW P i 2S AD HOCOOCK AKAN NODI 4 Specular Reflection YY Caliper per Chapter F Fluorescent Imaging 301 Black polystyrene microplates are recommended for in vitro fluorescent measurements Figure F 12 and Figure F 13 show that the black polystyrene microplate emits the smallest inherent fluorescent signal while the white polystyrene microplate emits the largest signal The clear polystyrene microplate has an autofluorescent signal that is slightly higher than that of the black microplate but it is still low enough that this type of microplate may be used Co
36. Delete Load Auto ROI Parameters Threshold Lower Limit s Minimum Size 20 G amp G Preview Use Bkg Offset Replace ROIs Restore Defaults Save Load Radiance p sec cm2 sr Color Scale Min 4 51e8 Max 4 96e9 4 Use the ring amp to move the create tool to the area where you want to draw the ROI then click Create The ROI and label appear on the image NOTE When drawing kinetic ROIs on kinetic data with multiple sources it is recommended that you start with the brightest source then the next brightest and so on in order to create ROIs that can be distinguished based on the signal strength 5 If itis necessary to adjust the ROI boundaries or change any of the auto ROI parameters use the slider or 4 arrows Threshold Specifies the minimum per cent of peak pixel intensity that a pixel must have to be included in an ROI identified by the software Click Done to hide the create tool The kinetic data playback starts and shows the ROI in each image NOTE After the ROIs have been created right click an ROI to view a shortcut menu of ROI commands Ctrl click for Macintosh users The shortcut menu provides easy access to many functions for managing ROIs and viewing ROI properties 7 To measure intensity in the ROI click the Measure button e805 in the Tool Palette The Kinetic ROI Measurements table appears Figure 8 16 8 To view ROI measurem
37. Figure 5 3 If you do not want to enter image information click Cancel Image acquisition proceeds During acquisition the Acquire button becomes a Stop button To cancel the acquisition click Stop When acquisition is complete the image window appears Figure 5 6 12 If this is the first image of the session you are prompted to enable the autosave function 52 Living Image Software User s Manual Figure 5 4 Autosave prompt FE Living Image 4 2 wea Do you want to enable auto saving of acquired data for this session This can be changed anytime from the Acquisition menu 13 To enable autosave click Yes in the prompt and choose a folder in the dialog box that appears If autosave is enabled all images acquired during the session are automatically saved to a user selected folder You can choose a different folder at any time select Acquisition Auto Save on the menu bar The acquisition proceeds and the control panel warns you that X ray radiation is being produced Figure 5 5 The image window and Tool Palette appear when acquisition is completed Figure 5 6 14 If you need to stop the acquisition click Stop in the control panel X Rays Energized Stop Figure 5 5 Control panel during X ray image acquisition Binning F Stop _ Excitation Filter Emission Filter a w System Status X Ravs E f ire Click to stop X ray energy warning Re
38. Moves the surface in the x or y axis direction eg Zooms in or out on the image To zoom in right click Cmd key apple key click for Macintosh users and drag the amp toward the bottom of the window To zoom out right click and drag the Q toward the top ofthe window Displays the x y z axis display in the 3D view window Displays coronal sagittal and transaxial cross sections through the subject in the 3D view window Displays a bounding box around the subject Displays a grid under the subject KAR wm fh Select this tool from the drop down list to change the view perspective top bottom left right front back or perspective view For examples of the views see Figure 14 35 page 229 k Select this tool from the drop down list to display the perspective view Rotates the 3D reconstruction results in the 3D view window 3D scene Click the or key to increase or decrease the rotation speed To stop the rotation click the 3D scene or the gh button a 4 Displays measurement cursors in the coronal sagittal or transaxial views pra Click this button then select a source or a point in a source to obtain source measurements total flux volume center of mass host organ in the 3D tools Source tab For more details see page 209 q Copies or pastes voxels or a source surface so that DLIT and FLIT reconstructions can be displayed on one surface For more
39. Sources FLIT Reconstructs the brightness and 3D location of fluorescent 201 SOUICES Table 2 4 IVIS Imaging System capabilities Acquire a Sequence for IVIS Imaging System Lumina Lumina XR 100 Series 200 Series Spectrum Kinetic Planar spectral image analysis optional optional optional yes yes optional Displaying multiple fluorescent or yes yes yes yes yes yes luminescent reporters Subtracting tissue autofluorescence yes yes yes yes yes yes using blue shifted background filters Spectral unmixing optional optional optional yes yes optional DLIT Analysis 3D reconstruction of no no no yes yes no bioluminescent sources FLIT Analysis 3D reconstruction of no no no no yes no Optional requires premium filters Y Caliper 2 4 Image Display amp Analysis Chapter 2 Overview of Imaging amp Image Analysis 11 Most of the tools for adjusting image display and analyzing images are located in the Tool Palette The Tool Palette automatically appears when acquisition is finished or when you open load image data Its contents depend on the type of active image data Figure 2 4 shows the tools that are available for an image or a kinetic sequence Figure 2 5 shows the Tool Palette for an example image sequence Figure 2 4 Tools available for a single image luminescent fluorescent X ray or a kinetic sequence Click a section of the Tool to show or hide the tools Overlay image E termososiaasso7_0
40. The image overlay window appears and shows the first photograph in the sequence To view a different photograph make a selection from the photograph drop down list Y Ca i a Chapter 7 Working With Optical Image Data 111 Figure 7 26 Image Overlay window ic C Image Overlay Window Sequence ARW20050826124002 SEO Photograph ARW20050826124002 001 v Fluorescent Images Units Radiant Efficiency v F ARW20050826124002_001 E ARW20050826124002_002 m Image Adjust Min L 9 38e7 je me z a olorScale Type Max 1 73e9 Re vy B Palette Label Opacity p 70 Reverse T Logarithmic Scales per Column 3 Seco oa To overlay all images click the button The overlay appears The photograph is at the bottom of the stack and the last fluorescent or luminescent image in the list is at the top of the stack Figure 7 27 Generated overlay is E Image Overlay Window Sequence ARW20050826 124002 SEO Photograph ARW20050826124002 001 v Fluorescent Images 4 ARW20050826124002_001 v ARW20050826124002_002 Units Radiant Efficiency v eo w Image Adjust mn D 9 38e7 ae one ame Max 1 73e9 Ged Palette Label Opacity B 70 Piece T Logarithmic Scales per Column 3 So E gt ea 150 100 107 a 50 Radiant Efficiency a pem Table 7 11 Image Overlay window Item Description U
41. acquisition parameters in the control panel fA Update Inserts a row above the currently selected row using the Insert information from the control panel add Adds a new row at the end of the sequence setup list Editing Image Parameters You can edit parameters in the sequence table or in the control panel To edit a parameter in the sequence table 1 Double click the cell that you want to edit Figure 4 26 Control panel and sequence table G IC Mss Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter Display Photographic Settings Subject Mouse Probes Mode Exposure Binning FStop Excitation Emission Structure FOV Height 1 DES a Auto Medium 1 Block 560 Yes C 1 50 2 EE Auto Medium 1 Block 580 No C 1 50 3 BS Auto Medium 1 Block 600 No C 150 4 Aut Medi 1 Block 620 N C 1 50 Field of View System Status it nud dau vi as Acquire Sequence 5 t Auto Medium v 640 No Cc 15 Mouse Imaging Shuttle Idle Subject height 1 50 alm Image Setup Focus use subjectheight v Temperature MTD Locked Initialize Number of Segments i Delay 0 0 min Apply to All X Remover A Update Insert Add 2 Enter a new value in the cell or make a selection from the drop down list 44 Living Image Software User s Manua
42. ee z Y Ca Pee Living Imagee Software User s Manual Version 4 2 2002 2011 Caliper Corporation All rights reserved PN 133026 Rev 00 Caliper Life Sciences 68 Elm Street Hopkinton MA 01748 USA 1 877 522 2447 US 1 508 435 9500 Fax 1 508 435 3439 E mail tech support caliperls com www caliperls com Discovery in the Living Organism IVIS Imaging System Living Image DLIT and FLIT are either registered trademarks or trademarks of Caliper Life Sciences Inc The names of companies and products mentioned herein may be the trademarks of their respective owners Apple Macintosh and QuickTime are registered trademarks of Apple Computer Inc Microsoft PowerPoint and Windows are either registered trademarks or trademarks of Microsoft Corporation in the United States and or other countries Adobe and Illustrator are either registered trademarks or trademarks of Adobe Systems Incorporated in the United States and or other countries Y Ca i oe Contents WeEICOMCG sa2eceateeetebente tee esa seedy ete bee 6 woe Boe ws 1 1 1 What s New In Living Image 4 2 Software 2 a l 1 2About This Manual se sa ekas 6644 58 56 pEi EER e bapae enua 2 1 3 Contacting Caliper Technical Support aoa aoa 3 2 Overview of Imaging amp Image Analysis a noaoono 0 08 2 eee eee ee 5 DA Workilow OVciView ss sestra eeb Se he ae eed BOG Bee Bee ee ee ee Gee 5 27 macine Modes s s ge eae gee Ba eee ea ee Me et boa eet e
43. s Manual Figure 2 5 Tool palette for an example luminescent image sequence Image sequence luminescent or fluorescent ROI Tools page 119 Image Adjust Tools page 88 e Tune the photograph brightness gamma similar to contrast or opacity e Measure counts or photons in a user specified region of interest ROI and compute measurement statistics for example average min max standard deviation e Set the image display color scale minimum and maximum e Select a color table for image display Planar Spectral Imaging page 157 Determines the average depth and total photon flux ofa luminescent point source In a user specitied region of interest Analyzes a sequence of luminescent images acquired using different emission filters Analyses Requiring an Image Sequence Surface Topography page 181 Analyzes structured light images to reconstructthe animal surface A surface is required for 3D reconstruction of luminescent or fluorescent sources inside a subject DLIT Reconstruction page 193 A 3 dimensional reconstruction of the subject that estimates the depth and intensity of a luminescent light emitting source FLIT 3D Reconstruction page 201 A 3 dimensional reconstruction of the subject that estimates the location and intensity of a fluorescent light emitting source e Measure efficiency in the ROI and compute measurement statistics for flu
44. 1 1 1 YY Caliper 4 TE Chapter 4 Optical Imaging 43 Table 4 5 Sequence table Item Description Starts the Imaging Wizard Gar Displays a dialog box that enables you to select and open a sequence setup xsq sequenceinfo txt or clickinfo txt Tile Displays a dialog box that enables you to save the information in the sequence table to a sequence setup file xsq Display Photographic Settings Choose this option to include the photograph exposure time binning and F Stop in the sequence table If a subject and probe are specified optional the software uses the information to automatically set parameters in the Surface Topography DLIT FLIT Spectral Unmixing and Planar Spectral Imaging tools If a subject or probe is not selected here the default parameters appear in the Tool Palette Number of Segments The sequence specified in the sequence table is called a segment Choose this option to set the number of segments to acquire and the time delay between segments This is useful for acquiring data for kinetic analysis Delay Specifies a time delay between each segment acquisition Applies the selected cell value to all cells in the same column Apply to All Remove Selected Deletes the selected row from the sequence E Remove table Remove All Removes all rows from the sequence table Updates the selected row in the sequence table with the
45. 107 o ms Click this button to draw and measure a rectangle on an image For more details eee see page 108 Click this button to display hide a scale on the x and y axis of the image window H Click this button to display hide a grid the image window 102 Living Image Software User s Manual Table 7 5 Tool palette Image Information tools continued continued Tool Units Description Choose the units cm or pixels for distance measurements in the image window Image Binning The binning applied to the image Note f soft binning is applied to the image data and the binning level is changed from 8 to 16 the new binning is indicated as 8x2 Width Height The FOV dimensions Note If Pixels is selected from the Units drop down list the dimensions are provided in terms of binned pixels Image X Y The x y pixel coordinates of the mouse pointer location in the image Image Data The intensity counts or photons at the pixel location of the mouse pointer Crop Distance ere The x y pixel coordinates at the upper left corner of the crop tool PARED i OR The x y pixel coordinates at the A end of the distance poi The x y pixel coordinates at the lower right corner of the crop tool OR The x y pixel coordinates at the B end of the distance The width and height of the image crop tool OR Ax y from the A to B end of the distance measurement cursor For more details
46. 2 eee eee ee ee 19 This chapter explains how to start the Living Image software and initialize the IVIS Imaging System After it is initialized the imaging system is ready to acquire images 3 1 Starting the Living Image Software The Living Image software on the PC workstation that controls the VIS Imaging System includes both the acquisition and analysis features The Living Image software on other workstations includes only the analysis features For information on installing the software see the Installation Guide included on the Living Image CD ROM By default the software is installed at PC 32 bit windows C Program Files Caliper Life Sciences Living Image PC 64 bit windows C Program Files x86 Caliper Life Sciences Living Image Macintosh Applications Caliper Life Sciences Living Image All components of the IVIS Imaging System should be left on at all times due to the long cooling time required to reach operating demand temperature It is also important to leave the system on to enable automatic overnight electronic background measurements Periodically rebooting the computer is permissible and does not affect the camera operation To start the software 1 PC Users Click the Windows Start button and select All Programs Caliper Life Sciences Living Image Alternatively click the Living Image software icon F on the desktop Macintosh Users Click the Living Image icon F on the desktop or run the
47. 25 M magnify image 98 manual focusing 264 save data 46 manual conventions 2 manual sequence setup 41 45 maximum intensity projection 255 maximum vs time graph tracking infusion 325 maximum vs time graph 70 measurement ROI automatically draw 127 129 measurement ROIs 120 125 129 free draw 129 measurements 107 measuring relative density 65 67 menu commands 327 miscellaneous material autofluorescence 301 302 Mouse Imaging Shuttle 245 multiple reporters per photograph 110 112 N normalization 293 NTF Efficiency 28 146 259 281 320 0 open image data 87 optical density 295 optical image data browse 83 87 optical properties for planar spectral imaging 311 organ atlas import 230 organ display 226 229 organ registration tools 225 231 overlay 280 overlaying images 110 112 P pan image 98 334 Living Image Software User s Manual PCA biplot 176 PCA explained variance 175 photon density 207 photon density map measured 208 simulated 208 photon density maps 207 photon radiance 283 photons 282 pixel 279 pixel data 102 planar spectral analysis optimizing precision 315 planar spectral image analysis 157 159 planar spectral imaging 309 315 diffusion model 310 intensity graph 161 linear fit graph 161 luciferase spectrum 311 optical properties 311 sequence requirements 157 tools 159 160 preferences 265 273 print images 114 116 pseudocolor image 279 0 quantification database 185 190
48. Background light emission from a female white furred Swiss Webster left and a female nude Nu nu mouse and a female nude Nu nu mouse right 0 O Photograph Luminesce Photograph Luminescent nt overlay overlay Usually only very low signals at the highest level of sensitivity require this type of background subtraction For more information on how best to handle these types of measurements please contact Caliper technical support 290 Living Image Software User s Manual This page intentionally blank YY Caliper Appendix F Fluorescent Imaging Description and Theory of Operation 2 ee ee ee ee 291 PHOS eile ct eee ea ee eho ee oo ee ee e Y 294 Working with Fluorescent Samples 2 0 0 8 eee eee eee 297 Image Data Display 2 2 a 298 Fluorescent Background 2 ee ee ee 299 Subtracting Instrument Fluorescent Background 0 6 304 Adaptive Background Subtraction 2 eee ee ee 305 Subtracting Tissue Autofluorescence Using Background Filters 305 F 1 Description and Theory of Operation System Components The VIS Spectrum IVIS 200 Series Imaging System and IVIS Lumina offer built in fluorescence imaging capability as standard equipment Figure F 1 Figure F 2 Figure F 3 The VIS Imaging System 100 and 50 Series use the XFO 6 or XFO 12 Fluorescence Option to perform fluorescence imaging The fluorescence equipment enables you to conveniently cha
49. Browse View Default Configure Load as Group Load Remove close _ Location C Share Caliper LS Caliper Data Sample Data FLIT CK20090729114835_SEQ SequencelInfo txt To preview data click a row Note A preview is only available if a snapshot was taken of the image or sequence thumbnails in the image window For more details on acquiring a snapshot see Chapter 4 page 25 3 To load data do one of the following Double click the data row Right click the data name and select Load on the shortcut menu Select the data row and click Load Double click the thumbnail The image s and Tool Palette are displayed Green rows in the browser indicate loaded data Figure 7 3 86 Living Image Software User s Manual Figure 7 3 Image sequences opened loaded Multiple data sets can be loaded at the same time Fee Eat View Teoh Windew Help Units Counts ahoa D lang imaget rown OD005O420 173030 EQ Chet Mamber EX Fite EM Fiter Murnenation hode User ID 20 CK20091209140055 SEQ x SE TLT20080510014512_5EQ TUT CRASMINTI_ SQ ox J Hede Browse ven Goose Preven Label Set Al IF Add to lst Dpape Vem Oefni Locator C Shere Caiger S Catper Data Sange Daa KNN TN LEOS tet Table 7 1 Living Image Browser Item Description Hide Browse View Closes the browser table Close Preview Closes the image preview box Label Set A drop down list
50. C using PC3M cells Optical Properties of Mouse Tissue Firefly Luciferase Spectra Ex vivo muscle tissue integrating sphere measurement PC3M Luc cells 1 1 2 z 07 Tissue Transmission 0 8 1 indow we In vitro 0 7 0 8 ln vivo 0 6 ae E 05 2 06 04 0 2 0 2 0 1 0 0 450 550 650 750 400 500 600 700 800 Wavelength nm Wavelength nm Diffusion Model of Light Propagation Through Tissue Light propagating through tissue undergoes scattering and absorption The diffusion model assumes that scattering is the predominant phenomenon and the reduced scattering coefficient u gt gt absorption coefficient u This is valid mostly for wavelengths in the red and near infrared part of the spectrum The model also assumes that the light is produced by a single point source and that the tissues are optically homogeneous Under these conditions if we model the animal surface as flat and infinite in extent and integrate the light that 1s collected over the animal surface the total integrated intensity I A is reduced to a relatively simple expression IO SKO exp Heyp 1 where S is the absolute total photon flux emitted by the luminescent source and d is the source depth The term pp is the effective attenuation coefficient It is determined by the tissue coefficient of absorption u and reduced scattering u that quantify the two main phenomena light undergoes in tissue The function K A i
51. Enables you to open and view DICOM data View Image Layout Window Opens the Image Layout window that enables you to paste an image of the active data In the window Tools gt 3D Animation Opens the 3D Animation window that enables you to view a preset animation or create an animation Tools Longitudinal Study Opens the Longitudinal Study window for side by side comparisons of DLIT or FLIT results Tools gt Well Plate Quantification for Opens the Well Plate Quantification window Tools gt Image Overlay for Opens the Image Overlay window for the active data Tools gt Colorize Opens the Colorized View tab for the active sequence Tools gt Image Math for Opens the Image Math window for the active data Acquisition Background Measure Dark Charge Opens a dialog box that enables you to acquire a dark charge measurement Acquisition gt Background Add or Replace Dark Charge Opens a dialog box that enables you to select an instrument luminescent background This background measurement is subtracted from luminescent images Acquisition gt Background Measure and Replace Dark Charge Measures the dark charge under the same conditions as the currently selected image When the measurement is complete the newly acquired dark charge image will be included in the dataset of the current image replacing any existing dark charge image that may b
52. Equation requires all three coefficients u4 H s and Uey as input The function K includes a term called the effective reflection coefficient to account for the reflection of light at the air tissue boundary due to a mismatch in the index of refraction The tissue index of refraction is generally assumed to be close to 1 4 The model assumes that the tissues are optically homogeneous and the Living Image software provides several factory set tissue optical property values to choose from G 3 Luciferase Spectrum Analyzing spectrally filtered images requires knowledge of the spectral dependence of luminescent light emission The luciferase luminescence spectrum was measured in vitro at 37 C and pH 7 in various cell lines This spectrum is used to normalize the photon flux values that the software measures at each wavelength Source spectra for several reporters are included in the database including firefly click beetle renilla and bacteria Figure G 1 The firefly luciferase spectrum is temperature and pH dependent The luciferase spectra included in the software are only valid for measurements performed at 37 C and pH 7 0 7 5 If you use other temperature or pH conditions for an experiment the associated luciferase spectral curve is required for planar spectral image analysis For more information on the pH and temperature dependence of the luciferase spectrum please contact Caliper Corporation 312 Living Image Software User s
53. F 11 Figure F 10 Illumination profiles for different FOVs on an IVIS Lumina measured from the center of the FOV Normalized Intensity FOV A 5cm FOV B 7 5cm FOV C 10cm FOV D 12 5cm 6 4 2 0 2 4 6 FOV cm y Caliper per Chapter F Fluorescent Imaging 299 Figure F 11 Fluorescent image data displayed in terms of radiant efficiency Choose Radiant Efficiency to units Radiantefficency v Display Overlay enable a more quantitative comparison of fluorescent signals 9 8 7 Radiant Efficiency secicm Sh pWiem Color Scale Min 5 58e6 Max 4 77e7 When Radiant Efficiency is selected the fluorescent image data is normalized divided by a stored calibrated reference image of the excitation light intensity incident on a highly reflective white plate The resulting image data is without units typically in the range of 10 2 to 10 NOTE On each IVIS Imaging System a reference image of the excitation light intensity is measured for each excitation filter at every FOV and lamp power The reference images are measured and stored in the Living Image folder prior to instrument delivery F 5 Fluorescent Background Autofluorescence Autofluorescence is a fluorescent signal that originates from substances other than the fluorophore of interest and is a source of background Almost every substance emits some level of a
54. Figure 6 6 Acquisition window Frame number 121 Current frame number Enter a new number or use the top slider to view another frame Start frame in a user specified data range Position of the displayed current frame A Position of the start frame a Position of the end frame j Kinetic Acquisition Control Panel Sele Display Photograph i 14 frames sec Acquisition Settings Luminescent v Overlay ma Dynamic Range Min 111 14 bit ba Max 15366 Exposure Time msecs 35 i 3 12000 l Binning ne 10000 4 F Stop 8000 1 6000 EM Gain 100 4000 Excitation Filter Block Emission Filter 2000 L oo FL Lamp Level Color Bar High Min 111 Photograph Light Level 7 Max 13486 140 7 C Accumulate Play hinas 0 00 17 A v j 444 a Color Scale Auto Maximum counts 110 9 MB 6 8 Time secs Time secs 17 05 Viewing Options After acquisition has been stopped right click the image to access a shortcut menu of viewing options Table 6 2 Kinetic view options Item Description Zoom Area To magnify a particular area draw a box around the area that you want to Zoom In on right click the area and select Zoom Area on the shortcut menu Zoom In Incrementally magnifies the view Zoom Out Incrementally reduces the magnification Reset Zoom Returns the image to the default display magnification Pan V
55. IVIS Kinetic Imaging System is ready to acquire kinetic data after the system is initialized and the CCD camera reaches operating demand temperature locked 6 1 Kinetic Acquisition NOTE Before setting the imaging parameters the IVIS Imaging System should be intialized and the temperature locked For more details see Chapter 3 page 15 1 If you are acquiring ventral images requires the subject be placed in the Ventral Imaging Chamber choose the VIC option in the control panel 2 When you are ready to begin imaging click Kinetics in the control panel The Kinetic Acquisition window appears Figure 6 1 Control panel top and Kinetic Acquisition window bottom 1 00 gt sec _ Medum Ifa foff Box open Medium v i6 v al Fed of view vIC Idle 12 5 im Subject height 0 00 cm Focus use subjectheight v 70 Living Image Software User s Manual 3 Select the type of data to acquire and set the acquisition parameters For more details on the acquisition parameters see Table 6 1 page 71 Figure 6 2 Kinetic Acquisition window 7 Kinetic Acquisition Control Panel me Som Display Overlay x 21 frames sec p After acquisition choose the type of data to display Select the type data to acquire luminescent or fluorescent and choose the Overlay option to acquire photographs 7 Overlay LAYI
56. Image Software User s Manual This page intentionally blank YW Caliper 1 1 Spectral Unmixing Image Sequence Requirements 2 ee ee ee ee ee ee 165 Performing Spectral Unmixing 3 bbs we 4 ee eee wR He Eee Ew 165 Spectra Window 4 4 48 KREBS EDS RE HSER H ESE RSE EE KEE EG 171 Spectral Unmixing Parameters 2 eee ee ee ee 173 Spectral Unmixing Options 2 eee a 174 The Living Image software applies spectral unmixing to distinguish the spectral signatures of different fluorescent or luminescent reporters and calculate the respective contribution of each on every pixel of an image Use spectral unmixing to e Extract the signal of one or more fluorophores from the tissue autofluorescence Images can be acquired using epi illumination excitation light above the stage or transillumination excitation light below the stage e Analyze luminescent or fluorescent images when more than one reporter is used in the same animal model 11 1 Image Sequence Requirements Use the Imaging Wizard to set up the image sequence that is required for spectral unmixing For more details on the wizard see Chapter 4 page 33 If you do not use the Imaging Wizard to set up the image sequence it is recommended that the image sequence include images acquired using several filters that sample the emission or excitation spectra at multiple points across the entire range Make sure that the band gap between the excitation
57. Manual G 4 An Example of Planar Spectral Imaging Melanoma cells were injected intravenously into the tail vein of nude mice After 13 days metastases developed in the lungs kidney and hind limb bone An image sequence was acquired on the IVIS Imaging System 200 Series using filters at six wavelengths from 560 to 660 nm in 20 nm intervals NOTE When using the 560 nm and 580 nm band pass filters tissue optics result in a larger attenuation of light due mainly to hemoglobin absorption A longer exposure time is recommended at these wavelengths Figure G 2 shows the metastasis sites The signals from the lungs and right kidney are well defined in both animals However in the lower back area of the left mouse the signals are in close proximity causing an artifact in the planar spectral analysis Figure G 2 Metastatic sites in nude mice Mice were imaged 13 days after a tail vein injection of 5x10 B16F10 melanoma cells Imaging parameters high sensitivity binning f stop 1 FOV C 13 cm exposure time 120 seconds at 560 and 580 nm exposure time 60 seconds at all other wavelengths This resulted in signals of 2000 counts on each image CK20031215150449_008 Units Photons Display Overlay k p sec em 2 sr Color Bar Min 5 69e4 Max 4 48e5 To perform the planar spectral analysis draw a measurement ROI that captures the entire signal of each site of interest without including a neig
58. Position Unlock Size os e se Co am ROT 3 25 2 088e 05 Vs Luminescence 1 G 2 1 068e 05 i 30000 20000 10000 Counts Color Scale Min 1999 Max 32995 Right click the measurement ROI and select an average background ROI from the shortcut menu 132 Living Image Software User s Manual Figure 8 14 Associate an average background ROI with measurement ROIs Method 3 Bd Soh don oP LE TET T RN r J Tl 14120050630142125 001 poaa E ROT Properties a jE Units Counts y Display Overlay Options _7 m a l Sa eet Label BKG 2 Shape Cirde kai Type Manual Background ROI Subject ROI 7 ROI 325 J 2 088e 05 tol E Use as BKG for future ROIs in JJH20050630142125_001 only Luminescence gt Entire sequence 30000 E Lock Position mg T Xc pix 193 30049 a BKG 1 5 986e 03 EKG 2 1 068e 05 ott en g a DA Re eee Resize Rotate Yc pix 95 17241 Angle deg 0 0000 Copy ROI Copy All ROIs E Lock Size Paste ROI i Width pix 34 28571 Duplicate ROI Height pix 34 28571 Hide ROI Tag Delete ROI Line Size 2 Delete All ROIs 2 Line Color aa Properties 4 Lock Position Lock Size 1 Right click a background ROI and select Properties on the shortcut menu 2 In the ROI Properties box that appears click th
59. Print Preview box that shows what will be printed File gt Recent Files Shows recently opened data sets Note The number of files displayed can be set in the Preferences box select Edit Preferences and click the General tab File Exit Closes the Living Image software Edit Copy Copies the active image window to the system clipboard Edit gt Image Labels oF Opens the Edit Image Labels dialog box that enables you to edit the label set information for the active data Edit gt Preferences Opens the Preferences box 328 Living Image Software User s Manual Table J 1 Menu bar commands and toolbar buttons continued Menu Bar Command ee Description Button View gt Tool Bar Choose this option to display the toolbar View Status Bar Choose this option to display the status bar at the bottom of the main window View gt Tool Palette Choose this option to display the Tool Palette View Activity Window Displays the Activity window at the bottom of the main application window The Activity window shows a log of the system activity View Image Information Displays the Image Information box that shows the label set and image acquisition information for the active data View ROI Properties Displays the ROI Properties dialog box View gt ROI Measurements Displays the ROI Measurements table View gt Volume Data Viewer
60. ROI tools click the ROI Measurements button W The ROI measurements table appears Figure 8 17 Plot Kinetic ROI Measurements tab Kinetic ROI Measurements Plot Kinetic ROI Measurements Plot Multiple ROI Measurements Measurement Unit Radiance Pho a Avg Radiance p s cm sr Vs Time a BI20080724144830 ROI 1 ee 4 04 Avg Radiance p s cm sr DA20081107162612 ROI 1 4 B120080724144830 W ROI 3 0 4 DA20081107162612 7I ROM zoif l at V Fao Configure Export Click the Plot Kinetic ROI Measurements tab Make a selection from the Measurement Unit and ROI Measurement drop down lists Select a data set and an ROI Click Plot ROI Measurements To add other ROI data to the graph repeat step 6 to step 7 a o et ee Y Caliper A por Chapter 8 ROI Tools 135 8 8 Managing ROIs In the ROI Properties box you can view information about an ROI change the position of the ROI on the image and edit the ROI label or line characteristics Viewing ROI Properties 1 To view ROI properties do one of the following e Double click an ROI in the image e Right click the ROI and select Properties from shortcut menu that appears e Select the ROI then select View Properties on the menu bar The ROI Properties box appears for more details see Figure 8 20 2 To view properties for another ROI click the ROI in the image Alternatively selec
61. ROIs to the system gt Image Information gt E ROI Tools O O W Mesweros X A L l Falete D Apply to Sequence e Measurement ROT Save ROIs Name ROT 3 KSA Delete Load Auto ROI Parameters Threshold Lower Limit Minimum Size 20 7 Preview Use Bkg Offset Replace ROIs Restore Defaults Save Load _ Surface Topography gt DLIT 3D Reconstruction gt 3D Multi Modality Tools eel nnii 2 Click Save The ROI s from the image are saved to the system and can be selected from the Name drop down list To load ROIs on an image 1 Open an image 2 In the ROI tools make a selection from the Name drop down list and click Load NOTE If you load ROI s onto an image then draw additional ROIs the Save button changes to Overwrite If you want to save this collection of ROIs using the existing name click Overwrite You can delete ROIs from an image or permanently remove ROIs from the system To delete ROIs from an image 1 In the ROI tools click the X button 144 Living Image Software User s Manual Figure 8 26 Removing ROIs from an image Delete Auto ROI Parameters All Measurements Threshold Lower Limit Minimum Size Z Preview _ Use Bkg Offset _ Replac
62. Reverse is chosen the source intensity photons sec from low to high is represented using the color scale from red to transparent Log Scale Applies a log scale to the color scale MIP When this option is chosen all maximum intensity voxels in the view are projected along the viewing direction into the viewing plane Copies the 3D View tab in the Longitudinal Study window to the system clipboard ti Opens a dialog box that enables you to export the 3D View tab to a graphic file for example png Enables you to select voxels for measurement Measurements are displayed in the Plots tab 14 8 Exporting a 3D Scene as DICOM The items in the 3D View comprise a 3D scene For example the 3D scene in Figure 14 26 includes a surface and voxels The 3D scene can be exported to DICOM format and viewed in the Living Image DICOM Viewer or third party software Figure 14 26 3D scene E Terenosezuiassor seq ismni S Die Spectre 218 Living Image Software User s Manual To export the 3D scene 1 Load the results that you want to export 2 Select File Export 3D Scene as DICOM on the menu bar 3 In the dialog box that appears set the export options and click Export For more details on the 3D Scene Exporter see Table 14 8 Figure 14 27 3D Scene Exporter dialog box Ez 3D Scene Exporter Save DICOM as Slice Orientation Parameters Slice Resolution
63. SPUM_7 Max 1 08e8 Max 3 09e7 Composite 7 To analyze an unmixed image double click the image The image appears in a separate image window and the Tool Palette is available This enables you to make ROI measurements and image adjustments that are saved with the image 8 To adjust the appearance of the composite image double click the composite image The composite image is displayed in a separate window 170 Living Image Software User s Manual Figure 11 5 Composite image window r TLT20060406164950_SEQ Sequence View I Units Radiancefphot Image List 7 TissueAF V UMX 2 Photograph Image Adjust Min 1 08e7 lit Brightness Spectra Unmixed Images TOEA Composite i g amp H Max 9 68e7 Color mi Logarithmic Scale Label TissueAF Table 11 3 Composite image window Item Description Units The type of data displayed in the composite image Image list A list of the images that comprise the composite background component s probe s and a photograph Min Max Sets the minimum and maximum count to display in the image Brightness Adjusts the brightness of the component signals Logarithmic Scale Choose this option to display signals using a logarithmic scale This may be useful when probe signal strengths differ significantly for example a bright source and a dim source Color Shows the colo
64. Scale Min f Max Color Scale Limits Auto Luminescence Individual Color Table 8000 Rainbow 4 Reverse E Logarithmic Scale Counts Color Scale Min 547 Max 8622 Color scale Min and Max Table 7 3 Image Adjust tools Item fa Description Click this button to incrementally zoom out on the image reduces the image dimensions in the image window Note The zoom tools are also available in the shortcut menu when you right click the image Ctrl click for Macintosh users Click this button to incrementally zoom in on the image incrementally magnifies the image in the image window m amp Click this button to magnify the area inside a rectangle that you draw using a click and drag operation Sets the dimensions of the magnified area equal to image window dimensions g Click this button to return the image to the default display magnification 98 Living Image Software User s Manual Table 7 3 Image Adjust tools continued Item T Description Click this button to move a magnified image pan in the image window For more details see page 99 E Click this button to hide or display the image min max information in the image window Click this button to hide or display the color scale in the image window B 4 Click this button to hide or display the color scale min max information in the image window Ph
65. Sequence Requirements Use the Imaging Wizard to setup the image sequence required for planar spectral image analysis For more details on the Imaging Wizard see Chapter 4 page 33 At a minimum the sequence must include a photographic and luminescent image at the first wavelength and a luminescent image at a second wavelength 560 660 10 2 Planar Spectral Image Analysis 1 Load the image sequence that you want to analyze Figure 10 1 Sequence for planar spectral image analysis a SSS ee C 11120050628145507 SEQ boa Tool Palette E Sequence View Spectra E Image Adjust If gt betas ROI Tools TS Counts a Euse Ses Cos Options 7 sino S 9 la 2 TE Imaging ROI 350 Analyze Properties Results _ Sequence 71720050624145507_SEQ Tissue Mouse Tissue Source Firefly Select Filters Filters ROI 4 50 J i ROT 1 50 ROT 2 50 J m ee Ii Min 2140 Max 36446 2 3 Imageset lt lt No active ROI selected gt gt ROI List x _none_ E Surface Topography L gt DLIT 3D Reconstruction 3D Multi Modality Tools gt Spectral Unmixing ROI 7 50 J ROI 8 50 ROI 10 50 Min 586 6 Min 1122 Max 10348 96 via a54 Max 1954 2 In the Tool Palette click Planar Spectral Imaging 3 In the Analyze tab select the
66. Shorter exposure times msecs enable faster frame rates longer exposure times provide greater sensitivity The14 bit dynamic range enables faster imaging by attaining a higher frame rate at the cost of a smaller dynamic range Binning Controls the pixel size on the CCD camera Increasing the binning increases the pixel size sensitivity and frame rate but reduces spatial resolution Using larger binning for a luminescent or fluorescent image can significantly improve the signal to noise ratio The loss of spatial resolution at high binning is often acceptable for in vivo images where light emission is diffuse For more details on binning see Appendix C page 2 6 Recommended binning 1 4 for imaging of cells or tissue sections 4 8 for in vivo imaging of subjects and 8 16 for in vivo imaging of subjects with very dim sources F Stop Sets the size of the camera lens aperture he aperture size controls the amount of light detected and the depth of field A larger f stop number corresponds to a smaller aperture size and results in lower sensitivity because less light is collected for the image However a smaller aperture usually results in better image sharpness and depth of field In kinetic mode the photographic and luminescent or fluorescent image are acquired at the same F Stop For more details on t stop see Appendix C page 275 EM Gain Multiplies the signal in real time This option is useful for boosting low signals
67. Table 11 6 Spectral unmixing options continued Option LP Description Sets a low pass filter for the spectrum Signal above the LP cut off frequency is forced to zero Choose N A to turn off the low pass filter Otherwise the value represents the cut off frequency of the low pass cut off frequency This constraint can help isolate components that are physically mixed and difficult to distinguish Sort Choose this option to automatically sort the unmixed spectra in ascending order of their center wavelength Force Denoise by PCA PCA Mode Choose this option to force the first component to non zero throughout the image Determines how much of the data will be filtered by principal component analysis Stronger denoising means fewer principal components will be used in the data and more details will be lost Stronger denoising also may slow down the unmixing Standard Principle component analysis performed on the original data Correlation Principle component analysis performed on the correlation matrix of the original data Covariance Principle component analysis performed on the covariance matrix of the original data n Explained Variance Click to display the PCA variance plot Figure 11 10 Biplot Click to display the biplot graph Figure 11 11 Update Click to redo the spectral unmixing results with updated constraints PCA Explained Variance The PCA Explained Varia
68. Total slices 256 Approx size 2 9 MB Export voxels using original resolution Low Z Slice spacing mm 0 3977 Pixel spacing mm 0 2891 es Single Frame DICOMs 7 Transasia Slice Hollow mesh Export 4 Inthe Browse For Folder dialog box that appears choose a folder for the DICOM files and click OK During the export operation the 3D View window displays the each slice in the export For example if Transaxial Slice is selected for export then the transaxial windowpane cycles through a display of each exported slice Table 14 8 3D Scene Exporter dialog box ltem Save DICOM as Description Single Frame DICOMs Exports multiple files that contain a single frame each Multi Frame DICOM Exports a single file that contains multiple frames Note Choose the Single Frame or Multi Frame DICOM option depending on the third party software you will use to import and view the 3D scene Some applications cannot reconstruct multi frame DICOM files Slice Orientation Choose transaxial coronal or sagittal slices for the export Export voxels using original resolution Choose this option to export source voxels without any smoothing or binning The original resolution of the source voxels is the resolution obtained after DLIT or FLIT reconstruction approximately 1mm resolution Slice Resolution Sets the number of slices required to accommodate the slic
69. Trans Ilumination For bioluminescent imaging an open filter image gives the most sensitivity since none of the emission light is being filtered This measurement technique is the most commonly used bioluminescent measurement This option selects the best excitation and emission filters Open Filter Filter Pair for a specific fluorescent probe The fluorescent signal is then detected on the surface of Planar Spectral the subject Spectral Unmixine Filter Scan Spectral unmixinc Imaging Wizard Bioluminescence options Imaging Wizard Fluorescence options NOTE The imaging options available in the Imaging Wizard depend on the IVIS Imaging System and the installed filter set Figure 4 17 Control panel and sequence table during acquisition Each row in the sequence table specifies the acquisition parameters for one image in the sequence For more details on the sequence table see Table 4 5 page 43 Exposure Time Binning F Stop Excitation Filter Emission Filter s no eo E Display Photographic Se Probes Block v a Subject X FStop Excitation Emission Structure FOV Height TD z Medium 1 Block 560 Yes C 150 Medium 1 Block 580 No C 150 Medium 1 Block 600 No Medium 1 Block 640 C 1 50 Mouse Imaging Shuttle Idle 134 om Subject height 1 50 EALL image Setup
70. You can choose a different folder at any time select Acquisition Auto Save on the menu bar Image acquisition proceeds During acquisition the control panel Acquire button becomes a Stop button To cancel the acquisition click Stop in the control panel The image window and Tool Palette appear when acquisition is completed Figure 4 8 page 28 YY Caliper A per Chapter 4 Optical Imaging 33 4 4 Acquire a Sequence Using the Imaging Wizard This section explains how to acquire a sequence of multiple images To acquire an image sequence first specify the acquisition parameters for each image in the sequence table Figure 4 15 The Imaging Wizard provides a convenient way to do this for some imaging applications Table 4 3 The wizard guides you through a series of steps prompting you for the information that the software needs to set up the sequence A sequence can also be set up manually for more details see page 41 Figure 4 14 Imaging Wizard EF mig mg igisi Tmp Meds fichiminascence MHA Sect the option fee maging bourmmesart oF Chemdinmescant raperters Bech as fret breraa dii b stia locferce rena ot bactaral lucheaee Huona Imaging Table 4 3 Imaging Wizard options Bioluminescence Description Page Open Filter Acquires an image at maximum sensitivity Planar Spectral Analyze the sequence to compute the average depth and total photon 15 7 flux of a luminescent point source
71. a structured light image an image of parallel laser lines scanned across the subject when you click Acquire The structured light image is used to reconstruct the surface topography of the subject which is an input to the Diffuse Luminescence Imaging Tomography DLIT algorithm that computes the 3D location and brightness of luminescent sources When this option is chosen the f stop and exposure time are automatically set to defaults for the structured light image f 8 and 0 2 sec respectively The spatial resolution of the computed surface depends on the line spacing of the structured light lines The line spacing and binning are automatically set to the optimal values determined by the FOV stage position and are not user modifiable Overlay If this option is chosen the system automatically displays the overlay after acquisition is completed for example luminescent image on photograph Lights Turns on the lights located at the top of the imaging chamber Fluor Lamp Level Sets the illumination intensity level of the excitation lamp used in fluorescent imaging Off Low High and Inspect The Low setting is approximately 18 of the High setting Inspect turns on the illumination lamp so that you can manually inspect the excitation lamp Note Make sure that the filters of interest are selected in the filter drop down lists before you select Inspect The Inspect operation automatically positions the selected fil
72. assess the depth of field at any f stop and FOV setting For more details on manual focusing see page 264 Generally f 1 is recommended for low light luminescent images and f 2 or f 4 is recommended for brighter luminescent or fluorescent images Figure C 2 Lens f stop positions Left lens wide open at f 1 right lens closed down at f 8 f 1 f 8 deep focus Image Exposure Time The image exposure time also affects sensitivity The number of photons collected is directly proportional to the image exposure time For example an image acquired over a two minute exposure contains twice as many detected photons as an image acquired over a one minute exposure Longer exposure times are usually beneficial when imaging very dim samples However this may not always be true because some types of background dark charge in particular increase with exposure time For more details on backgrounds see Appendix E page 285 An IVIS Imaging System has extremely low background that enables exposures of up to 30 minutes However animal anesthesia issues and luciferin kinetics limit practical exposure times for in vivo imaging to 5 10 minutes Field of View FOV C 2 Binning The FOV indirectly affects sensitivity Changing the FOV without changing the binning or the f stop does not significantly affect sensitivity However CCD pixels are effectively smaller at a smaller FOV higher magnification so that higher levels of binn
73. associated with the user ID selected at the start of a new session To view the user modifiable preferences after you log on select Edit Preferences on the menu bar Figure B 1 Preferences dialog box E Preferences Start Up Defaults Show Activity Window on Warnings Errors Save Settings E Save float corrected image Window Size Color Selections man 0a 55 E Folder Locators Height 65 E Dock Tool Palette Left Right Window Size amp Position Most Recently Used DatasetHistory 5 21 Apply Individual Color Scale for Sequences Display ROI Label As Measurement Show Transillumination Locations Counts Show Advanced Options Radiance Photons NOTE Any changes made to the Preferences are implemented at the start of the next session The Acquisition tab is only available in the Living Image software that controls the IVIS Imaging System 266 Living Image Software User s Manual B 1 General Preferences Figure B 2 General preferences Ez Preferences General Optical Properties Start Up Defaults E Dock Tool Palette Left Right Window Size Width 55 _ Restore Defaults Folder Locations Height 65 Apply Individual Color Scale for Sequences Display ROI Label As Measurement Show Transillumination Locations Show Advanced Options Show Activity Window on Warnings Errors Save Settings E Save float corrected image Color Sel
74. at the location marked by a green square E Table 4 2 Transillumination Setup box Item Description Move Motors to Selected _Transillumination motors will move the excitation light source to the Spot grid location selected in the Transillumination Setup dialog box Mask Grid points To When setting up a transillumination sequence choose this option to Subject automatically select only the grid locations within the subject boundaries Grid locations outside the subject are masked out The mask prevents the transillumination excitation source from selecting an uncovered hole Projecting light through an open hole would saturate the camera YY Caliper Chapter 41 Optical Imaging 31 Table 4 2 Transillumination Setup box continued Item Raster Scan Description If this option is not selected the software generates one image per transillumination location per filter pair For example a sequence setup that includes 20 locations using two filters will generate 20 images If the raster scan option is selected the software takes all of the images from the transillumination locations and adds them together into one image The raster scan option may be helpful when trying to determine the optimal excitation and emission filters for a particular fluorescent probe Grid Type Select a grid type from the drop down list 15x23 11x23 5x10 or 8x12 well plate Xenogen Sparse Mask 6x8x1cm Update Photogr
75. background subtraction 305 adjusting image appearance 97 animation 231 236 custom 234 235 edit an animation setup 236 preset 233 animation tools 231 236 auto exposure feature 19 autofluorescence 130 299 miscellaneous material 301 302 See tissue autofluorescence subtract using background filters 305 307 well plate 300 301 autoluminescence 130 automatically draw ROIs 127 129 average background ROI 120 130 B background adaptive fluorescent background subtraction 305 fluorescent 299 305 light on sample 286 288 tissue autofluorescence 305 307 background light from sample 288 289 on sample 286 288 background corrected signal 130 132 band gap 295 bandpass filter 294 batch mode 39 40 binning 100 276 278 browse optical image data 83 87 browser optical image data 86 volumetric data 241 C Caliper Corporation technical support 3 cascade images 88 classifying 3D volumetric data 250 control points 252 color table 279 colorize data 113 114 color opacity map 251 composite image 151 153 control panel 19 259 263 conventions 2 COpy 332 Living Image Software User s Manual ROI measurements 147 correction filtering tools binning 100 cosmic correction 100 dark background subtraction 99 flat field correction 100 smoothing 100 cosmic correction 100 cosmic ray corrections 284 counts 281 crop box 108 D dark background subtraction 99 dark charge 286 dark current 285 data graphic image 279 scientific ima
76. background subtraction on and off using this check box NOTE The fluorescence background also contains the read bias and dark charge Dark charge subtraction is disabled if the Sub Fluor Bkg option is checked YY Caliper REF nce Chapter F Fluorescent Imaging 305 Figure F 19 Comparison of dark charge bias subtraction left and fluorescent background subtraction right The autofluorescence from the nose cone and filter leakage have been minimized in the image on the right by using Sub Fluor Bkg option Color Bar Min 900 Max 40000 F 7 Adaptive Background Subtraction Adaptive background subtraction is a simple way to reduce the instrument fluorescent background by fitting and removing the background using the existing image for example the left image in Figure F 19 Unlike the method described in section F 6 Subtracting Instrument Fluorescent Background where you acquire an actual instrument fluorescent background image by removing the fluorescent subject from the imaging chamber to correct the background the new method uses software correction To perform adaptive background subtraction e Identify the fluorescent subject in the original image using the photo mask e The software automatically fits the instrument background to the whole image using the pixels outside of the subject e The software subtracts the fitted instrument background from the original image In most situations suc
77. but detected by the camera may occur outside the bandpass region The materials used in filter construction may also cause the filters to autofluoresce YY Caliper per Chapter F Fluorescent Imaging 295 Figure F 6 Typical excitation and emission spectra for a fluorescent compound The graph shows two idealized bandpass filters that are appropriate for this fluorescent compound 100 O Excitation 7 NS Emission Filter O gt lransmission O o ro 0 001 Wavelength nm Figure F 7 Typical attenuation curves for excitation and emission filters Separation 20 nm Excitation Optical Density Wavelength In Figure F 7 the vertical axis is optical density defined as OD log T where T is the transmission An OD 0 indicates 100 transmission and OD 7 indicates a reduction of the transmission to 10 For the high quality interference filters in the VIS Imaging Systems transmission in the bandpass region is about 0 7 OD 0 15 and blocking outside of the bandpass region is typically in the OD 7 to OD 9 range The band gap is defined as the gap between the 50 transmission points of the excitation and emission filters and is usually 25 50 nm There is a slope in the transition region from bandpass to blocking Figure F 7 A steep slope is required to avoid overlap between the two filters Typically the slope is steeper at shorter wavelengths 400 500 nm
78. called photon flux photons sec When cells occur in tissue photon emission from the tissue surface is called surface radiance photons sec cm2 sr Surface Radiance i Tissue gt Q Solid Angle units of steradians Flux Y Caliper Appendix D Image Data Display amp Measurement 283 Efficiency A steradian can be thought of as a three dimensional cone of light emitted from the surface that has a unit solid angle Much like a radian is a unit of arc length for a circle a steradian is a unit of solid angle for a sphere An entire sphere has 47 steradians Lens systems typically collect light from only a small fraction of the total 4m steradians When image data is displayed in radiance mode the units change to photons sec cm sr These are units of photon radiance on the surface of the animal A very important distinction between these absolute physical units and the relative units of counts is that the radiance units refer to photon emission from the subject animal itself as opposed to counts that refers to a measurement of photons incident on the detector Measurements in units of radiance automatically take into account camera settings for example integration time binning f stop and field of view As a result images of the same subject acquired during the same session have the same signal amplitude regardless of the camera settings because the radiance on the animal surface does not change The ad
79. control panel Figure 4 29 The stage moves to position C then you are prompted to remove the lens attachment 2 After you remove the Zoom Lens attachment click OK in the prompt Always store the lens wrapped in its protective container Figure 4 29 Control panel remove the check mark next to Zoom E YIS Acquisition Control Panel k Imaging Mode Exposure Time Binning F Stop Excitation Filker Emission Filter 1 d Field of view Jom System Status C FO 244 TE Acquire Sn Subject height 15 crm Sequence Setup Please remove the zoom lens by pulling down firmly from its magnetic attachment Click OK when done Canes 4 8 Manually Saving Image Data When you acquire the first image s of a session you are prompted to enable the autosave feature If autosave is enabled all images acquired during the session are automatically saved to this folder You can choose a different folder at any time select Acquisition gt Auto Save on the menu bar Y Caliper POL Chapter 4 Optical Imaging 47 This section explains how to manually save data if you do not want to use the autosave feature 1 Turn off the autosave feature select Acquisition on the menu bar and remove the check mark next to Auto Save After you acquire an image or image sequence click the Save button f l Alternatively select File Save on the menu bar 3 In the dialog box that appears select a directory and click OK
80. effective attenuation coefficient of and reduced scattering coefficient u s or usp Source Spectrum Choose this option to display the source spectrum for DLIT reconstructions Bioluminescent Spectrum Choose this option to display the spectrum of the bioluminescent source Fluorescent Spectrum Choose this option to display the spectrum of the fluorescent source Restore Defaults Click to restore the defaults in the Optical Properties tab 274 Living Image Software User s Manual This page intentionally blank YW Caliper Appendix C Detection Sensitivity CCD Detection Efficiency 1 ee 275 BHI s3 we Ge Goa eee ee eee Ee EGS EE Oe ee we eS 276 IOC 24 4 ERE PERE EOE ESE KRHA SER SRE ER EE ES 278 The parameters that control the number of photons collected signal and the image background noise determine the sensitivity of low light imaging To maximize sensitivity the goal is to increase signal and decrease background Several factors affect the number of photons collected including the lens f stop image magnification size and detection efficiency quantum efficiency of the CCD transport efficiency of the imaging optics and the image exposure time C 1 CCD Detection Efficiency Lens Aperture IVIS Imaging Systems use a back thinned back illuminated CCD cooled to 90 to 105 C depending on the system This type of CCD provides high quantum efficiency of over 80 across the v
81. emission filter wavelengths for the analysis Figure 10 2 It is recommended that you do not include a wavelength in the analysis if the signal is less than or equal to the autoluminescent background If autoluminescent background is a concern you can create a background ROI and link it to the measurement ROI 158 Living Image Software User s Manual prior to planar spectral analysis For more details see Measuring Background Corrected Signal page 130 Figure 10 2 Planar spectral imaging tools Analyze tab For more details on the Analyze tab see page 159 Tool Palette E3 gt Image Adjust Planar Spectral Imaging Properties Results Sequence 71720050624145507_SEQ Tissue Mouse Tissue Source Frefy Select Filters Filters 560 580 600 620 Imageset lt lt No active ROI selected gt gt Analyze ROI List _none_ _ Surface Topography gt DLIT 3D Reconstruction _ 3D Multi Modality Tools Emission filter wavelengths selected for the analysis 4 Inthe ROI List drop down select All or a particular ROI in an image for the analysis If there is no measurement ROI draw an ROI that includes the area for analysis For more details on drawing ROIs see page 120 You only need to draw the ROI s on one image in the sequence The software copies the ROI s to all other images of the sequence during the analys
82. es 275 C2 IMM 2aneeaueuvtoeeptandsedeeda Seen eeuateesbeesseueees beads 276 C 3 Smoothing aoaaa 278 Appendix D Image Data Display amp Measurement aaan aaa a 279 DA mae Dan es ecticea ee surer ranno eh ee eee eee ee eee eee ee 279 D2 QOuantityine Imace Data ss sss emar oe OE REE EAE Bee Ee ee 281 Dp Pla Fielding 2 46245 es SE RRSHRSESERREES SER EMDR ERE HOES eS 284 DA Cosmic Ray Corrections 0645422 4 4 84 4 8465 4 005464684 Sh 2 He 8 284 Appendix E Luminescent Background Sources amp Corrections 285 E 1 Electronic Background 2 1 sarietatik eaii srs rr ideaal 285 E 2 Background Light On the Sample aoao a aaa 286 E 3 Background Light From the Sample ooa aoaaa a 288 Appendix F Fluorescent Imaging aa aaao a 291 F 1 Description and Theory of Operation ooa ee 291 F2 Prop aese eaer nren eaa ae a eee eee ee eG 294 F 3 Working with Fluorescent Samples aoo aaa a 291 PA Image Data Display lt siss 6 e284 Heb GRE TEES SHE PERRO Eured HE EESS 298 F 5 Fluorescent Background aooaa a 299 F 6 Subtracting Instrument Fluorescent Background 0 0000 ee eee 304 F 7 Adaptive Background Subtraction 2 a aa 305 F 8 Subtracting Tissue Autofluorescence Using Background Filters 305 Appendix G Planar Spectral Imaging 2 0 008 eee eens 309 G 1 Planar Spectral Imaging Theory 2 2 0 0 0 ee 309 2 Optical Properties 24444 9 Sake HE ESE HE
83. filter Figure F 20 Example of the autofluorescent subtraction technique using a background excitation a primary excitation filter DsRed b blue shifted background excitation filter DsRed Bkg and c corrected data The corrected image was obtained by subtracting the scaled background filter image multiplied by 0 47 from the primary filter image The 6 week old female Nu nu mouse was injected subcutaneously with 1x108 HeLa luc PKH26 cells in the left flank 4 1x10 26x10 Primary Background Corrected excitation filter excitation filter image 200 iii m m m a a p E a 2 5 E aie 2 6x10 on z r E _ gt an 1 9x10 zi P 1 Signal Bkg 6 5 Signal Bkg 150 Minimum no of detected cells 1 5 x 105 Minimum no of detected cells 6 7 x 102 YY Caliper per Chapter F Fluorescent Imaging 307 Figure F 21 Spectral data describing the autofluorescent subtraction technique using a background filter The graph shows the excitation and emission spectrum of PKH26 and the autofluorescent excitation spectrum of mouse tissue Also included are the spectral passbands for the blue shifted background filter DsRed Bko the primary excitation filter DsRed and the emission filter used with this dye Bkg Ex Em Filter Filter Filter Excitation TE Emission ase Avitofluorescence Normalized Intensity 308 Living Image Software User s Manual This page intentiona
84. for all wavelengths are displayed Figure 14 15 2 To rotate the surface and view it from a different angle move the thumb wheel to the left or right Figure 14 15 Photon density maps Use the thumb wheel to rotate the surfaces 7 Photon Density Maps Eome Tool Palette Ed Wavelength Al Waves Angle of View Fiogsaie my amp Analyze Properties Results DLIT Results DLIT_45507 Loaded Key Value ss Final voxel size mm 1 25 Number of voxels 115 Reduced Chi2 9 73e 05 Index Of Refraction 1 40 Angle limit deg 70 Damping reduce 100 of samples 200 Show samples 0 Starting voxel size 5 Sampling method Uniform a Photon Density Maps a Export Results Save Results Name DLIT_45507 x gt 3D Multi Modality Tools gt 3D Tools gt Spectral Unmixing 3 Select a wavelength from the drop down list The photon density profiles at the crosshairs location are displayed In a good reconstruction the simulated red photon density curves closely resemble the measured blue photon density curves 208 Living Image Software User s Manual Figure 14 16 Simulated and measured photon density plots E Photon Density Maps Wavelength 620 ka photons mm 4 He Photon density photons rer 3 5 3 0 2 5 2 0 1 5 1 0 0 5 0 0 5 Fosition rer Position 620 nm wavelength Angle o
85. frame in the sequence select the key frame and click the or arrow 5 To update a key frame a Select the key frame and adjust the 3D view b Click the 4 button 6 To delete a key frame a Select the key frame that you want to remove b Click the button and select Delete Current Y Caliper Chapter 14 3D Reconstruction of Sources 237 14 14 DLIT FLIT Troubleshooting Issue Solution e In DLIT or FLIT this can occur if the surface is not correct That is if No sources in solution a surface is imported into the 3D View from another source other than from the Structured Light Analysis e The most common source of spiky surfaces are folds in the animal skin or fur which corrupt the desired smooth lines projected on the animal from the laser galvanometer Choose the Fur Mouse option for Subject Smoothing the surface by using the Smooth feature in the Surface Topography tools can help improve the surface Surfaces are spiky Tool Palette Ey COO SssC lt CSs s s SCS 1 gt Planar Spectral Imaging sid 7 Surface Topography sd Surface Reconstruction Subject Nude Mouse Mouse Reconstruct Reconstruct Orientation Dorsal Surface Smoothing Level Low x Smooth Restore Save Results Name SURFACE 1 Delete Load Overwrite gt DLIT 3D Reconstruction gt 3D Multi Modality Tools i gt 3D Toos gt gt o gt Spectr
86. functionality for Auto ROI parameters e Additional export and import option for 3D surfaces and voxels Show Activity Window on A drop down list of options for when to display the activity log Figure B 3 YY Caliper per Appendix B Preferences 267 Table B 1 General preferences continued Item Description Save Settings Save float corrected image Saves an image after all corrections are applied read bias subtraction flat field correction cosmic correction Color Selections Applies the color settings of the active image data to subsequently opened image data Folder Locations Sets the default folder path to the current folder path setting Click the Export button ey in the image window to view the current folder path setting Figure B 3 Window Size amp Position Applies the active image window size and position settings to subsequently opened image data Most Recently Used Dataset History Applies the active image window size and position settings to subsequently opened image data Display ROI Label As Sets the type of measurement in counts radiance photons or Measurement efficiency to show in the ROI label Some of the general preferences specify how the main application window is organized To undock the Tool Palette click on the palette title bar and drag it a distance greater than its width To dock the Tool Palette in the main window drag the palette to the right or left side o
87. histogram to the system clipboard cs Opens the print dialog box Line Profile The line profile plots intensity y axis at each pixel x axis along a user specified line in the image The line profile is automatically updated when you change the line position NOTE In the Overlay display mode the line profile plots the luminescent data To obtain a histogram of the photograph select Photograph from the Display drop down list To display the line profile 1 Open an image and in the Image Information tools click the Line Profile button A line appears on the image and the Line Profile window appears Figure 7 20 Viewing a line profile of pixel intensities Tool Palette fe J E t1120050624145507 005 fe jlo ss units Counts ar Display Options info gy g NO r E owes Image nind Binning amp Width 12 6 cm Height 12 6 cm uminescence 10000 Image X Y 12 349 10 750 an Image Data 6 counts Pi 2000 Crop Distance 6000 4000 2000 Counts Color Scale Min 586 Max 10348 Line Orientation Horizontal Width 1 2 Position 137 L ei g X Min 0 IX Max 239 iy Min 25 HVY Max 61 5 V Full Scale C Logarithmic Scale 4 TLT20050624145507_005 Overlay x10 Counts 1 00 0 60 0 40 0 20 0 00 pixels 2 To view the line profile at another location in the image put the mouse pointer over the line When the pointer becomes a drag t
88. illuminated to help determine the source location 288 Living Image Software User s Manual In Figure E 1 the pen appears very bright due to phosphorescent emission that is also illuminating the portion of the hemisphere next to the pen If the pen had been outside the field of view it would not have been imaged and the source of the phosphorescence would be less obvious However the illumination of the hemisphere would still be very apparent and indicative of a light pollution problem OL ALERT Handle the High Reflectance Hemisphere by its black base plate while wearing cotton gloves provided by Caliper Skin oils can phosphoresce and will contaminate the hemisphere Latex gloves and the powder on them may also phosphoresce If the hemisphere becomes contaminated contact Caliper technical support for a replacement There are no known agents that can clean the hemisphere To check the hemisphere for contamination take several images of the hemisphere rotating it slightly between images A glowing fingerprint for example will rotate with the hemisphere while a glowing spot due to external illumination most likely will not E 3 Background Light From the Sample Another source of background is the natural light emitted from a sample that is not due to emission from the source of interest in the sample This type of background may be due to a material associated with the experimental setup For example the cell culture medium may p
89. image on photograph na Fluorescent An exposure of the subject illuminated by filtered light The target fluorophore emission is captured and focused on the CCD camera a g Fluorescent image data can be displayed in units of counts or photons absolute calibrated orin Fluorescent image terms of efficiency calibrated normalized For more details on fluorescence image data see Appendix F page 291 Overlay Fluorescent image on photograph Y Caliper Chapter 2 Overview of Imaging amp Image Analysis 9 Table 2 2 Image types optical data continued Imaging Mode Structure Description A structured light image of parallel laser scanned across the subject The surface topography of the subject is determined from the structured light image Example 5 K oF as X ray An exposure of the subject using the X ray energy source on the Lumina XR The X ray image Is displayed as a grayscale image X ray image Overlay Fluorescent image on X ray image Kinetic A series of images captured on the IVIS Kinetic Imaging System that enables visualization of luminescent or fluorescent signals in real time Play back kinetic data In real time or view a particular frame s 10 Living Image Software User s Manual 2 3 About Image Sequences A sequence is a collection of images that are grouped together in a single folder A sequence may include images tha
90. in a region of interest ROI Spectral Unmixing Analyze the sequence to determine spectral signature of different 165 reporters in the same image and calculate the contribution of each reporter on each pixel in the image DLIT Apply the DLIT algorithm to the sequence to reconstruct the 3D 193 surface topography of the subject and the position geometry and strength of the luminescent sources Fluorescence Description Page Filter Pair Choose this option to acquire measurements of one or more fluorescent probes Spectral Unmixing Analyze a sequence to extract the signal of one or more fluorophores 165 Filter Scan from the tissue autofluorescence Helps you determine the optimum excitation and emission filter for a probe FLIT Apply the FLIT algorithm to the sequence to reconstruct the 3D 201 surface topography of the subject and the position geometry and strength of the fluorescent sources For details on acquiring a sequence on the Lumina XR that includes X ray images see Chapter 5 page 49 34 Living Image Software User s Manual Sequence Setup Using the Imaging Wizard 1 Click Imaging Wizard in the control panel Figure 4 15 NOTE If necessary click Restart to show the first page of the wizard 2 In the wizard that appears a Choose Bioluminescence or Fluorescence imaging b In the next wizard screen choose the type of image sequence that you want to acquire see Table 4 3 page 33
91. in the reconstruction slice The coordinates mm of the position are displayed The coordinates are updated when you press and hold the mouse button while you drag the cursor e Coronal plane Displays the x y coordinates e Sagittal plane Displays the y z coordinates e Transaxial plane Displays the x z coordinates Figure 14 19 Viewing y z coordinates in the sagittal plane 212 Living Image Software User s Manual Displaying Slices Through a Reconstruction 1 Click a location on a source Alternatively click the ioe toolbar button draw a box around a source then click Center of mass in the 3D Source tools 2 Click the jg toolbar button The Coronal Sagittal and Transaxial windowpanes show a slice through the surface taken by the associated plane Figure 14 20 Planes cutting a reconstruction 3 To move a plane put the mouse cursor over a line in the coronal sagittal or transaxial windowpane When the cursor becomes a ji or arrow drag the line The view is updated in the windowpanes as you move the line Figure 14 21 Moving the coronal plane Y Caliper per Chapter 14 3D Reconstruction of Sources 213 14 6 Displaying Luminescent and Fluorescent Sources on One Surface If an experiment includes luminescent and fluorescent reporters DLIT and FLIT reconstructions can be displayed on one surface NOTE If the DLIT and FLIT image seque
92. infusion when acquisition stops even though the pump may not be stopped Figure 1 4 Tracking infusion inad szue aay i MEO 8 amp amp amp iby Fee Dannie Fro toe l Time secs 3 Time secs 25 45 1 3 Closing the Infusion Pump Control Panel 1 Close the kinetic control panel 2 Click Acquisition Infusion Pump Setup on the menu bar The check mark is removed and the panel closes 326 Living Image Software User s Manual This page intentionally blank y Caliper LifeSciences Appendix J Menu Commands Tool Bar amp Shortcuts Figure J 1 Living Image toolbar s AHRS R Table J 1 Menu bar commands and toolbar buttons Menu Bar Command File Open Toolbar Button a ei Description Displays the Open box so that you can select and open an image data file File gt Browse BX Displays the Browse For Folder box so that you can select and an image data folder The selected folder is displayed in the Living Image Browser File gt Browse 3D Volumetric Data Displays the Browse For Folder box so that you can select and a volumetric data folder for example DICOM format TIF data The selected folder is displayed in the 3D Browser File gt Save Saves overwrites the AnalyzedClickInfo text file to update the analysis parameters but the original image data files are not altered File gt
93. is active Color Table C Click the drop down arrow to select a color table for the image data For more details on color tables see Pseudocolor Images page 279 Reverse Choose this option to reverse the selected color table Logarithmic Scale Choose this option to apply a log scale to the relationship between numerical data and the color range in the color table A log scale increases the range of meaningful numerical data that can be displayed Magnifying or Panning in the Image Window To incrementally zoom in or out on an image Click the or S button Alternatively right click the image and select Zoom In or Zoom Out on the shortcut menu To magnify a selected area in an image 1 Click the button Alternatively right click the image and select Area Zoom on the shortcut menu YY Caliper DET nce Chapter 7 Working With Optical Image Data 99 2 When the pointer becomes a draw a rectangle around the area that you want to magnify The selected area is magnified when you release the mouse button To reset the magnification remove magnification Click the 4 button Alternatively right click the image and select Reset Zoom on the shortcut menu To pan the image window Panning helps you view different areas of a magnified image If the image has not been magnified you cannot pan the image 1 Click the Hi button 2 When the pointer becomes a p click and hold the pointer while
94. l gt Surface Topography a Coronalfz 19 7 Sagittal x 1 5 DUT Reconstruction Analyze Properties Results Tissue Properties Mouse Tissue nA a Source Spectrum Firefly x Plot Tissue Properties z Luminescent Calibration St mm 19 7 mm Result not found f Transaxialfy 10 3 lt i mm Wavelength nm 3D Multi Modality Tools 3D Tools z we e _ Spectral Unmixing E i Subject Height 26 3 mm Perspective Select tissue and Reconstruct and view source measurements source properties LifeSciences Chapter 14 3D Reconstruction of Sources 193 14 1 Reconstructing Luminescent Sources General Considerations Animal Requirements The best surface topography reconstruction is obtained from nude mice It is possible to perform 3D imaging on white or light colored furred mice if the fur is reasonably smooth over the mouse surface Therefore it is recommended that you comb the fur before imaging to eliminate any fluffy areas that may trigger artifacts during the surface topography reconstruction In this case it is recommended that you shave the animals or apply a depilatory 3D reconstructions are currently not possible on black or dark colored furred mice Luminescent Exposure vs Luciferin Kinetic Profile It is important to consider the luciferin kinetic profile when you plan the ima
95. loading onto other images For more details see Saving ROIs page 142 Auto ROI Parameters Parameters that specify how the auto ROI tool draws an ROI Threshold If the Auto All or Auto 1 method is selected the Threshold specifies the minimum percent of peak pixel intensity that a pixel must have to be included in an ROI identified by the software After ROIs are drawn on an image if you modify the Threshold move the slider or enter a new value the software automatically updates the ROls Note The following Auto ROI parameters are only available if Show Advanced Options is selected in the general preferences For more details on setting Preferences see Appendix B page 265 Lower Limit Specifies a multiple 1 to 10 of the color scale minimum that sets the lower threshold for identifying an ROI For example if the lower limit 2 and the color scale minimum 1000 counts then the auto ROI tool will only draw an ROI on areas of 2000 counts or greater This helps create ROIs only in the visible range Minimum Size Sets the minimum size of an ROI measured in pixels For example if the minimum size is set at 50 then ROIs created on the image must be greater than 50 pixels in size Preview f this option is chosen the software draws the ROI each time a parameter is changed ROI parameters can be saved without drawing the ROI Use Bkg Offset Choose this option to measure background corrected signal Th
96. ltem Description Units Select the measurement units for the image display from this drop down list The available units depend on the type of image data For more details on measurement units see page 281 Use Saved Choose this option to display the image data using the color table that was Colors image specified in the Preferences at the time of acquisition If this option is not sequence selected image data are displayed using the color table currently specified in the Preferences 90 Living Image Software User s Manual Table 7 2 Image window continued Item Description Options Layout Choose a display option for the images in a sequence Default Dynamic image For example ee em Strip mode sequence tae esnadi i othe TS Fo Sort by Options for ordering images in the sequence window Default Order in which the images are stored in the folder TimeStamp Ascending order of the image acquisition time UserlD Ascending alohanumeric order of the user ID Display Choose the types of information to display with each image Layout j m Jace Mma i d Taai Pea ator 0 Display o yo pomire Tim bs Labe HO yo Bimming Factor Enititiin Mber Eminion fitter I burbs Field of Views Sehect All Clear All ening pacar 8 Twit gt Ee In this example exposure time and binning factor are displayed on each image Lablels Enables you to select the information
97. of the available label sets which specify image information column headers that is displayed in the Living Image Browser Add to List If this option is chosen the selected in the Browse for Folder box is added to the Living Image Browser If this option is not chosen the data selected in the Browse for Folder box replaces the contents of the Living Image Browser except for loaded data Browse Opens the Browse For Folder box View The name of the Living Image Browser configuration the column headers and their order in the browser Configure Opens a dialog box that enables you create and save custom Living Image Browser configurations Note lo reorder a column in the browser click the column header then press the mouse key while you drag the header left or right Release the mouse key to set the new position YY Caliper Chapter 7 Working With Optical Image Data 87 Table 7 1 Living Image Browser continued Item Load as Group De on Enables you to select particular images that you want to view as a sequence The images may be acquired during different sessions To select adjacent images in the browser press and hold the Shift key while you click the first and last file in the selection To select non adjacent images in the browser PC users Press and hold the Ctrl key while you click the images in the browser Macintosh users Press and hold the Cmd key apple key while you click the ima
98. of the crop box drag a handle I at a corner or side of the box 4 To delete the crop box from the image click the 7 7 button Table 7 10 Crop box position amp dimensions Description x y coordinates at the upper left corner of the box x y coordinates of lower right corner of the box Box width and height Distance Length of the diagonal from the upper left to lower right corner of the box 7 8 Creating a Transillumination Overview The transillumination overview tool combines the images of a FLIT sequence a fluorescence sequence acquired in transillumination mode into a single image All of the individual fluorescent signals are stacked over one photograph and the intensity is summed One overview is created per filter pair If two filter pairs were used during acquisition then two overview images will be created All transillumination locations are displayed simultaneously a tool tip displays the transillumination position when you mouse over a transillumination point An overview image is displayed in radiant efficiency and can be analyzed using the tools in the Tool Palette If you choose the Raster Scan option in the Transillumination Setup box the overview image is automatically generated For more details see page 30 1 Load a sequence that was acquired in fluorescence transillumination mode 2 Click the button Alternatively select Tools Transillumination Overview for lt name
99. or after acquisition If you do not want to enter image information click Cancel 8 If this is the first image of the session you are prompted to enable the autosave function Figure 4 3 Autosave prompt E Living Image 4 2 z Do you want to enable auto saving of acquired data for this session This can be changed anytime from the Acquisition menu 9 To enable autosave click Yes in the prompt and choose a folder in the dialog box that appears If autosave is enabled all images acquired during the session are automatically saved to the user selected folder You can choose a different folder at any time select Acquisition Auto Save on the menu bar Image acquisition proceeds During acquisition the control panel Acquire button becomes a Stop button To cancel the acquisition click Stop in the control panel The image window appears when acquisition is completed Figure 4 4 24 Living Image Software User s Manual Figure 4 4 Overlay luminescent image on photograph in the image window 7 fie Edt View Toots Acquisition Window Help 18x ah amp e AET Unite Counts gt Display Crveriay Tool palette Lurenescenve 100009 6000 4000 Table 4 1 Image window Item Description Units Select the measurement units for the image display from this drop down list The available units depend on the type of image data F
100. see page 107 and 108 Viewing X Y Coordinates amp Intensity Data 1 Open an image and the Image Information tools choose Cm or Pixels from the Units drop down list 2 Put the mouse pointer over a location of in the image The x y coordinates and intensity data are displayed in the Tool Palette Figure 7 18 x y coordinates and intensity data at the mouse pointer location E TLT20050624145507_005 Units Counts n _ eee Tool Palette v Display Overlay Options z Info w _ Image Adjust gt L gt Corrections Filtering le Image Information w A BY if E Units Cm Image Binning 8 Width 12 6 cm Height 12 6 cm Luminescence 10000 gt ROI Tools _ Planar Spectral Imaging gt Surface Topography gt DUT 3D Reconstruction gt 3D Multi Modality Tools gt Spectral Unmixing Counts Color Scale Min 586 Max 10348 v i ET Y Caliper Chapter 7 Working With Optical Image Data 103 Image Histogram The image histogram plots a frequency distribution of the pixel intensities in an image The software sorts the intensities into groups or bins x axis and plots the number of pixels per bin y axis To view the image histogram 1 Open an image and in the Image Information tools click the Image Histogram button
101. select a color table for luminescent and fluorescent image data Choose the Reverse option to reverse the min max colors of the selected color table Y Ca i ermi Appendix B Preferences 271 Table B 5 Image view preferences continued Item Description Use saved color palette while loading datasets If this option is chosen data are displayed using a user specified color palette For example after you load data specify a color table in the Image Adjust tools and save the data The user specified color table is automatically applied whenever the data are loaded Background amp Text Color Sets the colors for the background and text in the image window shown here To change a color click the button that opens the color palette r E7 TLT20050624145507_005 GEI Units Counts v Display Overlay cA Options 7 Info i Luminescence 10000 8000 6000 4000 2000 Counts Color Scale Min 586 Max 10348 ROI Color Sets the colors for the ROI outline To change a color click the a button that opens the color palette Luminescent Color of the ROI outline on a luminescent image Fluorescent Color of the ROI outline on a fluorescent image Restore Defaults Click to apply the default settings 272 Living Image Software User s Manual Figure B 8 3D view preferences Preferences Solid Color Gradien
102. that the tissue excitation spectrum is much broader than the excitation spectrum of the fluorophore of interest and that the spatial distribution of autofluorescence does not vary much with small shifts in the excitation wavelength Figure F 20 shows an example of this technique using a fluorescent marker In this example 1x10 HeLa luc PKH26 cells were subcutaneously implanted into the left flank of a 6 8 week old female Nu nu mouse Figure F 21 shows the spectrum for HeLa luc PKH26 cells and the autofluorescent excitation spectrum of mouse tissue It also shows the passbands for the background filter DsRed Bkg the primary excitation filter DsRed and the emission filter DsRed Figure F 20 shows the IVIS images using the primary excitation filter the background excitation filer as well as the autofluorescent corrected image The corrected image was obtained using a background scale factor of 1 4 determined by taking the ratio of the autofluorescent signals on the scruff of the animal The numbers shown in the figures are the peak radiance of the animal background within the region of interest In the corrected image the RMS error is used to quantify the background The signal to background ratio of the original fluorescent image DsRed filter is 6 5 The ratio increases to 150 in the corrected image an improvement factor of 23 This improvement reduces the minimum number of cells necessary for detection from 1 5x105 to 6 7x103
103. the What s This tool Ls l Click this button then click an item in the user interface to display information about the item NOTE Macintosh users use the Cmd key apple key instead of the Ctrl key Y Ca ais Index Numerics 3D intensity signal 106 3D Multi Modality tools adjusting image resolution 254 classifying 3D volumetric data 250 color opacity map 251 control points 252 fiducial registration 245 gradient illumination 255 loading data 241 243 manual registration 247 249 maximum intensity projection 255 requirements 239 volume display options 254 3D quantification database 185 190 create 186 189 manage results 189 samples 185 3D reconstruction fluorescent sources 201 203 luminescent sources 193 200 reconstruct particular regions 199 3D reconstruction results DLIT or FLIT 204 205 3D surface 177 183 3D tools 220 236 Animate tab 231 236 Registration 225 231 Source tab 222 224 Surface tab 220 222 3D Volumetric Browser 241 3Dsurface export or import 183 generate 178 179 manage 182 view perspective 181 182 A accumulate signal 72 73 acquire image sequence using Imaging Wizard 33 36 image sequence with X ray images 54 61 kinetic data 69 sequences in batch mode 39 40 single fluorescent image epi illumination 25 28 single fluorescent image transillumination 28 32 single luminescent image 21 25 single X ray image 49 54 Index 331 activity window 19 adaptive fluorescent
104. the start of animation Key frame 3 Time stamp 50 frame occurs 5 0 seconds after the start of animation Key frame 4 Time stamp 75 frame occurs 7 5 seconds after the start of animation Key frame 5 Time stamp 100 last frame of the animation Presets A drop down list of predefined animation setups Key frame A 3D view The software interpolates the key frames to create intermediate frames in real time then generates an animated sequence from all of the frames Each successive key frame in a sequence should differ slightly from the preceding one so that motion is smoothly depicted when the frames are shown at a proper frame rate frames second The Living Image software provides preset key frames or you can specify the 3D views for the key frames Preset Key Frame Factor Determines how many key frames are used to generate one revolution in a spinning animation No of frames 4 x Key Frame Factor 1 Increasing the key frame factor reduces the time period between key frames and creates the appearance of finer movement Decreasing the key frame factor increases the time period between key frames and creates the appearance of coarser movement Frames displayed per second in the animation sequence Creates a new key frame from the current 3D view Updates the selected key frame to the current 3D view Deletes a selected or all key frames from the key frame box Moves a selecte
105. to exposure times greater than five minutes The signal is linear with respect to exposure time over the range from 0 5 to 10 minutes Integration times less than 0 5 seconds are not recommended due to the finite time required to open and close the lens shutter Binning Controls the pixel size on the CCD camera Increasing the binning increases the pixel size and the sensitivity but reduces spatial resolution Binning a luminescent image can significantly improve the signal to noise ratio The loss of spatial resolution at high binning is often acceptable for in vivo images where light emission is diffuse For more details on binning see Appendix C page 276 Recommended binning 1 4 for imaging of cells or tissue sections 4 8 for in vivo imaging of subjects and 8 16 for in vivo imaging of subjects with very dim sources F stop Sets the size of the camera lens aperture he aperture size controls the amount of light detected and the depth of field A larger f stop number corresponds to a smaller aperture size and results in lower sensitivity because less light is collected for the image However a smaller aperture usually results in better image sharpness and depth of field A photographic image is taken with a small aperture f 8 or f 16 to produce the sharpest image and a luminescent image is taken with a large aperture f 1 to maximize sensitivity For more details on t stop see Appendix C page 275 Excitation Filter
106. tool X axis crop tool Y axis crop tool Z axis crop tool Y Caliper Chapter 15 3D Multi Modality Tools 249 ifeSciences b Click and hold a control point while you move the crop plane As you move the crop plane the slice views are updated Release the mouse button to crop the data c To reset the crop planes click the TA button When finished cropping press the Tab key to turn off the crop tool 4 Click the Manual Registration button IE The transformation tool appears Figure 15 14 The tool has three modes that enable you to translate scale or rotate the 3D volumetric data press the Tab key to change the tool mode The slice views are automatically updated when you use the tool Figure 15 14 Manual registration tool transformation modes a b Usa Tab bey ho pach beteen rarioa boe r ine Teb hey to pch bebrasen monasi maton hooks z Line Y Y a Z beys io restrict scaling bo anhy one ane Seting On YZ Translate Moves the volume in the Scale lIncreases or decreases scale x y or z axis Drag the tool to adjust the the size of the volume drag ared cube at position of the volume a corner of the volume To restrict scaling to a particular axis press the X Y or Z key then drag a red cube Line iah key bo veich bot eer rater Ene Rotate lo rotate the volume on the x y Or z axis click the blue green or red circle and drag the mouse arrow in the direction of in
107. you move the mouse 7 6 Correcting Optical Image Data Use the Corrections Filtering tools to subtract background or apply corrections to the optical image data For more details on sources of background see Appendix E page 285 You can also apply smoothing and soft binning to the image data For more information on binning and smoothing see Appendix C page 275 Figure 7 15 Tool palette Corrections Filtering tools Read Bias Subtraction Read Bias Subtraction and Flat Hat Field Correction Field Correction are default ee mandatory corrections in photons Fluorescent Background Subtraction mode In counts mode these corrections can be cleared Adaptive FL Background Subtraction Binning Eom Smoothing None t gt ROT Tools Roto OOOO O O Table 7 4 Tool palette Corrections Filtering tools Tool Description Read Bias Select this check box to subtract dark background from the image data If Subtraction Dark a dark charge image is available for the imaging conditions the dark Charge Subtraction background image including read bias noise will be subtracted Otherwise only read bias noise will be subtracted For more details on background see Appendix E page 285 Note In Radiance Photons mode dark background or read bias subtraction is a mandatory default In counts mode the check box can be cleared 100 Living Image Software User s Manual Table 7 4 Tool pa
108. 0 5 675e 05 i ROI 7 50 3 349e 05 l top a 4 ROT 6 50 7 273e 6 50 J 7 273e 05 ROI 8 50 1 763e 05 ROI 10 50 3 025e 05 i P a hy hy fi gt it fit r Min 586 Max 10348 f 6 4 To adjust the ROI boundaries change any of the auto ROI parameters using the slider or 2 arrows Figure 8 4 122 Living Image Software User s Manual Figure 8 4 Auto ROI parameters Tool Palette Name ROI_1 KSA Delete Threshold Specifies the minimum per cent of peak pixel intensity that a pixel must have to be included in an ROI identified by the software After the ROIs have been created right click an ROI to view a shortcut menu of ROI commands Ctrl click for Macintosh users The shortcut menu provides easy access to many functions for managing ROIs and viewing ROI properties 5 Click the Measure button esas in the ROI tools to show the ROI Measurements table Figure 8 5 ROI Measurements table ROI Measurements CIE ROI Measurements Refresh Refresh Image Number ROI Image Lay Total Coun Avg Count Stdew Cour Min Count Max Count v5 TLT20050624145507_001 ROI1 Overlay 6 536e 03 2 334e 02 4 395e 01 1 723e 02 3 301e 02 3 TLT20050624145507_002 ROI 2 Overlay 2 653e 04 6 982e 02 1 402e 02 4 730e 02 9 393e 02 TLT20050624145507_003 ROIL 3 Overlay 8 290e 05 2 961e 04 5807e 03 2 209e 04 4 280e 04 TLT20050624145507
109. 03 wets Canis Opies Overlay Image Adjust Tools page 97 e Tune the photograph brightness gamma similar to contrast or opacity e Set the image display color scale minimum and maximum e Select a color table for image display Tool Palette Palette 7 Image Adjust aq QM Q amp E Photo Adjustment toeloes 8 nto A Color Scale min Max Color Scale Limits Auto Individual Color Table Reverse Image Informati Corrections Filtering Tools page 99 e Subtract dark background from the image data e Apply flat field correction to the image data e Specify pixel binning e Smooth the pixel signal Corrections Filtering E Logarithmic Scale Image Information Tools page 101 e Display x y coordinates and intensity data ata user selected location on the image e Display a histogram of image pixel intensities e Plot the intensity y axis at each pixel x axis along a user specified line in the image e 3D plot of intensity signals e Measure distance in an image Kinetic sequence ROI Tools page 119 e Measure counts or photons in a user specified region of interest ROI and compute measurement statistics for example average min max standard deviation Measure efficiency radiant efficiency or NTF efficiency in the ROI and compute measurement statistics for fluorescent images only 12 Living Image Software User
110. 0414101005_001 v For Sequence EL20090414101005_SEQ Click EL 20090414101005 001 v Fluorophore Type Fluorophore Type Measurement Measurement Sample Wells 3D 3A O set 9 Sample Wells 3D 3A E Background Wells 1 Set F Background Wells Set 4s C Apply to Sequence V Apply to Sequence Well Plate Quantification Plots Results Well Plate Quantification Plots Results Set position and enter dilution values in cells Set position and enter dilution values in cells D Cc B A 500000 250000 125000 62000 1 2 3 4 5 6 For Click EL20090414101005_001 Quantify For Click EL20090414101005_001 9 Enter the dilution values in the table cells NOTE The values must be entered as dimensionless numbers For example for a picomole of dye molecules enter 6 022e11 10 To delete a dilution value select the table cell and press the Delete key Alterrnatively right click the number to view a shortcut menu of edit commands for example cut copy paste 11 Choose the Apply to Sequence option 12 Choose the Background Wells option 13 In the well plate table select the background wells and click Set Clicking a row or column header selects the entire row or column To remove the background well designations click the button 188 Living Image Software User s Manual Figure 13 5 Set the background wells For Sequence EL
111. 1 DLIT FLIT Troubleshooting 0 ee eee ee ee es sss 97 The Living Image software provides algorithms which analyze 2 dimensional image data to reconstruct 3 dimensional 3D luminescent or fluorescent sources located inside an animal tomographic analysis For more details on the DLIT or FLIT algorithm see Appendix H page 317 3D Reconstruction Description Algorithm Diffuse Tomography DLIT provides a complete 3D reconstruction of the 194 DLIT luminescent source distribution within the subject DLIT places no constraints on the geometry or spatial variation of the source strength throughout the volume DLIT is well suited for analyzing complex and spatially extended luminescent sources The 3D reconstruction is presented as voxels If a luminescent calibration database is available the number of cells per source can be determined in addition to source intensity photons sec Fluorescent Tomography FLIT provides a complete 3D reconstruction of the 201 FLIT fluorescent source distribution within the subject The 3D reconstruction is presented as voxels If a fluorescent calibration database is available the number of fluorophore molecules or cells per source can be determined in addition to the total fluorescence yield The input data to the DLIT algorithm for a 3D reconstruction of luminescent light sources includes e A structured light image that is analyzed to generate a surface for the imaging s
112. 141e 02 1 020e 03 287 HX20070420121444 001 ROI 2 Overlay 1 489e 06 3 309e 02 6 061e 02 8 642e 00 2 545e 03 4500 2 880e 05 4 168e 02 9 001e 02 2 82 HX20070420121444 001 ROI3 Overlay 1 676 06 3 637e 02 5 988e 02 1 056e 01 2 545e 03 4608 2 949e 05 4 168e 02 1 152e 03 2 84 HX20070420121444 002 ROI1 Overlay 7 261e 06 2 123e 03 3 445e 03 2 181le 01 1 031e 04 3420 2 189e 05 4 141e 02 1 020e 03 2 87 HX20070420121444 002 ROI2 Overlay 7 572e 06 1 683e 03 3 107e 03 2 181e 01 1 031e 04 4500 2 880e 05 4 168e 02 9 001le 02 2 82 _ 4 tu p Customized Selections Measurements Types Image Attributes ROI Dimensions Copy Select All Counts x Al Possible Values y Pixels v Configure Export Close 148 Living Image Software User s Manual oY To include image information in the ROI table make a selection from the Image Attributes drop down list To include ROI dimensions in the table select units Pixels or cm from the ROI Dimensions drop down list Creating a Custom ROI Table Configuration A table configuration specifies the column headers in the ROI table Several preset configurations are available selected from the Measurements Types drop down list in the ROI table Figure 8 30 You can also create a custom table configuration NOTE Preset table configurations cannot be edited You can modify a preset configuration and save it to anew name l In the ROI Measurement
113. 145507_006 x Units Counts Options vY Info ij Y Display Overlay X Luminescence Color Scale Min 1122 Max 19546 3 Use the ring amp to move the create tool to the area where you want to draw the ROI then click Create The ROI appears on the image and the ROI label displays the intensity signal y Caliper A por Chapter 8 ROI Tools 129 4 To draw another ROI on the image repeat step 2 to step 3 For information on how to save ROIs see page 142 Drawing an ROI Using the Free Draw Method 1 Open an image and in the ROI tools select the type of ROI that you want to draw from the Type drop down list 2 Click an ROI shape button Circle _ Square O _ or Contour and select Free Draw from the drop down list In this example the Contour shape was selected for the free draw method The ROI shapes that are available depend on the type of ROI selected 3 If you selected lt or O Use the pointer to draw the ROI Use the pointer to click around the area of interest and draw line segments that define the ROI Right click when the last point is near the first point in the ROI Figure 8 11 Drawing an ROI using the free draw method E7 TLT20050624145507_006 Co El _ Toot Palette op Units Counts v Display Overlay v Options v Info it gt Image Adjust le M Corrections Filtering gt _
114. 20 Level of Detail slider evel Of Detail D D 2 Performance Resolution 0 5x 1x 2x oa 3x Table 15 3 Example volume with 512 slices at 1x resolution Volume Resolution No of Slices Displayed 0 5x 256 1x original resolution 512 1 5 768 2X 1024 ax 1280 3X 1536 Maximum Intensity Projection MIP MIP projects all maximum intensity voxels in the view along the viewing direction into the viewing plane Gradient Illumination Gradient Illumination is based on the idea that light is reflected at boundaries between different voxel intensities but is not affected when passing through homogeneous regions Choosing this option illuminates the voxels at boundaries more than voxels within a homogeneous region The boundaries are based on the gradient magnitude between heterogeneous regions or the change in intensities between neighboring voxels in heterogeneous regions Choosing this option enhances the variation in tissue properties and may be helpful for visualizing the boundaries of different tissues Editing Volume Slices You can modify the pixel and slice spacing of the volume Changing the pixel or slice spacing modifies the volume resolution 1 In the Volume tab click the Edit Space and Orientation button im 2 In the dialog box that appears Figure 15 21 edit the pixel or slice spacing Figure 15 21 Volume information dialog box A Volume Information Slice Information Bae
115. 20090414101005_SEQ Click EL20090414101005_001 7 Fluorophore Type Dr mdenies Cds Measurement Sample Wells 3D 3A C Set e Wee 0 64 Eya C Apply to Sequence Well Plate Quantification Plots Results Set position and enter dilution values in cells For Click EL20090414101005_001 Quantify 14 Click Quantify The results are displayed Figure 13 6 Example fluorescence quantification plot and results For Sequence EL20090414101005_SEQ Click EL 20090414101005_001 v For Sequence EL20090414101005_SEQ Click EL 20090414101005_001 v Fluorophore Type Fluorophore Type Dyenelaies cele Dye mlases Ca Measurement Measurement Sample Wells 3D 3A C Set Sample Wells 3D 3A C Set 7 Background Wells 6D 6A 7 Background Wells 6D 6A V Apply to Sequence V Apply to Sequence Results Ck A E Excitation Emission Extinction Coeff Cross Section nm nm Qe M cm 1000 Qoa mm x10 a ROI vs well plate population f 6 0 Total Efficiency cm Linear Fit j 2919e 07 1 115e 08 1 262e 07 4 821e 09 5 894e 06 2 251e 09 2 230e 06 8 518e 10 Database 2 0 3 0 i i WPQUANT_1 v Name WPQUANT_1 well plate population Delete Load Delete Load 15 Check the linear fit of the data for each image in the quantification plot A good straight line fit gives confidence to the results values Large deviations fro
116. 25 pmol uL 2 u CF750 dye in pillows Location C Share Caliper LS Caliper Data Sample Data IVIS200 data PC3M JJH20050630142125_SEQ JJH20050630142125_002 ClickInfo txt Images loaded in the browser as part of a sequence highlighted yellow in this example These images can also be selected for grouping into another sequence 2 In the browser select the images that you want to group together 118 Living Image Software User s Manual To select adjacent images in the browser press and hold the Shift key while you click the first and last file in the selection To select non adjacent images in the browser e PC users Press and hold the Ctrl key while you click the images of interest in the browser e Macintosh users Press and hold the Cmd key apple key while you click the images of interest in the browser 3 Click Load as Group The image thumbnails are displayed together in an image window 4 To save the images as a sequence a Click the Save button f l Alternatively select File gt Save on the menu bar b In the dialog box that appears select a folder and click OK YY Caliper 8 ROI Tools ADOULROIS 262464 84648 SRR ERE RARE SE HERES EEG BOS OS BG 119 Quick Guide Drawing Measurement ROIs on an Image or Sequence 120 ROIL TOUS ias ee BRERA RE REREAD ee Ee 123 Measurement ROIs 1 1 ee ee a 125 SUHJ RUIS s es ce ee RRR ERS Ee ERD EHO we gt Oo De SS 129 Measuring Backgr
117. 6 Living Image Software User s Manual Select an excitation and emission filter from the drop down lists For more information about the standard filter sets see Table F 1 page 296 3 Put a check mark next to Photograph Make a selection from the Field of View drop down list For more details on the field of view see page 261 Nn Set the proper Focus by choosing one of the methods below The focal distance to the camera is set at stage z 0 for each field of view To focus at the top of the animal the stage will move down so that the top of the animal is at z 0 You can either enter the height of the animal using the use subject height option or let the software determine the animal height by choosing the scan mid range option e Select use subject height and use the nes arrows or the keyboard arrows to specify a subject height cm e Select manual focus For more details on manual focusing see page 264 e Select scan mid range available on the IVIS 200 or Spectrum Imaging System only The imaging system determines the subject height using a laser scan This value is automatically entered in the subject height box and is used to set the focal length To acquire an overlay image coregistered images for display after acquisition put a check mark next to Overlay NOTE To view the subject s inside the chamber before image acquisition take a photograph Uncheck the Fluorescent option choo
118. 62e 07 4 821e 09 3 465 560 5 894e 06 2 251e 09 4 465 580 2 230e 06 8 518e 10 Wavelength nm Sequence Database Name WPQUANT_1 v Name WPQUANT_1 v D Save Delete Load Save _ 3D Multi Modality Tools 7 Saves the results with Saves the results to a database that is the image sequence available for DLIT or FLIT analyses 190 Living Image Software User s Manual Table 13 2 Managing quantification results ltem Description Sequence Delete Removes the active quantification results from the image Name PC3M_GFP v sequence wae Load Opens quantification results from the sequence path Save Saves the quantification results with the selected image sequence Overwrite Saves the results with the selected image sequence and overwrites previous results Database Delete Deletes the database from the system A Load Opens quantification results from the system path Save Saves the quantification results to a system database that is available for DLIT or FLIT reconstruction Overwrite Saves the results to the selected database name and overwrites previous results Exporting Quantification Results Right click the results table to view copy and export options e Copy Copies the selected rows to the system clipboard e Select All Selects all rows in the results table e Export Results Opens a dialog box that enables you to export the selected resu
119. 99e 05 4 741e 04 4 660e 04 4 673e 04 4 484e 04 Customized Selections Measurements Types Image Attributes ROI Dimensions Copy Select All al Posse aes a eal 146 Living Image Software User s Manual Figure 8 29 ROI Measurements table kinetic ROIs a ROI Measurements So Il f S ROT Measurements Kinetic ROI Measurements Plot Kinetic ROI Measurements Click DA20081107162612 ROI ROIi AllFrames lt gt Refresh Image Numb ROI ImageLayer FrameTime Total Flux Avg Radiance Stdev Radiance Min Radiance Max Radiance Threshold UserID z p s p s cm sr Frame 001 ROIL Overlay 0 000 1 379e 10 4 301e 09 1 559e 09 1 948e 09 7 381e 09 26 DA Frame 002 ROI1 Overlay 0 162 4 391e 09 1 370e 09 1 371e 09 1 808e 08 5 248e 09 26 DA Frame 003 ROI1 Overlay 0 324 3 620e 09 1 129e 09 1 036e 09 2 068e 08 5 004e 09 26 DA Frame 004 ROI1 Overlay 0 486 3 953e 09 1 233 09 1 397e 09 2 056e 08 5 170e 09 26 DA gt q m 7 Customized Selections Measurements Types Image Attributes ROI Dimensions Copy Select All Radiance Photons v al Possible Values i _none_ Y Configure Export Close Table 8 4 ROI Measurements table Item Measurement Types None Counts luminescence Radiance Photons fluorescence Radiant Efficiency fluorescence Efficiency epi fluorescence NTF Efficiency t
120. Animal Number Animal Strain Animal Model Sex View Cell Line Reporter Treatment Luc Injection Time 4 84 4 4 4 4 4 4 5 4 4 4 IACUC Number 3 In the Edit Image Labels box enter information about the image and click OK If you do not want to enter image information click Cancel 4 If this is the first image of the session you are prompted for an autosave location All images acquired during the session are automatically saved to this folder You can choose a different folder at any time select Acquisition Auto Save on the menu bar To select a folder for autosaved data click Yes in the prompt and choose a folder in the dialog box that appears Figure 5 12 Autosave prompt appears if this is the first acquisition of the session FE Living Image 4 2 Do you want to enable auto saving of acquired data for this session This can be changed anytime from the Acquisition menu Image acquisition proceeds The Sequence View window appears and displays the images as they are acquired The Tool Palette appears when acquisition is completed Figure 5 13 5 To stop acquisition click the Stop in the control panel To pause acquisition click Pause in the control panel Y Caliper 4 per Chapter 5 X Ray Imaging 59 Figure 5 13 Image sequence and Tool Palette Double click an image in the sequence to open it in a separate image window File Edit View Tool
121. Co http www pdcorp com healthcare frs html 30000 10000 Color Bar 2000 x 45000 YY Caliper per Chapter F Fluorescent Imaging 303 Animal Tissue Autofluorescence Animal tissue autofluorescence is generally much higher than any other background source discussed so far and is likely to be the limiting factor in in vivo fluorescent imaging Figure F 17 shows ventral images of animal tissue autofluorescence for the GFP DsRed Cy5 5 and ICG filter set in animals fed regular rodent food and alfalfa free rodent food Harlan Teklad TD97184 Animals fed the regular rodent diet and imaged using the GFP and DsRed filter sets show uniform autofluorescence while images taken with the Cy5 5 and ICG filter sets show the autofluorescence is concentrated in the intestinal area The chlorophyll in the regular rodent food causes the autofluorescence in the intestinal area When the animal diet is changed to the alfalfa free rodent food the autofluorescence in the intestinal area is reduced to the levels comparable to the rest of the body In this situation the best way to minimize autofluorescence is to change the animal diet to alfalfa free rodent food when working with the Cy5 5 and ICG filter sets Control animals should always be used to assess background autofluorescence Figure F 17 Images of animal tissue autofluorescence in control mice Nu nu females Animals were fed regular rodent food top or alfalfa free rode
122. D EERE EEK ERE Be S 311 G 3 Luciferase Spectrum 2 311 G 4 An Example of Planar Spectral Imaging 2 ee 312 G 5 Optimizing the Precision of Planar Spectral Analysis 0 000000 315 Appendix H DLIT amp FLIT Reconstruction of Sources 2 317 H 1 Determining Surface Topography 2 317 H 2 Converting Light Emission to a Photon Density Map 0 0 0000 319 H 3 Defining the Linear Relationship Between a Source and Photon Density or NTF Efficiency 320 H 4 Determining the Best Approximate Solution to the Linear System 320 H S Source amp lisse Properties lt s sss be ee eee ee ue et eee eee OSE we YES Es 320 Appendix I IVIS Syringe Injection System a noaoono a a 323 I 1 Controlling the Infusion Pump 2 0 2 ee 323 I 2 Tracking Infusion in the Maximum vs Time Graph 2 2 000000 eas 325 I 3 Closing the Infusion Pump Control Panel 2 2 2 2 ee 325 Y Ca per Contents v Appendix J Menu Commands Tool Bar amp Shortcuts 327 vi Living Image Software User s Manual This page intentionally blank Y Caliper LifeSciences 1 Welcome What s New In Living Image 4 2 Software 2 2 ee ee ee ee eT POOG TING Manual s bw ee wee ES PRK SEE REGO LKR GOS LAS KEK KE Contacting Caliper Technical Support 2 08254 2 eee i The Living Image software controls optical image acquisition o
123. Datasets TE Volume Data Viewer a lar mouse 3_3_ 20100528 140051 mi J RAW mouse 3_3_20100528 140051 State BA Shared TIF File Date taken Specify date taken To view a particular slice move the Starts playback of Click to display the slider or enter a slice number the DICOM files DICOM file header information Appendix A IVIS Acquisition Control Panel Control Panel gt s s a a ne ee a ee ee ee eS er eee a a a a eee 259 Manually Setting the FOCUS 445 2 6 ee ae E SEES ERE a 264 A 1 Control Panel The control panel provides the image acquisition functions Figure A 1 Figure A 1 IVIS acquisition control panel auto exposure selected To acquire an image using auto exposure click the arrow and select Auto eal Luminescence T VIS Acquisition Control Panel roe ame i ma g i n g S ett i n g S Imaging Mode Exposure Ti Time Binning F Stop Excitation Filter Emission Filter Mel ec v vedum fs foe open Fluorescence imaging settings EM medum vje v Photographic imaging settings F Field of view System Status Structured light iore i l g Mouse Imaging Shuttle de maging settings Gee iss jm OOO meorowad Subject height 1 50 uw E cm lid setup X ray imaging settings NOTE The options available in the IVIS acquisition control panel depend on the selected imaging mode the imaging system and the filter whee
124. Focus use subject height Temperature I Locked Initialize Number of Segments 1 Delay 0 0 min Apply to All X Remover Update J insert C Add Acquire the Image Sequence 1 Confirm that the VIS Imaging System is initialized and the CCD temperature is locked For more details see page 17 2 When you are ready to acquire the images click Acquire Sequence in the control panel The Edit Image Labels box appears 36 Living Image Software User s Manual Figure 4 18 Edit Image Labels box A Edit Image Labels Fes UserID KSA Living Image Universal Saved Labels LABELS_1 A E Check any 5 fields for display V User X 7 Group v He In the image label Figure 4 20 Commenti Comment2 Animal Number Animal Strain F Animal Model E Sex Cell Line __ Reporter The information entered here appears Time Point View Treatment Luc Injection Time 4 4 4 4 4 4 4 4 4 4 4 IACUC Number 3 4 In the Edit Image Labels box enter information about the image and click OK If you do not want to enter image information click Cancel If this is the first image of the session you are prompted to enable the autosave function Figure 4 19 Autosave prompt Oe Living Image 4 2 Ea Do you want to enable auto saving of acquired data for this session This can be changed anytime from the
125. Folder box that appears select a data folder and click OK The Living Image 3D Volumetric Browser appears Figure 15 2 NOTE Only DICOM or TIFF data can be added to the 3D Volumetric browser For details on loading other data types raw or vox files see page 243 Figure 15 2 Opening the 3D Volumetric Browser File Edit View Teek Megion Window Help EIA es ES e tare n MN Felder Path i cet hehe slick BE Menang Et A Moute 3 Day 28 148522 C Shaen Cabper L3 Caliper Data CoReg Dema S82 236x256 CT E BegSub Jb Cofeg Dems Jk dem 20110810364 d DUT Dataset emfitert gt fd waoana EQ 3D Volumetric Browser The next time you start the Living Image software and open the Browse For Folder box the software automatically returns to the last folder visited The 3D Volumetric Browser automatically previews a playback of the data along with other information about the data Figure 15 3 sch DICOM file Ik TIFF file To load volumetric data with the optical data a Confirm that the Load in a new window option is not selected If this option is selected the volumetric data are loaded in a new window b Double click the data row in browser Alternatively select the data row and click Load 242 Living Image Software User s Manual The 3D volumetric data appears in the 3D View window of the optical data Figure 15 4 The software converts loaded volumetric data in
126. I ID information There are two ways to move an ROI on an image e Drag the ROI to a new location e Edit the settings in the ROI Properties box An ROI cannot be moved if it was created using the auto ROI tool or if the ROI position is locked To drag an ROI 1 Put the mouse pointer over the ROI so that it becomes a lt 4 arrow 2 Drag the ROI 3 Release the mouse button when the ROI is properly positioned To move an ROI using the ROI Properties dialog box 1 Double click the ROI in the image e o Y Caliper _ Chapter 8 ROI Tools 139 The ROI Properties box appears and displays the position and dimensions of the selected ROI Figure 8 21 ROI Properties dialog box Use as BKG for future ROIs in TL720050624145507_005 only Entire sequence xc pix 11700218 f Position of the ROI Ye pix 97 93839 selected in the image Angle deg 0 0000 Lock Size Width pix 22 06161 Height pix 20 43172 Line Size 7 Line Color D 2 To set ROI position enter new coordinates for the center of the ROI Xc pix and Yc pix values in the ROI Properties box 3 To rotate the ROI clockwise enter the degrees in the Angle deg box and click outside the box 4 To lock the current ROI position choose the Lock Position option NOTE The ROI position cannot be changed until the Lock Position option is cleared Editing ROI Dimensions There are two ways to resize a circle or squar
127. I from a measurement ROI The software computes Background corrected intensity signal Average signal in the measurement ROI Average signal in the average background ROI NOTE This is an optional background correction that is applied in addition to the electronic dark charge and read bias corrections that are applied to the raw CCD data The Image Adjust tools and zoom feature are helpful for selecting an appropriate area for an ROI By setting the image minimum close to zero and zooming in on a background area in the image you can determine where naturally occurring background luminescence or autofluorescence is present For more details on the Image Adjust tools and the zoom feature see Adjusting Image Appearance page 97 and Magnifying or Panning in the Image Window page 98 To measure background corrected signal 1 Draw one or more measurement ROIs on the subject For more details see page 129 2 Draw an average background ROI on the subject a Select Average Bkg ROI from the Type drop down list b Click the Square H or Circle button and select 1 The ROI is added to the image For more details on adjusting the ROI position or dimensions see page 138 and page 139 NOTE The average background ROI and measurement ROI do not need to be the same shape or size because the software computes the average intensity signal in each ROI 3 Use one of the following methods to associate the average background
128. IT or FLIT results Overwrite If you reanalyze saved results click to save the new results and overwrite the previous results Managing 3D Reconstruction Results Figure 14 14 3D analysis results Tool Palette ROI Tools E gt Surface Topography Value Final voxel size mm 125 Number of voxels 115 Reduced Chi2 9 73e 05 Index Of Refraction 1 40 Angle limit deg Damping reduce of samples Show samples Starting voxel size Uniform Save Results Name DLIT_6 Delete Photon Density Maps H Export Results Results name 206 Living Image Software User s Manual To save 3D results 1 Inthe Results tab of the DLIT FLIT 3D reconstruction tools confirm the default name or enter a new name 2 Click Save The results are saved to the sequence click number folder and are available in the Name drop down list To open 3D results 1 In the Results tab make a selection from the Name drop down list 2 Click Load The 3D results appear in the 3D View window To copy user specified results 1 In the Results tab select the text of interest 2 Right click the results table and select Copy from the shortcut menu that appears The selected results are copied to the system clipboard To copy all results 1 In the Results tab right click the results table and choose Select All from the shortcut menu that appears 2 Right click the results table again and select Copy from t
129. Image Browser displays when data are selected For more details on the browser see page 83 Preview picture of the selected data tat OS ee a ee el ee ee ee ee ee ee Close Prewiew Label Set All F add tose Eros ews Deta cenigure Losies wao Load Remove d Location Cr Shane Caliper LS Cainer atajin Data i200 data iraran TT 45507_at 5 3 Acquire Multiple Sequences in Batch Mode In batch mode you can set up multiple separate sequences which will be automatically acquired one after another without manual intervention To setup and acquire sequences in batch mode 1 In the Control Panel click Sequence Setup 2 Choose the Batch Sequences option Figure 5 14 Control Panel F Stop Excitation Filter Emission Filter Field of View E xFov 24 E Zoom Service 10 cm aa Eo Sa z Terooore M e MS Acquisition Control Pane Exposure Time Binning F Stop _ _ Excitation Filter Emission Filter F Display Photographic Setti mocro open Ea Meam fi y Ce BS oma Field of View Cc v E XFov 24 E Zoom E m imagna Ward aipctape lL Ble Acquire Sequence Batch Sequences option Focus use subjectheight v Temperature TS Locked T Number of Segments i 4 Delay 0 0 1 min Apply to All x Remover Hj Update Insert C Add 62 Living Image Software User s Manual 3 To set up the first sequence do
130. Image Data 95 3 Edit the label information To add information to the image label 1 Click the aA toolbar button Alternatively select Edit Image Labels on the menu bar 2 In the Edit Image Labels box that appears select information and or enter a comment Figure 7 11 If a single image is active changes are applied to that image only If a sequence is active changes are applied to each image of the sequence Figure 7 11 Edit Image Labels A Edit Image Labels DS UserID TLT Living Image Default Saved Labels LABELS _1 a Series Male Nn nu hd Experiment DOB 03 21 05 v Label kidney bd Comment dorsal id Analysis Comment v Apply To Sequence x J cane 3 When finished click OK The image information is updated 4 Save the image to save the updated image label select File Save or File Save As on the menu bar 7 4 Adding Comments or Tags to an Image Adding Comments Comments can be added to an image and saved with the image 1 Open an image 2 Right click the image and select Insert Comment on the shortcut menu Enter comments in the yellow box that appears Figure 7 12 To reposition a comment 1 Position the mouse pointer over the comment 2 When the hand tool appears use a click and drag operation to move the comment box then click the mouse to set the location To remove a comment s e To remove a comment right click the comment and s
131. Image Information a ROI Tools O Ek e Yf Measure ROIs x a Apply to Seque Auto All ype Measuremer v Luminescence Auto 1 Save ROIs Name ROI_3 KS Free Draw E Auto ROI Parameters Threshold 15000 10000 5000 gt Surface Topography DLIT 3D Reconstruction gt 3D Multi Modality Tools Counts Color Scale Min 1122 Max 19546 8 5 Subject ROIs A subject ROI identifies a subject animal in an image It provides a convenient way to automatically associate link a measurement and average background ROI for background corrected ROI measurements when there is significant autoluminescence or autofluorescence For more details on background corrected ROI measurements see page 130 Using a subject ROI is optional To draw a subject ROI using the auto ROI feature 1 Select Subject ROI from the Type drop down list 2 Click the G button 130 Living Image Software User s Manual 3 Select Auto All To manually draw a subject ROI 4 Select Subject ROI from the Type drop down list 5 Click the O button and select 1 6 Position the subject ROI so that it includes the measurement ROI s and the associated average background ROI 8 6 Measuring Background Corrected Signal If a subject has significant autoluminescence or autofluorescence you can obtain a background corrected ROI measurement by subtracting an average background RO
132. Image software is loaded the tissue type will be listed in this drop down list Source Spectrum Drop down list of luminescent sources Plot Results tab Spectral Results Tissue Properties Click to display graphs cm vs nm of the absorption coefficient effective attenuation coefficient 4 and reduced scattering coefficient H a Source Spectrum Click to display the spectrum of the selected luminescent source intensity versus wavelength normalized to one ROI Name of the analyzed ROI Depth mm Estimated depth of the point source Total Flux phot s Estimated total photon flux from the point source Plot Linear Fit Displays a graph of normalized intensity versus the effective attenuation coefficient Ho the optical property of the tissue selected in the Tissue Properties drop down list along with the linear fit to these data determined by the spectral analysis code Plot Intensity Displays a graph of normalized intensity versus wavelength Intensity is normalized by the selected source spectrum and filter transmission properties Export Opens a dialog box that enables you to save the results to a text file txt Save Results Name A drop down list of saved results Includes the default name for new unsaved analysis results Splm_ lt name gt Delete Deletes the selected results Load Opens the selected results Save Saves the analysis results results
133. Imaging With the Optical Zoom Lens Attachment 63 5 5 Measuring Relative Density oaoa ee 65 6 Kinetic IMAGING s 6s2ienuw twee antaa HD HERDER ER ERD Rew Es 69 Ol Kinetic Acquisito s e is stes bae abea 4446 6 oe Oe See be ee oe ee we Boe amp x 69 6 2 High Resolution Imaging With the Optical Zoom Lens Attachment 74 6 3 Viewing amp Editing Data in the Kinetic Acquisition Window 004 75 6 4 Saving Kinetic Data 2 242 sue eee E SE RO ee ERE EH ES BH RES 77 6 5 Playing Kinetic Data 2 4426 22 4442 bee e eee Ee Ee ede 78 Living Image Software User s Manual 7 Working With Optical Image Data 0 20 00 008 os 83 7 1 Opening Optical Image Data aooaa 83 7 2 About the Image Window amp Tool Palette 2 2 ee 89 7 3 Viewing Image Information 2 0 0 2 93 7 4 Adding Comments or Tags toanImage aaa a 95 7 5 Adjusting Image Appearance ooo a a 97 7 6 Correcting Optical Image Data oaoa a 99 7 7 Viewing Intensity Data amp Making Measurements ooa aa a 101 7 8 Creating a Transillumination Overview oaoa a 109 7 9 Overlaying Multiple Images aoaaa 110 7 10 Rendering Intensity Data in Color aoaaa a 113 7 11 Exporting or Printing Images oaoa 114 142 Editing an Imac Scquciice s s pr ia kh eRe eB EE SER a EE eee E a 116 7 13 Creating an Image Sequence from Individual Images 04 117 6 AOMTOOIS Geax a Gee ae
134. Is 5000 Counts Color Scale Min 1122 Max 19546 3 Adjust the ROI position a Place the mouse pointer over the ROI When the pointer becomes a click the ROI b Drag ROI s NOTE To move multiple ROIs at the same time press and hold the Shift key while you click the ROIs and then drag them to a new location Contour ROIs S cannot be moved using this method 4 Adjust the ROI dimensions a Place the mouse pointer over the ROI When the pointer becomes a click the ROI b Place the mouse pointer over an ROI handle W so that it becomes aX Drag the handle to resize the ROI NOTE You can also change the ROI position or size using the adjustment controls in the ROI Properties box see Moving an ROI page 138 and Editing ROI Dimensions page 139 5 Click the Measure button MeeueRois The ROI measurements and table appear For more details on the table see Managing the ROI Measurements Table page 145 For information on how to save ROIs see page 116 Y Caliper Bet Chapter 8 ROI Tools 127 Automatically Drawing Measurement ROIs The Living Image software can automatically identify all of the ROIs in an image or image sequence that meet the auto ROI parameter thresholds or draw one ROI at a user specified location 1 Open an image or image sequence and in the ROI tools select Measurement ROI from the Type drop down list
135. Kange 14bit Exposure Time msecs 35 F MIP on be bebe i Z To adjust a setting using the thumb wheel put the mouse arrow on the wheel then click and hold the mouse button while you move the mouse arrow left or right FL Lamp Level Hian Photograph Light Level 77 2 hr Accumulate Dsi h mm ss Frame No data available 0 4 Click the Record button to start acquisition After acquisition begins the button changes to a Stop button s To stop acquisition click the Stop button m so The maximum vs time graph appears when kinetic acquisition begins and plots the maximum intensity signal in each frame The graph provides a convenient way to look for signal trends or select particular frames for viewing Y Ca i i Chapter 6 Kinetic Imaging 71 Figure 6 3 Maximum vs time graph e Click a point in the graph to view the corresponding image graph display options Kinetic Acquisition Control Panel SE Acquisition Settings Display Photograph 14 Frames sec Luminescent Overlay ees Dynamic Range Min 111 14 bit Max 15366 Exposure Time msecs 35 s 12000 Mee Binning 10000 ha F Stop 3000 F EM Gain 100 4000 Excitation Filter Block Emission Filter Open FL Lamp Level Color Bar High Min 111 Photograph Light Level Max 13488 io CUCU 4 6000 2000 Counts
136. Manually Saving Image Data a a a a a a 46 Exporting Image Data ae a ee a a a a ee ne 47 Optical imaging measures the light emitted by bioluminescent or fluorescent light producing reporters such as luciferase or fluorescent proteins The IVIS Imaging System is ready to acquire optical images after the system temperature is initialized and the CCD camera reaches operating demand temperature the system is then locked This chapter explains how to acquire optical image data a bioluminescent or fluorescent image or an image sequence 4 1 Bioluminescent Optical Imaging This section explains how to acquire a single bioluminescent optical image For information on acquiring an image sequence see Acquire a Sequence Using the Imaging Wizard page 33 NOTE Before setting the imaging parameters the IVIS Imaging System should be intialized and the temperature locked For more details see Chapter 3 page 15 1 In the control panel put a check mark next to Luminescent and select Auto exposure click the arrow When you select Auto exposure the software automatically determines the binning and F Stop settings Alternatively you can manually set the exposure binning and F Stop For more details on these control panel settings see page 259 Figure 4 1 Acquisition Control Panel zora Time Binning F Stop Excitation Filter Emission Filter mocone open medum v8 Field of View System Status E Mouse Imag
137. OI label r E TLT20050624145507_005 BES Luminescence 10000 Counts BKG 1 2 270e 03 8000 6000 ROT 7 50 3 349e 05 ff 4000 BROT S150 1 763e 05 o 2000 Color Scale Min 586 Max 10348 Units Counts v Display Overlay X Options X Info aT bts tree rere py E RT 7 ROI Properties Edit the ROI Contour label here Auto 50 Background ROI Subject ROI Image Number TLT20050624145507_005 v IROI _none_ J Lock Position Xc pix 106 81818 Width pix Height pix 11 00000 Line Size 2 ine color I C Done To edit the ROI label 1 Double click the ROI of interest Alternatively right click the ROI Ctrl click for Macintosh users and select Properties on the shortcut menu 2 In the ROI Properties box that appears edit the name in the ROI Label box and click Done Figure 8 24 Saving ROIs The software automatically saves ROIs with an image The ROI measurements are saved in the AnalyzedClickInfo txt file associated with the image ROIs are saved per user and can be applied to other sequences Additionally ROI parameters can be saved per user and applied to other sequences To save ROls to the system 1 In the Name drop down list confirm the default name or enter a new name for the ROI s Y Caliper a ai Chapter 8 ROI Tools 143 Deleting ROIs Figure 8 25 Name and save the
138. Q by measuring the displacement Ax The software utilizes fast numerical methods to rapidly evaluate Ax over the entire image to determine the surface topography The surface topography determination is limited to the topside of the object facing the lens Y Caliper Appendix H DLIT amp FLIT Reconstruction of Sources 319 H 2 Converting Light Emission to a Photon Density Map The input data to the FLIT algorithm for 3D reconstruction of fluorescent light sources includes e A surface that defines the surface of the subject e A sequence of images acquired at different transillumination source positions using the same excitation and emission filter at each position Use the Imaging Wizard to acquire the images The input data to the DLIT algorithm for a 3D reconstruction of luminescent light sources includes e A surface that defines the surface of the subject e A sequence of two or more images of the light emission from the surface of the subject acquired at different filter bandwidths Table H 1 Use the Imaging Wizard to acquire the images Table H 1 IVIS System filters for luminescence and fluorescence tomography IVIS Imaging System Filters Bandwidth 200 Series 6 emission filters 550 670 nm 20 nm Spectrum 10 excitation filters 415 760 nm 30 nm 18 emission filters 490 850 nm 20 nm The IVIS Imaging System 200 Series and the IVIS Spectrum are absolutely calibrated so that the electron counts on each CCD pixel can be
139. ROI with one or more measurement ROIs e Method 1 Draw a subject ROI around the average background ROI and the measurement ROI s Figure 8 12 e Method 2 Right click the measurement ROI and select a background ROI from the shortcut menu that appears Figure 8 13 e Method 3 Set the background ROI in the ROI properties dialog box Figure 8 14 Y Ca i a Chapter 8 ROI Tools 131 a JJH20050630142125 001 v Display Overlay v Subject 1 li i Units Counts BKG 1 5 986e 03 IROI 2 25 J 3 803e 05 ROI 1 25 1 164e 06 oe is Options Info i Luminescence 30000 20000 10000 Counts Color Scale Min 1999 Max 32995 Figure 8 12 Associate the average background ROI with measurement ROIs Method 1 Draw a subject ROI that includes the measurement ROI and the average background ROI For details on how to draw a subject ROI see page 129 Figure 8 13 Associate an average background ROI with measurement ROIs Method 2 JJH20050630142125 001 Units Counts Display Overlay oe ie Resize Rotate Copy ROI Copy All ROls Paste ROI Duplicate ROI Set Ekg ROI to none Set Ekg RO to BKG 1 Set Bkg ROI to BKG 2 ROT 2 25 3 60ge 05 ROT 1 25 1 164e 06 Set Subject ROI to none Set Subject ROI to Subject 1 M Hide ROI Tag Delete ROI Delete All ROIs Properties Unlock
140. Re ERER 7 2 3 Agut Image SCQUCNCES 2422522542246 SOR SESE eH ERE Ee EEG 10 2 4 Image Display amp Analysis 1 2 ee 11 3 Getting Started 64 seek eet eects se EH EES SEEKER BES EG 15 3 1 Starting the Living Image Software 2 0 0 0 0 2 ee 15 3 2 Initializing the IVIS Imaging System 2 2 2 2 2 00 eee ee ee ee ee 17 3 3 Checking the System Temperature 2 oaa a 18 3 4 About the IVIS Acquisition Control Panel amp Auto Exposure Feature 19 3 5 Tracking System and User Activity aoaaa ee 19 4 QOoucallimaging 2e5 2s cen Geena ree etre eee e 21 4 1 Bioluminescent Optical Imaging oaoa a a a a 21 4 2 Fluorescent Optical Imaging With Epi Ilumination 0000 25 4 3 Fluorescent Optical Imaging With Transillumination 004 28 4 4 Acquire a Sequence Using the Imaging Wizard 2 1 a 33 4 5 Acquire Multiple Sequences in Batch Mode 1 2 ee ee 39 4 6 Manually Set Up an Image Sequence 2 2 0 ee 41 4 7 High Resolution Imaging With the Optical Zoom Lens Attachment 45 4 8 Manually Saving Image Data 2 a 46 49 Exporting Image Data e saec aaar be ae Bee Hee ew Ee Sem BER EHR ES 47 5 X Ray Imaging 2 1 ee ee 49 5 1 Acquire an X Ray Image 2 49 5 2 Acquire an Image Sequence That Includes X Ray Images 54 5 3 Acquire Multiple Sequences in Batch Mode 2 2 ee 61 5 4 High Resolution
141. Sagittal windowpane does not display a sagittal slice or the data appears flipped with respect to the surface derived from the IVIS Spectrum In such cases you can e Invert the data along the x y or z axis e Manually rotate the data using the Transformation tool for more details see page 249 Y Caliper a Chapter 15 3D Multi Modality Tools 245 To invert the subject orientation 1 Click the Edit Spacing amp Orientation button Er 2 In the dialog box that appears choose a Subject Orientation option and click OK Fi Volume Information Slice Information Pixel Spacing Row Column 0 1000 0 0016 mm Slice Spacing 06 0016 mm Subject Orientation El Invert X Axis Invert Y Axis E Invert Z Axis Dea Figure 15 8 Volume Information dialog box 15 3 Registering Multi Modal Data Two multi modal registration methods are available e Automatic fiducial registration For experiments in which the Mouse Imaging Shuttle was used to transfer the animal between the IVIS Spectrum and the Quantum FX uCT instrument The Living Image software automatically registers the optical surface reconstruction with 3D volumetric data acquired on the Quantum FX uCT instrument e Manual registration Use the 3D Multi Modality tools to register 3D volumetric data with a 3D surface reconstruction Automatic Fiducial Registration Automatic fiducial registration is available for CT data acquired on the Qua
142. Save As Displays the Browse For Folder box so that you can specify a folder in which to save the image data The original data is not overwritten File gt Import 3D Surface Opens a dialog box that enables you to import a surface Note This menu item is only available if Show Advanced Options is selected in the Preferences see page 266 File gt Import gt 3D Voxels Opens a dialog box that enables you to import a source volume Note This menu item is only available if Show Advanced Options is selected in the Preferences see page 266 File gt Import Organ Atlas Opens a dialog box that enables you to import an organ atlas iv dxf stl File gt Export gt Image Sequence as DICOM Opens the Browse for Folder dialog box that enables you to export the active image data to DICOM format dcm File Export 3D Surface Opens a dialog box that enables you to save the 3D surface of the active data to a file such as Open Inventor format iv File Export 3D Voxels Opens a dialog box that enables you to save the voxels from the active data File gt Export 3D Scene as DICOM Opens a dialog box that enables you to save a 3D reconstruction and or surface in DICOM format The Multi Frame DICOM option supports 3D CT reconstruction in third party software File gt Print Displays the Print box File gt Print Preview lor Milt Displays the
143. Spectral Imaging gt Surface Topography gt 3D Multi Modality Tools 3 Click the Contour button and make a selection from the drop down list Image or image or sequence Select Auto All from the drop down list Kinetic data Select Kinetic ROI YY Caliper 4 per Chapter 8 ROI Tools 121 The software automatically draws measurement ROIs on all images The ROI label shows the total intensity in the ROI and the Threshold Figure 8 3 NOTE Auto ROIs are created and numbered in order from highest to lowest maximum signal within the ROI ROI 1 contains the highest maximum signal You may want to arrange the ROIs in a known order for easier comparison between images To renumber the ROIs ascending order from right to left right click the image and select Sort ROIs on the shortcut menu If the Apply to Sequence option is selected in the ROI tools choose Sort ROIs in Sequence to sort all of the ROIs in the sequence The sort options are only available if the ROIs have not been sorted Figure 8 3 ROI intensity measurements r E7 TLT20050624145507_SEQ Catet Sequence View Spectra Units Counts v E Use Saved Colors Options Info R C ROI 3 50 8 290e 05 ROI 1 50 6 536e 03 ROI 2 50 2 653e 04 i ROL 415001 2 6870 05 iN ROI 4 50 2 687e 05 g Ay Ay 1 IF Min 2140 Max 36446 A ROI 5 50 1 621e 06 i ROI 9 5
144. The wavelength range of the luminescent images in the sequence The two sliders determine the lower and upper end of the filter range Only the parts of the image that are within the selected wavelength range are colorized By default the entire filter range is selected Log Scale If this option is chosen the dynamic range of the brightness in the image is compressed using a log scale This improves the visibility of dark areas in the image Real Color If this option is chosen the colors are rendered using the wavelengths that directly correspond to the camera setup For example GFP appears green using real color rendering If this option is not chosen the original wavelength range of the image is modified to include the entire visible wavelength range of the camera setup This helps improve the color contrast Click this button to copy the colorize view to the system clipboard Click this button to export the colorize view as a graphic file for example jpg ce Click this button to print the colorize view 7 11 Exporting or Printing Images The Image Layout window Figure 7 30 provides an alternative way to e Annotate and export an image for example bmp e Print an image e Copy an image to the system clipboard 1 To open the Image Layout window select View Image Layout Window on the menu bar 2 To paste the active image into the Image Layout window click the x button To resize the image drag a ha
145. Tool Palette choose FLIT 3D Reconstruction The Analyze tab shows the images that the algorithm automatically selects for the reconstruction based on an appropriate signal level Figure 14 2 For more details about the Threshold see page 198 202 Living Image Software User s Manual Figure 14 10 FLIT 3D Reconstruction tools Analyze tab Tool Palette EmWL Threshold 55 L 63 55 5 6 Images selected 57 for reconstruction 61 5 4 Type of image used in the reconstruction If no NTF data are available only Radiance is available BEESEEEEE 52 8 Select an algorithm 4 Select the type of image used in the reconstruction Radiance or NTF Efficiency Figure 14 10 5 In the Properties tab make a selection from the Tissue Properties and Source Spectrum drop down lists Figure 14 3 Figure 14 11 FLIT 3D Reconstruction tools Properties tab Tool Palette Fluorescent Quantification The selected plot type Tissue Properties Plot is displayed below YW Caliper Chapter 141 3D Reconstruction of Sources 203 feSciences 6 To view the tissue properties U4 Ug H s for the tissue you selected make a selection from the Plot drop down 7 To include the number of fluorescent molecules source in the results select a fluorescent calibration database For details on generating a luminescent calibration database see page 185 In
146. Tools see page 220 231 For details on managing results for example save load or delete see page 205 Figure 14 5 DLIT reconstruction results 3D View toolbar Tool Palette 2 E TLT20050624145507_SEQ gt ROI Tools lel Sequence View 4 3DView Spectra Planar Spectral Imaging la _ Surface Topography A DLIT 3D Reconstruction Gronal z 16 5 Sagittal x 2 8 H Help Analyze Properties Results DLIT Analysis Results Unsaved D Key Value Final voxel size mm 1 25 Number of voxels 98 Reduced Chi2 1 22e 06 Index Of Refraction 1 40 Angle limit deg 70 Damping reduce 100 of samples 200 Show samples 0 Starting voxel size 5 Sampling method Uniform val Photon Density Maps Export Results Save Results Name DLIT_6 kd Delete Load Save c Transaxial y 17 3 3D Multi Modality Tools C gt 3D Tools _ Spectral Unmixing photo nsisec Source Intensity Subject Height 26 4 mm Perspective Y Ca i i Chapter 14 3D Reconstruction of Sources 197 Figure 14 6 3D View toolbar SmE 2 a V T Table 14 3 3D View tools Tool Image Tools op fae Description A drop down list of tools for viewing and working with the surface or DLIT results fy or b Rotates or spins the surface in the x y or z axis direction b
147. Tools 257 feSciences 15 7 Volume Information and Results The Results tab displays information about the loaded data taken from the DICOM file header Figure 15 23 Figure 15 23 Volume information Tool Palette NameOfVolume Mouse3_3_Day 28_145522_00 PatientsName Mouse3 Modality cT Manufacturer Rigaku BitsAllocated 16 SamplesPerPixel 1 Rows 256 Columns 256 NumberOfFram PixelSpacing 0 236 0 236 SliceThickness 0 236 ImagePositionP 30 208000 30 208000 60 4160 ImageOrientati 1 0 0 0 1 0 PatientsBirthDate 20101018 mW r Save Results Name MULTIMODALITY_4 x Delete Load Overwrite gt 3D Tools gt Spectral Unmixing a You can save the registered and classified data This provides a convenient way to share data The software saves the following Level of detail setting Color tables for the opacity map and slices Histogram tool control settings and the resulting color opacity map Multi modal registration settings Crop settings Managing Results To save registered results 1 In the Results tab confirm the default name in the Name drop down list or enter a name 2 Click Save The registered 3D volumetric data along with the color opacity settings appear in the 3D View window NOTE The results are saved in XML format in the optical data set location The results can only be accessed from the same optical data set
148. Total Flux phot s ROL1 1 250 0 105 4 36e6 3 09e5 pecwceecccses seeweesesuseeresesssessssnssssesenssneseensseneresssessesssensesesd a After the analysis is completed p pesis click a button to display graphical results E Plot Intensity Name Splm_2 Delete Load i 25 gt Surface Topography DLIT Plot of Linear Fit Results DAOR PS Units Photons Display Overlay EimageAdust E ROI 1 3 091e 06 e a A Plot of Intensity Vs Lambda a Linear Fit Plot for ROI 1 Log Norm Flux K 12 mueff cm 1 10 A X Image Min 1 26e3 Max 9 76e5 60 50 40 x10 3 0 2 0 10 p sec em 2s Color Bar Min 6 97e4 Mas 6 44e5 Coe Intensity vs Lambda Plot for ROI 1 1 00 Normalized Flux a u 600 4 620 Wavelength nri 314 Living Image Software User s Manual To estimate the cell count divide the absolute photon flux by the flux per cell This is best determined by making independent in vitro measurements of the cell line used in the experiment The Plot of Linear Fit Results is weighted by the uncertainty of the measured images and takes into account the uncertainty in the determination of the optical properties The precision of the method is largely determined by the known precision of the optical properties In most cases the relative uncertainty in the depth determination is equal to the re
149. Units Select the measurement units for the image display from this drop down list The available units depend on the type of image For more details on measurement units see page 281 Use Saved Choose this option to display the image data using the color table that was specified Colors in the Preferences at the time of acquisition If this option is not selected image data are displayed using the color table currently specified in the Preferences 38 Living Image Software User s Manual Table 4 4 Sequence View window continued ltem Options Description Layout Choose a display option for the images in a sequence Default Dynamic or Film Strip For example here is Film Strip mode C TLT20050624145507_SEQ Aae Sequence View Spectra Units Counts KA EF Use Saved Colors options x Info R 2 A a Default Dynamic Display gt Y Film Strip Sort by Options for ordering images in the sequence window This option only applies to images that were opened using the Load as Group function in the Living Image browser Default Order in which the images are stored in the folder TimeStamp Ascending order of the image acquisition time UserlD Ascending alphanumeric order of the user ID Display Choose the types of information to display with each image CG Sequence View Lis Ee eci oo ne SS ely m Eppes 2 00060600 Ere Time ous Tine L Be
150. Use as BKG for future ROIs in 71720050624145507_005 only Entire sequence Brightness slider F Lock Position 4 Select Color E Ne pix 117 00218 Basic colors on SD TTT Tell tt tt fF Cross hairs in Angle deg 0 0000 EEHEHE mi the custom ma a haa Lean color field E Lock Size ot ft Width pix 22 06161 m m E m u Height pix 20 43172 Custom colors Hue 104 Red 109 Line Size 2 F 5 ry Sat 199 gt Green 255 Line Color Val 255 2 Blue 56 G Add to Custom Colors Done G 2 To edit the ROI line thickness enter a new value in the Line Size box Alternatively click the arrows 3 To change the ROI line color a Click the Browse button _ b The Select Color box appears c To select a basic color for the ROI line click a basic color swatch and click OK d To define a custom color drag the crosshairs in the custom color field adjust the brightness slider and click Add to Custom Colors e To select a custom color for the ROI line click a custom color swatch and click OK 142 Living Image Software User s Manual Move or Edit the ROI Label To move the ROI label 1 Put the mouse pointer over the ROI label 2 When the pointer becomes a fh drag the label and then click to release the label at the new location Figure 8 24 Figure 8 24 Move or edit the R
151. Y Ca i oh or 2 Overview of Imaging amp Image Analysis Workflow Overview aaa a a a a a a a a aaa aD megma MOEG s sas ss srira ee EEE ER EG a eH eR EGE KG About Image Sequences 2 ee ee ee ee ee ee ee ee 10 Image Display amp Analysis 1 ee ee ee ee ee ee 17 This chapter provides a brief overview of imaging and image analysis Images acquired on an IVIS Imaging System are called optical data 2 1 Workflow Overview The Living Image software provides image acquisition viewing and analysis functions for IVIS Imaging Systems Figure 2 1 shows the steps to acquire an image Figure 2 2 shows an example sequence acquisition workflow Figure 2 1 Steps to acquire a luminescent or fluorescent image Start the Living Image software and initialize the IVIS Imaging ystem inthe control panel D MIS Acquniben Control Panel 6 imogng Mode Exposure Time F Stop Erusaon Her 100 foes ee Ce Ce e Select the imaging modes for example luminescent and photograph e Set the imaging parameters Feti of View IC e When you are ready to acquire the a score images click Acquire ron ireorg wise Rahegi 1 50 gt on Sequerxe Sehp Focus use subjectheight reroe one ED 3 0 inseize Pua Enter image label information optional The image window and Tool Palette appear when acquisition is finished 6 Living Image Software User s Manual F
152. _003 ROI 4 Overlay 2 687e 05 4 405e 03 9 302e 02 3 042e 03 6 048e 03 TLT20050624145507_004 ROIS Overlay 1 621e 06 4 053e 04 8 599e 03 2 976e 04 5 899e 04 0050624145507 004 ROIG Overla e 0 996 2d Customized Selections Measurements Types Image Attributes ROI Dimensions Select All _som_ _ sonst The ROI Measurements table displays data for all ROIs created in images or sequences during a session one ROI per row The table provides a convenient way to review and export ROI data For more details on the table see Managing the ROI Measurements Table page 145 Y Caliper 8 3 ROI Tools Chapter 8 ROI Tools 123 Table 8 2 provides brief explanations for the ROI tools The ROI tools that appear in the Tool Palette depend on the type of ROI selected from the ROI Type drop down list and whether an image or sequence is active Some ROI parameters are only available if Show Advanced Options is selected in the General Preferences Figure 8 6 ROI tools Tool Palette Tool Palette imatt a CoE rections Filtering O O i Q W Mareos X i Type Measurement ROI x 7 ROI Tools Save ROI Bes F gt O Y Measure ROIs x RW 1 ROA r r r r Delete Apply to Sequence a pe MeasurenentROL Threshold fa 50 4 Save ROIs Name ROI 3 KSA _ Planar Spectral Imaging Delete Load 6 gt Surface T
153. a Factor 3 8 j Duplay E y Labels 4 Dinning Factor Gocitation filter Frnevann filter I Humber Field of Viiv Select All y Hin 2140 In this example exposure time and binning factor are displayed on each image Click to show or hide the image label information Figure 4 20 Opens all of the images in the sequence Closes all open images Opens the Edit Sequence dialog box that enables you to add or remove images from the sequence Enables you to export the active image as a graphic file for example ong dcm Y Caliper _ Chapter 41 Optical Imaging 39 Table 4 4 Sequence View window continued Item q Description Creates a preview picture snapshot of the image or thumbnails that the Living Image Browser displays when the data are selected For more details on the browser see page 83 eat b F m Foe x Tree iaa SEQ Chek Mumber EX Filter EM Fler Hurunihi Madr Lier BD Lhe Group Experenscrt REG CHQOMQOGSIMLOSO SEQ i i iK i i CFTSO dye in pillows MES CKIO0SLBOSIE0056 500 K Tina LMOOSTIL DLIT subjecta phantom gt fg TLNOSOSEOT ASL SEQ it E Da 9 row ie cha Hose Prewew Lobel set AI 7 JF add to List romge Views Default cenigure Load as Groce uod Remove cose Laratan C Shane Caliper LS Caine Duain Dais T5200 data irrin TT ase 4 145507 S80 eurer ini be Preview picture of the selected data 4 5 Acquire Multiple Sequences in Batc
154. ab Tool Palette Sequence M7200064 145507 SEQ Tissue Mouse Tissue Source refy Select Filters Save Results Imageset TLT20050624145507_001 Name SpIm_1 ROI List All Delete 6 Make a selection from the Source Spectrum drop down list Firefly in this example 7 Click Analyze in the Analyze tab The Results tab displays the computed average depth mm and total flux photon sec of the luminescent point source in the specified ROI s For more details on the results see page 160 Table 10 1 Planar spectral imaging tools ltem Description Analyze tab Sequence Name of sequence used for the analysis Tissue Source The tissue properties and source spectrum selected in the Properties tab Select Filters In the Filter box select the acquisition wavelengths for the images in the selected sequence To select non adjacent wavelengths press and hold the Ctrl key while you click the wavelengths Macintosh users press and hold the Cmd key while you click the wavelengths ROI List A drop down list of the ROIs in the active image Analyze Click to perform the spectral analysis 160 Living Image Software User s Manual Table 10 1 Planar spectral imaging tools continued Item Properties tab Tissue Properties Description Drop down list of the absorption and scattering properties for Mouse Muscle or XPM 2 XFM 2 mouse phantom Note f a result from an earlier version of Living
155. able associated with the data A photograph is a gray Overlay mode pseudocolor image of the scale pseudoimage luminescent data Is overlaid on a photograph A pseudocolor scheme is typically used to display the numerical contents of scientific image data like the luminescent or fluorescent images acquired on an IVIS Imaging System The pseudocolor scheme makes it easy to see areas of bright light emission The amount of light emission can be quantified using measurement ROIs For more details page 120 Measurement data is independent of the colors displayed in the pseudocolor image You can change the appearance of the image data without affecting the underlying numeric pixel values For example you apply a different color table to the data or adjust the range of numeric values associated with the color table Measurements that quantify pixel data produce the same results independent of the appearance of the pseudocolor display A pseudocolor image can be converted to an RGB color code and saved as an RGB image The RGB image looks like a pseudocolor image but does not include the numerical information derived from the light detected in each pixel Therefore the amount of light in an RGB image cannot be quantified In the overlay display mode the pseudocolor luminescent or fluorescent image is displayed over the associated grayscale photographic image Figure D 1 Pixels in the luminescent or fluorescent image that are less
156. above the background noise For kinetic imaging the EM gain may be set to 50 100 or 250 For conventional 16 bit still image acquisition EM gain may be set to Off 50 100 or 250 Excitation Filter A drop down list of fluorescence excitation filters For fluorescent imaging choose the appropriate filter for your application GFP DsRed Cy5 5 or ICG For luminescent imaging Block is selected by default Emission Filter A drop down list of fluorescence emission filters For fluorescent imaging choose the appropriate filter for your application GFP DsRed Cy5 5 or ICG For luminescent imaging Open is selected by default FL Lamp Level Sets the illumination intensity level of the excitation lamp used in fluorescent imaging Off Low or High Low This setting is approximately 18 of the High setting Note Make sure that the filters of interest are selected in the filter drop down lists before you select Inspect The Inspect operation automatically positions the selected filters in the system before turning on the lamp Subsequent changes to the filter popup menus will have no effect until another Inspect operation is performed Photograph Light Controls the brightness of the lights at the top of the imaging chamber Level that are used to acquire photographic images Accumulate Select this option to view the cumulative intensity signal in real time When this option is chosen the software computes and visualize
157. ackground ROI selected from the drop down list at the top of the dialog box The Background ROI tab shows a drop down list shows all average background ROIs in the click number selected above that can be linked to a user specified measurement ROI or subject ROI selected from the drop down list at the top of the dialog box ID User entered information about a subject ROI Label Label name of the selected subject ROI Lock Position Choose this option to lock the position of the ROI selected in the image XC X axis coordinate at the center of the ROI selected in the image Yc y axis coordinate at the center of the ROI selected in the image Lock Size Choose this option to lock the dimensions of the ROI selected in the image Width Width pixels or cm of the ROI selected in the image for more details on setting the units see ROI Dimensions page 147 Height Height pixels or cm of the ROI selected in the image Line Size Specifies the ROI line thickness To change the line thickness enter a new value or click the up down arrows Line Color Specifies the color of the ROI line To select a line color click the Browse button bel Done Click to close the ROI Properties box and apply any new settings including e Linkage between a measurement ROI and subject ROI for more details see Drawing an RO Using the Free Draw Method page 129 e ROI size dimensions or position e Subject RO
158. ackground corrected signal is displayed 7 To view the mathematical result overlay mode in a separate image window click Display Result For Measuring 156 Living Image Software User s Manual If necessary use the Color Scale Min and Max sliders in the Image Adjust tools to adjust the image display To save the new image 1 Click the Save button fA Alternatively select File Save on the menu bar 2 In the dialog box that appears select a directory and click Save A folder of data is saved to the selected location AnalyzedClickInfo txt ClickInfo txt luminescent and photographic TIF images To export the new image to a graphic file 1 Click the Export button ig 2 In the dialog box that appears select a directory enter a file name and select the file type from the Save as type drop down list 3 Click Save Y Ca i a 1 0 Planar Spectral Image Analysis Image Sequence Requirements aaao a 2 a ee a a a a 157 Planar Spectral Image Analysis aaoo a a 157 Planar Spectral Image Analysis aaoo oaa a a 157 Viewing Graphical Results a a a 161 Managing Planar Spectral Imaging Results aoaaa a a a a a 162 The Living Image software applies planar spectral image analysis to a sequence to determine the average depth and total photon flux of a luminescent point source in a user specified region of interest For more information on planar spectral image analysis see Appendix G page 309 10 1 Image
159. ading isiti eading the acquisition Imaging Wizard 7 seconds remaining Sequence Setup Temperature M Locked Initialize WW Caliper Chapter 5 I X Ray Imaging 53 Figure 5 6 Overlay fluorescent image on X ray image in the image window E 120090623143901_001 Units Counts v Display Overlay v Image EL20090623143901_001 Tue Jun 23 2009 14 39 09 Level High Em GFP Ex GFF 7 FOV 10 f8 1s Camera IVIS 11209 Andor iK rc C ea ee S a User Group HT 1080 Experiment subcutaneous Commenti Animal Number 1 Tool Palette Image Adjust a a m A Photo Adjustment Brightness lel Contrast Opacity x Color Scale Tool palette Epi fluorescence 1000 1008 Color Scale Limits Auto Full Manual Color Table lot J m _ Reverse l Logarithmic Scale gt Corrections Filtering gt Image Information L gt ROI Tools Check the image min and max in the color scale to determine whether the signal of interest is above the noise level and below CCD saturation Counts Color Scale Min 314 Max 1008 It may be necessary to use the Image Adjust tools to optimize the overlay display Use the Opacity control to adjust the appearance of the overlay For more details on adjusting image appearance see page 97 Table 5 2 Image window Item Descripti
160. aging 51 e Select Manual focus from the Focus drop down list For more details on manual focusing see page 264 If you want to acquire a photograph set the Photograph image settings a Put a check mark next to Photograph b Enter an exposure time or choose the Auto option c Confirm the binning and f stop defaults or enter new values If necessary click mage Setup in the control panel to operate in single image mode NOTE In single image mode the Sequence Setup button appears in the control panel Click this button to set up Sequence acquisition For more details on setting up a Sequence see page 54 10 When you are ready to acquire the image click Acquire The Edit Image Labels box appears Figure 5 3 Edit Image Labels box Fas 7 Edit Image Labels zz UserID KSA Living Image Universal Saved Labels LABELS_1 A E x Check any 5 fields for display g User v Group The information you enter here appears I fs ee in the image label Figure 5 6 Commenti Comment2 F Time Point Animal Number Animal Strain Animal Model Sex View Cell Line Reporter Treatment Luc Injection Time 4 4 4 4 4 4 4 4 4 4 4 IACUC Number Apply To Sequence 11 In the Edit Image Labels box enter information about the image and click OK
161. aging Subject Background Signals Tissue Autofluorescence E Food Signal Frobe Information Choose the components to unmix Choose the number of components to unmix Pick the significant background signals first then add the probe information to the table if itis not listed If you are unclear about the probe or itis notin the library choose Mouse v Match Probe Labels Use Constraints Number of components to unmix 2 1 Choose a subject type from the drop down list 2 Choose one or both types of background signals 3 Ifthe probe does not automatically appear in this list select the probe s If you are not sure about the probe that was used choose Unknown NOTE Select at least two but no more than four components to unmix Table 11 2 Spectral unmixing wizard choose the components to unmix ltem Imaging Subject Description A drop down list of subject types Background Signals Any undesired fluorescence that the camera detects for example autofluorescence from the animal food or instrument background Probe Information Specify the probes ala Click to add a probe ba Click to remove the selected probe Match Probe Labels If the probe names are specified the software attempts to automatically match the unmixed spectra with the specified probe names Note A correct match is not guaranteed due to the complexity of the in vivo spectra and filter sections es
162. al Unmbing 9 gt Spectral Unmixing e The optical properties or source spectrum may have been chosen erroneously For example for mice Mouse Tissue optical property is appropriate while XPM 2 is only appropriate for the Caliper phantom Bad Photon Density fit 238 Living Image Software User s Manual This page intentionally blank y Caliper LifeSciences 1 5 3D Multi Modality Tools 3D Multi Modality Tools Requirements 2 28 239 Loading Data for Registration aooaa a a amp eee ee ee a 241 Registering Multi Modal Data aoaaa a a a 2 eee eas 245 Classifying 3D Volumetric Data aoaaa a a ee es 250 Volume Display Options 2 a 254 MIGWING A SOE ska SRG aw wR REE KDR REE eS we HE GS ew 8 256 Volume Information and Results 0 0 2 eee eee ee ee 257 Volume Data Viewer 2 1 2 ee 258 The Living Image 3D Multi Modality tools enable you to co register 3D reconstructions of luminescent or fluorescent sources optical imaging data with CT or MRI images 3D volumetric data Registering multi modal data optical and volumetric data provides an anatomical context for interpreting biological functional information Figure 15 1 shows the steps to register multi modal data in the Living Image software 15 1 3D Multi Modality Tools Requirements The Living Image 3D Multi Modality tools require a separate license Additionally the graphics processing
163. al Unmixing Wizard Select the data mask Data Mask Options Photograph Threshold Draw Mask Rectangle i Default data mask applied by the software Data mask manually applied to a user 7 wake Spectral Unmixing Wizard ss Select the data mask Data Mask Options J5 Photograph Threshold Ellipse Draw Mask Rectangle Ellipse Cancel lt Bad Next gt Cancel selected area 4 If you do not want to analyze the entire subject draw a data mask on a user selected area using the data mask options Table 11 1 Data mask options Option Description Photograph If this option is chosen the software automatically draws the data mask so that it includes the entire photograph Threshold e If necessary use the threshold slider or s arrows to adjust the mask so that it matches the unerlying subject photograph as closely as possible without including any area outside the subject image Draw Mask Choose this option to manually draw a data mask on an area of the photograph Rectangle Specifies a rectangular shape for the manual data mask Ellipse Specifies an elliptical shape for the manual data mask 5 Click Next in the wizard In this screen you will select the subject type and signals components to unmix 168 Living Image Software User s Manual Figure 11 3 Choose the component to unmix Ez Spectral Unmixing Wizard Tips mown Im
164. ale Provides a reference for the pixel intensities in a luminescent or fluorescent image Pixels less than the color scale minimum do not appear in the image Pixels single Image i i i i greater than the color scale maximum are displayed in the maximum color Tool Palette The Tool Palette appears when you open an image or sequence The options available in the Tool Palette depend on the type of active image data Figure 7 7 Tool palette For an overview of the tools see Figure 2 4 on page 11and Figure 2 5 on page 12 Tool Palette 3 Tool Palette 2 J Image Adjust Click to expand a tool a a mM Q amp E E Photo Adjustment oe Brightness fa 100 Contrast f 15 Opacity 100 Color Scale Min fa 586 gt Max H 10348 Color Scale Limits Auto Full Manual Individual Color Table Rainbow 4 g Reverse E Logarithmic Scale gt Corrections Filtering gt Image Information gt ROI Tools _ Planar Spectral Imaging _ Surface Topography _ DLIT 3D Reconstruction gt 3D Multi Modality Tools gt Spectral Unmixing YY Caliper per Chapter 7 Working With Optical Image Data 93 7 3 Viewing Image Information At acquisition the software captures image information that includes all of the text information that is associated with an image for example camera param
165. alog box Ez Import Organ Atlas hu Organ Files Select Skin Mesh emma Generate Mesh Co efficients Select Organ Files Look in organ atlas IW files gt amp ex Fee bladder iv E stomach iv cx i uterus iv My Recent Documents My Network File name Places Files of type Import Organ Atlas Organ Files Select Skin Mesh uterus bladder iv bones iv brain iv colon iv eves iv Fat iv heart iv kidneys iv liver iv lungs iv Generate Mesh Co efficients Organ Atlas Name muscle iy 45507 4dd Organ Files Save Organ Atlas Qr Y Caliper Chapter 14 3D Reconstruction of Sources 231 4 In the next dialog box that appears select all of the files iv dxf stl that you want to include in the atlas one file per organ and click Open 5 In the Select Skin Mesh drop down list select the skin organ file which must include skin in the file name 6 Click Generate Mesh Coefficients 7 Enter a name for the atlas and click Save Organ Atlas The organ atlas atlas is created and is added to the Organ Atlas drop down list in the 3D tools Registration tab 14 13 3D Animation The Living Image software can create an animation from a sequence of 3D views key frames For example an animation can depict a rotating 3D scene Figure 14 37 The animation series of key frames can be recorded to a movie file mov
166. an ROI page 138 and Editing ROI Dimensions page 139 YY Caliper 4 REF nce Chapter 5 I X Ray Imaging 67 7 Click the Measure button 9 The ROI intensity measurements appear in the X ray image and the ROI measurements table appears For more details on the table see Managing the ROI Measurements Table page 145 For information on how to save ROIs see page 142 Figure 5 21 Measurement ROIs on X ray image i I EL20090623143901_001 ka a Too Palette Units Counts v Display XRay Options Info ij gt Image Adjust _ Corrections Filtering X Ray gt Image Information ROI Tools gt OC HE Uf Measure ROIs T Apply to Sequence Type Measurement ROI Save ROIs ROI 1 1 565e 06 6000 Name ROI_3_KSA Auto ROI Parameters resha 4 4000 2000 ROI 2 2 012e 06 0 00 Absorption Color Scale Min 0 Max 7971 68 Living Image Software User s Manual This page intentionally blank Y Caliper _ 6 Kinetic Imaging Kinetic Acquisition 6 fd wx HED PRR THKARHE RED a 69 High Resolution Imaging With the Optical Zoom Lens Attachment 74 Viewing amp Editing Data in the Kinetic Acquisition Window 75 Saving Kinetic Data gt s ss es ee Rok De HEKARDRG EE HES He OH OH He HA 77 Playing Kinetic Data ek te od eee a eH Re BEER we 78 The
167. and emission filters is sufficiently large for example gt 35 nm so that the excitation light does not leak through the emission filter where it can be detected by the CCD 11 2 Performing Spectral Unmixing 1 Load the image sequence In the example in Figure 11 1 the fluorophore is Quantum Dots 800 Images were acquired using a 675 nm excitation filter and emission filters from 720 to 820 nm in 20 nm increments 166 Living Image Software User s Manual Figure 11 1 Sequence for spectral unmixing o amp s Tool Palette E TLT20060406164950_SEQ Sequence View Spectra Units Radiance Photons Mi EF Use Saved Colors Options z Info j 3 A a Analyze Options Results Select Wavelenaths Excitation wavelength Emission wavelengths of the sequence Max 1 1008 C 3 i q Min 1 05e6 Wax 2 10e7 2 In spectral unmixing tools click the Analyze tab and put a check mark next to the emission wavelengths that you want to include in the analysis 3 Click Start Unmixing The Spectral Unmixing Wizard appears and shows the purple data mask that specifies the analysis area By default the data mask includes the entire subject Y Ca i a Chapter 11 Spectral Unmixing 167 Figure 11 2 Selecting the data mask For more details on the data mask options see Table 11 1 6 Spectr
168. aph Click to acquire a new photographic image If the chamber door is opened during transillumination setup you are prompted to acquire a new photograph Clear Selections Clears selected highlighted transillumination locations on the grid 5 Confirm that the Lamp Level is set to High a The lamp may be set to Low for certain applications such as long wavelength data through thin tissue Make a selection from the Field of View drop down list For more details on the field of view see page 261 Set the Focus A Select use subject height and use the 4 arrows or the keyboard arrows to specify a subject height cm Select manual focus For more details on manual focusing see page 264 Select scan mid range available on the VIS 200 or Spectrum Imaging System only The imaging system determines the subject height using a laser scan This value is automatically entered in the subject height box and is used to set the focal 8 If necessary click mageSetus in the control panel to operate in single image mode NOTE In single image mode the Sequence Setup button appears in the control panel Click this button to set up Sequence acquisition 9 To acquire an overlay image coregistered images for display after acquisition put a check mark next to Overlay NOTE To view the subject s inside the chamber before image acquisition take a photograph Uncheck the Fluorescent option choo
169. ar Spectral Imaging DLIT or FLIT tools Tool Palette 33 E Image Adjust 5 ROI Tools lal _ Planar Spectral Imaging B 7 1 gt Surface Topography sid _ DLIT 3D Reconstruction Analyze Properties Results A Preferences l r AEREA P jes al isition Optical Properties Tissue Properties Mouse Tissue v oe 7 02 l Source Spectrum Firefly x Preview Tissue Properties Mouse Tissue z Plot Tissue Properties e m Ha EPEE Bioluminescent Firefly 39 em a pet Luminescent Calibration Source Spectrum Result not found Fluorescent Source Spectrum AEGEA Plot Tissue Properties z Restore Defaults Wavelength nm cancel apy Table B 7 Tissue properties preferences Item Tissue Properties Description Choose a default tissue type that is most representative of the area of interest This tissue type will be used if a Subject Type is not selected in the Imaging Wizard and saved during acquisition Source Spectrum Choose the default luminescent source This Source Spectrum will be used if a Subject Type is not selected in the Imaging Wizard and saved during acquisition for DLIT sequences Plot Tissue Properties Choose this option to display a graph of the absorption coefficient u
170. arting the system See system initialization steradian 283 subject ROI 120 129 surface 177 183 export or import 183 generate 178 179 manage 182 view perspective 181 182 syringe injection system 323 325 system initialization 17 optics autofluorescence 304 temperature 18 system activity 19 T tag an image 96 technical support 3 temperature 18 threshold angle 205 tile images 88 tissue autofluorescence 305 307 eliminate by spectral unmixing 165 176 subtracting with background filters 153 156 tissue properties 205 tool palette 92 3D tools 220 236 correcting filtering 99 100 ROI tools 123 toolbar 327 transillumination overview 109 troubleshooting DLIT FLIT 237 U units See display units user activity 19 user preferences 265 273 V ventral imaging chamber 69 volume slices information and results 257 volumetric data classify 250 252 color opacity map 251 display options 254 information and results 257 voxel 320 VSIZe starting 204 Index 335 W well plate autofluorescence 300 301 X X ray acquire image 49 54 acquire sequence 54 61 Z Zoom Lens attachment 45 46 63 64 74 75 Index This page intentionally blank
171. ate tab e If setting up the sequence manually click the EA button in the sequence table to add a new tab then proceed with manual set up in the new tab NOTE Sequence tabs can be renamed Double click a tab name to edit it Alternatively right click the selected name to view a shortcut menu of edit commands for example Cut Copy Paste Figure 4 22 Multiple sequence tabs In this example three sequences are specified Adds a new tab use with Sequence tabs Removes the active tab and its sequence manual sequence setup o Mode Exposure Binning FStop Excitation Emission Structure FOW Height 1 BES auto Medium 1 Block 560 Yes c laso 2 Auto Medium 41 Block 580 Mo a 1 50 3 i m Auto Medium 1 Block 600 No cC 150 4 EE Auto Medium 1 Block 620 No Cc i50 5 Auto Medium 1 Block 640 No a 150 Fl Number of Segments 1 oeley 0 0 min na ud Renews a l A 5 To remove a sequence click the sequence tab and then click the E button 6 Click Acquire when you are ready to capture the sequences Image acquisition proceeds with no intervening time delay between sequences NOTE If the check mark is removed next to the Batch Sequences option Figure 4 21 only the sequence in the active tab will be acquired v i J Y Caliper _ Chapter 41 Optical Imaging 41 4 6 Manually Set Up an Image Sequence This section explains how to set up an image sequence if you do not use the Imaging Wizar
172. ath 155 Figure 9 4 Draw measurement ROI on an area that represents background signal E TLT20060510114512_008A o ees Units Counts X Display Overlay X Options x Info iw Epi fluorescence 20000 15000 ELTTI 10000 5000 Counts Color Scale Min 1214 Max 22777 Select Tools Image Math for lt name gt _SEQ on the menu bar 4 Inthe Image Math window that appears select the primary image in box A Select the background image in box B For more details on items in the Image Math window see Table 9 1 page 153 5 Select the math function A B k in the Result drop down list Figure 9 5 Select a math function and view the mathematical result g n EE Image Math Window co amp 3 tet_m20060510114512 008 Display Overlay Sequence KSA20110305104918 SEQ A 7L120060510114512_ 008A TLT20060510114512 010A Epi fluorescence 6000 B TLT20060510114512_008A TLT20060510114512_010A f Counts Color Scale Limits for A and B Full Auto Result Color Scale Limits Full 5 Auto E Min 0 Counts Result A B k M k 0 787938 Compute k from ROI Color Scale Min 1773 Max 6087 7 with Photo from A X Display Result For Measuring 6 Click and select the ROI created in step 2 from the drop down list The b
173. atial size doubled Spatial size quadrupled At binning 2 four pixels that comprise a 2 2 group on the CCD are summed prior to read out and the total number of counts for the group is recorded Figure C 3 This produces a smaller image that contains one fourth the pixels compared to an image at binning 1 However due to summing the average signal in each pixel is four times higher than at binning 1 At binning 4 16 pixels are summed prior to read out Binning significantly affects the sensitivity of the VIS Imaging System Binning at higher levels for example gt 4 improves the signal to noise ratio for read noise an electronic noise introduced into the pixel measurement at readout If four pixels are summed before readout the average signal in the summed pixel super pixel is four times higher than at binning 1 The read noise for the super pixel is about the same as it was for the individual pixels Therefore the signal to noise ratio for the read noise component of the image noise is improved by a factor of four Read noise is often the dominant source of noise in in vivo images so a high binning level is a very effective way to improve the signal to noise ratio Unfortunately binning reduces the spatial resolution in an image For example at binning 2 a super pixel is twice as wide as a pixel at binning 1 This results in a factor of two loss in image spatial resolution However for in vivo imaging the added sensitivi
174. ation J Display Subject Surface a Opacity 100 Display Photon Density Map Color Table Transaxial y 14 0 Subject Height 22 9 mm ff Perspective _ Spectral Unmixing 180 Living Image Software User s Manual Figure 12 5 3D View toolbar Table 12 1 3D view tools Tool Image Tools oS DA Description A drop down list of tools for viewing and working with the surface Select b to e Click and display measurement dimensions in the coronal sagittal or transaxial view in the 3D view window e Drag a measurement cursor in the coronal sagittal or transaxial view and display measurement dimensions For details on measurement cursors see page 107 select eg to zoom in or out on the image use a click and drag operation Select aad to move the subject in the window use a click and drag operation Select d to rotate the subject around the x y or z axis use a click and drag operation Click to hide or show the x y z axis display in the 3D view window Click to hide or show coronal sagittal and transaxial planes through the surface in the 3D view window Click to show or hide a bounding box around the surface He WR Le Click to show or hide a grid under the surface EF 4 Select this tool from the drop down list to change the view perspective top bottom left right front back or perspective view F
175. ax edit box Click to reset the X and Y Min and Max values to the defaults Full Scale Select this option to display the full X and Y axis scales Logarithmic Scale Select this option to apply a log scale to the y axis 106 Living Image Software User s Manual Viewing 3D Signal Intensity 1 Open an image and then click the Plot 3D button in the Image Information tools A 3D representation of all signals in the image is displayed in the 3D Plot window Figure 7 21 Figure 7 21 3D intensity signal 7 3D Plot Window men For Click TLT20050624145507_005 Plot Full Image v ZMax 240 Ba Refresh Help 2000 0 Max 10348 Perspective 2 To change the display make a selection from the Plot drop down list and click the Refresh button amp refresh Table 7 8 3D Plot Window Item Description Plot Full Image Displays all signals in the image ROI lt ROI number or name gt Displays the signal within the selected ROI All ROIs Displays the signal within all ROIs in the image Z Max Height of the z axis Use the up down arrows to change the height of the z axis Click to reset the z axis to the default setting Ba Copies the 3D window to the system clipboard EJ Opens a Print dialog box that enables you to print the 3D window YY Caliper per Chapter 7 Working With Optical Image Data 107 Mak
176. background wells that contain diluent only 09 Place the well plate on the IVIS stage positioning it so that it is centered and squared in the field of view NOTE All of the wells must be within view in the image For wells containing fluorosphores FOV D is recommended to reduce shadows from well walls and ensure more uniform excitation of the wells 4 Acquire the images Bioluminescent samples Acquire one Open filter image of the well plate Fluorescent samples Acquire reflectance illumination Filter Scan images using the appropriate excitation and emission bandpass filters The well plate in Figure 14 1 contains a dilution series of a sample at four concentrations The image sequence is a filter scan set of images with the excitation filter centered at 465 nm for all the images and emission filter images centered at 520 nm 540 nm 560 nm and 580 nm Figure 13 1 Well plate data GE Emdal sto ese eT Sequence View Seectre g units RadantEfiice Pse Seved cok opsons o o sino l 186 Living Image Software User s Manual 13 2 Creating a Quantification Database 1 Load the well plate image sequence 2 Select Tools gt Well Plate Quantification for lt name gt _SEQ on the menu bar The Well Plate Quantification window appears 3 For fluorescent samples choose the Dye molecules or Cells option Figure 13 2 Well Plate Quantification window
177. bit non negligible autofluorescence The chart in Figure F 15 compares the autofluorescence of miscellaneous laboratory materials to that of black Lexan For example the autofluorescence of the agar plate with ampicillin is more than 180 times that of black Lexan Such a significant difference in autofluorescence levels further supports the recommended use of black polystyrene well plates 302 Living Image Software User s Manual It is recommended that you take control measurements to characterize all materials used in the IVIS Imaging System Figure F 15 Comparison of autofluorescence of various laboratory materials to that of black Lexan 200 180 160 140 120 100 80 60 40 Autofluorescence Relative to Black Lexan 20 Despite the presence of various background sources the signal from most fluorophores exceeds background emissions Figure F 16 shows the fluorescent signal from a 96 well microplate fluorescent reference standard TR 613 Red obtained from Precision Dynamics Co Because the fluorescent signal is significantly bright the background autofluorescent sources are not apparent Figure F 16 96 well plate fluorescent reference standard TR 613 Red The fluorescent signal is strong enough to exceed background emissions Imaging parameters DsRed filter set Fluorescence level Low Binning 8 FOV 15 f 1 Exp 4sec Reference standard TR 613 Red is available through Precision Dynamics
178. c i hot h TIF Choose the Show All _PhstesPhicimss ee s saved luminescent corrected image luminescentFloatCorrected TIF Sections option to 3 structure image structure TIF display all categories P reference image reference TIF of image information Camera System Info Key val Analysis Comment dorsal DOB 03 21 05 Label kidney Male Nn nu Comment Experiment Series 4 To view particular information select a category in the upper box to show the associated information in the lower box For example select luminescent image in the upper box to show the luminescent image acquisition parameters Editing the Image Label You can edit image label information or add information to the label after acquisition To edit the image information 1 Open an image or sequence 2 Click Info to display the image label Figure 7 10 Image information e Fa y E ter20050628145507 005 l T eana Units Counts v Display Overlay v Options vY 2 Info i Image TLT20050624145507_005 Series Male Nn nu Fri Jun 24 2005 07 56 46 Experiment DOB 03 21 05 Em Filter 640 Bin M 8 FOV 12 6 f2 1s Label kidney Camera IVIS 200 Beta II SI620EEV Cie ae Edit an entry For example revise the comment Luminescence 10000 8000 6000 4000 2000 Color Scale Min 586 Max 10348 YY Caliper per Chapter 7 Working With Optical
179. ce Surface m Click to open the color palette from which you can select a display color for the source surface Opacity Adjusts the source surface opacity Display Voxels Choose this option to display the sources reconstructed using DLIT Maximum Intensity Projection Choose this option to project all maximum intensity voxels in the view along the viewing direction into the viewing plane Threshold DLIT FLIT only Choose this option to apply a minimum threshold intensity to the voxel display Gradation DLIT FLIT only Use this slider to set a threshold for the percentage voxel intensity above which voxels are opaque and below which voxels will gradually face to transparent The percentage voxel intensity is the percentage relative to the maximum intensity Voxel size The 3D grid spacing size for interpolation of the reconstructed source Smoothing The smoothing box filter size Display voxels as The voxel display mode cubes spheres points or texture 224 Living Image Software User s Manual Table 14 11 Source tools continued ltem Color Scale Description Color Scale Min 2 10e3 Color Table Red E Reverse Log Scale Min Use the slider or up down arrows to set the minimum value of the source color scale Voxels with intensities less than the color scale minimum are not displayed in the reconstruction Color Table Color
180. ce set format which stores all of the vertex information first then stores the triangle information in terms of indexes into the vertex list AutoCAD DXF dxf Drawing exchange format that is compatible with most yes yes DXF file viewers VRML 1 0 wrl VRML 1 0 wrl Virtual reality modeling language yes no format that is compatible with most VRML viewers Open Inventor iv The ASCII version of the IV file format which is yes yes supported by all IV viewers STL stl or ASCII Stereo lithography binary format compatible with most yes yes format STL viewers binary 184 Living Image Software User s Manual This page intentionally blank Y Ca i oh or 1 3 3D Quantification Database Preparing amp Imaging the Samples a a ee ee ee ee es 185 Creating a Quantification Database 2 0508 eee a 186 Managing Quantification Results 2 0 2 2 eee ee es 189 It is possible to determine the number of cells in a DLIT source or the number of dye molecules or cells in a FLIT source if a quantification database is available The database is derived from an analysis of images of known serial dilutions of luminescent cells or fluorescent cells or dye molecules 13 1 Preparing amp Imaging the Samples 1 Prepare a well plate 4 x 6 6 x 4 8 x 12 or 12 x 8 well format that contains a dilution series of luminescent cells or fluorescent dye at four or more concentrations x Include at least four
181. ced to an organ atlas and the organ from the atlas that is closest to the source will be reported here This information is available if you select and register an organ atlas with the reconstruction For more details see page 230 Export Voxels Enables you to export the voxel measurements in their x y and z coordinates and source intensities csv file Center of mass Click to compute the center of mass for the source selected with the Measure Source tool A For details using this tool see page 209 YY Caliper 14 12 3D Tools Registration Chapter 14 3D Reconstruction of Sources 225 Use the registration tools to e Display organs in the surface page 226 e Manually adjust the location or scale of organs in the surface page 227 e Check the organ fit page 228 e Import an organ atlas page 230 You can check the organ fit in the 3D View window page 228 displayed Figure 14 31 3D registration tools and surface with fitted organs skin not E 6 wee m s Dy Registration Tools Safa B colon WB eyesExterior photonsisec Eoaea Reniy Table 14 12 Registration tools Description Use this tool to manually adjust the scale of location of organs For more details see page 227 Fits the organs to the surface using a linear transformation that keeps the shape of the atlas surface Fits the organs to the surface using linear transformation a
182. cified in the Preferences 60 Living Image Software User s Manual Table 5 4 Image window Sequence view continued ltem Options Description Layout Choose a display option for the images in a sequence For example here is Film Strip mode Sort by Options for ordering images in the sequence window Default Order in which the images are stored in the folder TimeStamp Ascending order of the image acquisition time UserID Ascending alphanumeric order of the user ID Display Choose the types of information to display with each image Units Counts Use Saved Colors Qotons Info e 7 amp Pooare Teen ee el ee mee Tere 1 Layout gt Duplay gt V Exposure Time A Labels b y Binning Fector is txettation fiter Emission fitter f Number Field of View Select All In this example exposure time and binning factor are displayed on each image Click to show or hide the image label information Figure 5 13 Opens all of the images in the sequence Closes all open images Opens the Edit Sequence dialog box that enables you to add or remove images from the sequence Enables you to export the active image as a graphic file for example ong dcm W Ca Pe cs Table 5 4 Image window Sequence view continued Chapter 5 I X Ray Imaging 61 Item Description Creates a preview picture snapshot of the image or thumbnails that the Living
183. citation filter B background image acquired using the background filter k primary signal background signal The background signal is obtained from a measurement ROI that is located in an area where no fluorophore signal is present The scale factor k accounts for different levels of 154 Living Image Software User s Manual tissue autofluorescence due to different excitation wavelengths and filter transmission characteristics After you acquire an image sequence that includes a primary and background image use the image math tool to subtract tissue autofluorescence For more details on acquiring an image sequence see Chapter 4 page 33 To subtract tissue autofluorescence 1 Load the image sequence that includes the primary and background fluorescent images Figure 9 3 Image sequence Sect puns J510491 KSA20110305104918_SEQ Sequence Vie Spectra unis E Use Saved Cors ELE TLT20060510114512_0084 TLT20060510114512_ 0104 Min 1214 n amp 2 Open either the primary or background image and a Optimize the image display using the color scale Min and Max sliders in the Image Adjust tools b Draw a measurement ROI on an area of the animal that represents background signal area where no fluorophore signal is present NOTE You only need to draw the ROI on one of the images The software copies the ROI to the other image Y Caliper ifeSciences Chapter 9 Image M
184. clipboard Select All Selects all rows in the sequence table Delete row s Deletes the selected row s from the sequence table Replace Row s Replaces the row s selected in the sequence table with the rows in the system clipboard Note The Replace function is only available when the number of rows in the system clipboard is the same as the number of rows selected in the sequence table Paste Row s Adds copied rows to end of the sequence YP Caliper per Chapter 4 Optical Imaging 45 Removing Images From a Sequence Method 1 1 Select the row s that you want to delete 2 Click and choose Selected from the drop down list Method 2 Select the row s of interest and right click the sequence table to view a shortcut menu of edit commands Figure 4 27 4 7 High Resolution Imaging With the Optical Zoom Lens Attachment The optional Optical Zoom Lens attachment enables close up and high resolution imaging on the IVIS Lumina VIS Lumina XR and IVIS Kinetic Imaging Systems When the Zoom lens attachment is installed only the Z field of view setting 2 6 cm is available for single image or sequence acquisition The imaging system is set to the Z field of view position until the Zoom lens attachment is removed If the Zoom lens attachment is installed when the Living Image software is closed the stage will move to the Z position when the system is initialized NOTE When installing or removing the Op
185. cquire 6 Click Acquire when you are ready to capture the image NOTE If necessary click mage Setup jin the control panel to operate in single image mode In single image mode the Sequence Setup button appears in the control panel Click this button to set up sequence acquisition For details on sequence setup see page 33 y Calipe 4 per Chapter 41 Optical Imaging 23 Figure 4 2 Enter image label information optional Information entered here Sree ee appears in the image label SSL Lr Z Edit Image Labels Ea Image TLT20050624145507_005 Series Male Nnjnu UserID KSA v Living Image Universal ri Jun 24 2005 07 56 46 Experiment DOB 03 21 05 Filter 640 Bin M 8 FOV 12 6 f2 1s rar kidney ia s f x V r Saved Labels LABELS 1 aa amera IVIS 200 Beta II SI620EEV eh as Image label Check any 5 fields for display V User X V Group v x Luminescence V Experiment X 10000 V Commenti 8000 V Comment2 6000 Time Point Y Animal Number v Animal Model Y F Sex v F View v 2000 Cell Line v Reporter v C Treatment v Counts ICY Luc Injection Time a Color Scale E IACUC Number Min 586 Max 10348 Apply To Sequence OK Cancel 7 In the Edit Image Labels box that appears Figure 4 2 enter information about the image and click OK NOTE You can enter image label information at any time during
186. create 186 189 manage results 189 samples 185 R radiance photon 283 radiance units 282 read bias 285 reconstruct 3D fluorescent sources 201 203 reconstruct 3D luminescent sources 193 200 reconstruct particular pixels 199 reduced Chi2 204 registering multi modal data fiducial registration 245 loading data 241 243 manual registration 247 249 ROI 119 3D signal 106 automatically draw 127 129 background corrected signal 130 132 delete 143 edit dimensions 139 free draw 129 managing 135 manually draw 125 126 measurement ROI free draw 129 measurement ROIs 125 129 Measurements table 122 move 138 move or edit label 142 quick guide 120 ROI line 141 save 142 subject 129 tools 123 124 ROI kinetic quick guide 132 133 ROI Measurements table 145 147 configure 147 149 copy or export 149 ROI properties 135 138 ROI types average background 120 130 measurement 120 subject 120 S save data manually 46 ROI 142 save kinetic data 78 save kinetic data 77 scientific image data 279 segment 43 sequence requirements DLIT 193 FLIT 201 planar spectral imaging 157 spectral unmixing 165 smoothing 100 278 software help 20 source depth 210 source spectrum 205 Spectra window 171 172 spectral imaging See planar spectral imaging spectral unmixing 165 176 options 174 175 parameters 173 174 PCA biplot 176 PCA explained variance 175 results 169 sequence requirements 165 starting the software 15 Y Ca ie st
187. ct Measurement ROI from the Type drop down list Figure 5 20 Figure 5 20 X ray image with absorption correction 7 EL20090623143901_001 oo amp z Tool Palette Units Counts Display XRay gt Options v Info iw _ Image Adjust i _ Corrections Filtering S L gt Image Information ROI Tools O O amp Qf Measure ROIs x E Apply to Sequence Type Measurement RO Sav Measurement ROI 6000 Name Average Bkg ROI Subject ROI Auto ROI Parameters 4000 2000 0 00 Absorption Color Scale in 0 Max 7971 4 To select the ROI shape a Click the Circle or Square OJ button b On the drop down list that appears select the number of ROIs that you want to add to the image The RO s and intensity label s appear on the image If you are working with a Sequence open an image to show the ROI intensity 5 Adjust the ROI position a Place the mouse pointer over the ROI When the pointer becomes a click the ROI b Drag the ROI 6 Adjust the ROI dimensions a Place the mouse pointer over the ROI When the pointer becomes a click the ROI b Place the mouse pointer over an ROI handle so that it becomes a Drag the handle to resize the ROI You can also change the ROI position or size using the adjustment controls in the ROI Properties box see Moving
188. d You can save the sequence parameters in the sequence table to a Living Image Sequence Setup file xsq For details on image acquisition see Acquire the Image Sequence page 35 NOTE To create an image sequence it may be convenient to edit a sequence setup generated by the Imaging Wizard or an existing Sequence setup xsq Save the revised sequence setup to a new name 1 Click Sequence Setup in the control panel The sequence table appears 2 If necessary click the Remove button X Rm and select All to clear the sequence table Figure 4 23 Opening the sequence table F Stop Excitation Filter Emission Filter s oa Cocor open Auto mm Medum is Sequence table Focus Temperature M Locked Initialize E Number of Segments a Delay 0 0 H min x Remover Al Update Insert Add 3 Choose a subject and probe from the drop down lists Figure 4 24 42 Living Image Software User s Manual Figure 4 24 Choose a subject and probe lt Display Photographic Settings Subject Probes E seq _ Bioluminescent Probes gt Fluorescent Probes gt Unselect Probes Bacteria CBGreen CBRed Firefly hRenilla Tritium Bead 5 XPM 2 LED ZnS Unselect Probes E Number of Segments a H Delay 0 0 E min x Remove J Update Insert had 4 In
189. d background Note Using this type of ROI is optional If the animal has significant Identifies a subject animal in an image Note Using this type of ROI is optional It provides a convenient way to automatically associate Description l link a measurement and average autoluminescence or background ROI for background autofluorescence you can determine corrected ROI measurements a background corrected signal in a when there is significant measurement ROI by subtracting an autoluminescence or average background ROI from a autofluorescence measurement ROI e Manual e Manual e Manual Available ROI Drawing Methods e Automatic e Free draw e Free draw e Automatic e Free draw Available Shapes Circle square grid or contour Circle or square Square 8 2 Quick Guide Drawing Measurement ROls on an Image or Sequence These steps provide a quick guide on how to apply a measurement ROI to an optical image or image sequence For more details about measurement ROIs see page 125 1 Open an image or sequence and click ROI Tools in the Tool Palette 2 In the ROI tools select Measurement ROI from the Type drop down list Tool Palette fROToos o l Cl 8 Y Measure ROIs i Veo ET efa avii Eee ame Average Ekg ROI Subject ROI Delete I Auto ROI Parameters Threshold o DUT 3D Reconstruction gt Spectral Unmibxing Figure 8 2 Select a type of ROI gt Planar
190. d an image sequence 2 Generate or load a surface using the Surface Topography tools For details on generating the surface see page 181 3 In the Tool Palette choose DLIT 3D Reconstruction The Analyze tab shows the data that the algorithm automatically selects for the reconstruction Figure 14 2 For more details about the Threshold see page 198 Figure 14 2 Analyze tab Tool Palette gt ROI Tools gt Planar Spectral Imaging ll Analyze Properties Results Sequence 72720050624145507_SEQ Tissue Mouse Tissue Source Firefly Filter Threshold 4 In the Properties tab make a selection from the Tissue Properties and Source Spectrum drop down lists Figure 14 3 aA Y Caliper Chapter 14 3D Reconstruction of Sources 195 Figure 14 3 Properties tab Tool Palette gt ROI Tools gt _ Planar Spectral Imaging Surface Topography s DLIT 3D Reconstruction Result not found The selected plot type is displayed below L Normalized Amplitude 5 To view the tissue properties u4 Heys W s for the tissue and source you selected make a selection from the Plot drop down 6 To compute the number of cells per source select a luminescent calibration database For details on generating a lu
191. d at two or more wavelength filters spanning the luciferase emission spectrum for example firefly luciferase 560 660 nm To reconstruct fluorescent sources the software requires a structured light and fluorescent images obtained using the same excitation and emission filters at different transillumination source positions on the IVIS Spectrum To localize and quantify the light sources in a subject the software e Reconstructs the subject surface topography surface from structured light images The surface is defined by a set of connected polygons or surface elements e Maps the surface radiance photons s cm2 steradian to the photon density photons mm3 just beneath the surface of each element of the surface For NTF Efficiency data from normalized transmission fluorescence data the NTF Efficiency 2D data is mapped to the 3D surface e Divides the interior of the subject into a solid surface of volume elements or voxels Each voxel is considered to contain a point light source at its center that contributes to the photon density at each surface element e Defines equations that relate the source strength of each voxel to the measured data photon density or NTF Efficiency at each surface element e Determines the optimum approximate solution to the system of linear equations to reconstruct the source strength in each voxel H 1 Determining Surface Topography The software determines the surface topography from a structured light
192. d key frame up in the key frame box 4 Hi 3 Moves the selected key frame down in the key frame box Total Duration The total time of the animation sequence Play Click to view the animation sequence defined by the current key frames and animation parameters Record Displays a dialog box that enables you to save the current animation to a movie mov mp4 or avi mpg Animation Setup Load Displays a dialog box that enables you to open an animation setup xml Save Displays a dialog box that enables you to save the current key frames and animation parameters to an animation setup xkf A _ YW Caliper Chapter 14 3D Reconstruction of Sources 233 Viewing a Preset Animation Preset animations are factory loaded animation setups They include predefined key frames which are used to generate the animation To view a preset animation 1 Open an image sequence and load 3D reconstruction results 2 Select properties to display in the 3D View window for example organs sources surface or photon density maps W Select View 3D Animation on the menu bar In the 3D Animation tools that appear a Clear the key frame box if necessary click the button and select Delete All b Make a selection from the Presets drop down list See Table 14 13 page 232 for a description of the preset animations After a preset animation is selected a list of the key frames appea
193. d size in the adaptive gridding scheme Nsurf best The number of surface element data analyzed per wavelengths images Total surf samples The total number of surface element data analyzed for all wavelengths images Y Caliper Chapter 14 3D Reconstruction of Sources 205 Table 14 5 DLIT or FLIT 3D reconstruction results continued ltem Threshold angle Description The angle that the object surface normal makes with the optical axis The optical axis can be considered to be a line perpendicular to the stage The default setting for this limit is 70 for IVIS Spectrum or IVIS 200 data Image Threshold The percentage of the minimum radiance at each wavelength DLIT or source location FLIT is of the maximum radiance This defines the minimum intensity included in the data Tissue Properties The tissue properties for modeling the photon propagation Source Spectrum The emission spectrum of the type of luminescent source Photon Density Maps Click to view the photon density maps For more details see page 207 Export Results Opens a dialog box that enables you to save the results csv Save Results Name The default name for the active DLIT or FLIT results Select results from this drop down list Delete Click to delete the selected DLIT or FLIT results Load Click to load the selected DLIT or FLIT results Save Click to save the active DL
194. details see page 213 aK Enables you to save the 3D view to a graphic file for example jpg 198 Living Image Software User s Manual Data Preview Window The Data Preview window shows the image data that the algorithm automatically selects for reconstruction In special cases you may want to include or exclude particular data from this default selection There are two ways to do this e Change the Threshold value Applying a Threshold value excludes or includes some pixels from the reconstruction The software computes the minimum and maximum pixel values of an image based on an histogram of pixel intensities If Threshold 0 5 then pixels with intensity less than 0 5 of the maximum intensity value are excluded from the reconstruction The Threshold can be edited for individual images The Data Preview window is updated when you change the Threshold value e Region selection Use the pencil tool to mark particular regions to include in the reconstruction This may be useful for noisy images with high intensity pixels where changing the Threshold value is not helpful You can also use this method to focus on particular sources to reconstruct and ignore others To change the Threshold for a selected image 1 Click Start in the Analyze tab Figure 14 7 The Data Preview window appears 2 Click an image in the Data Preview window Changes to Threshold are applied to the selected image only To appl
195. e Restore Defaults Save Ld All Autos All BKGs All Subjects BKG1 ROI7 ROI8 2 In the drop down list that appears select a delete command The specified ROIs are deleted from the image NOTE This does not delete ROIs saved to the system global save To permanently remove ROls from the system 1 Select the ROI s that you want to delete from the drop down list of saved ROIs 2 Click Delete Figure 8 27 Figure 8 27 Removing ROIs from the system Restore Defaults gt 3D Multi Modality Tools a i z2 FY 4 Preview _ Use Bkg Offset _ Replace ROIs Save Load Caliper y p Chapter 8 ROI Tools 145 ifeSciences 8 9 Managing the ROI Measurements Table The ROI Measurements table shows information and data for the ROIs created during a session The ROI measurements can be displayed in units of counts or photons or in terms of efficiency For more details see Quantifying Image Data page 281 Viewing the ROI Measurements Table 1 Click the button Alternatively select View ROI Measurements on the menu bar Figure 8 28 Opening the ROI Measurements table Column headers in the table include ROI information ROI measurements and dimensions and information about the image recorded at acquisition Tool Palette Save ROIs Name ROI_1_KSA Delete
196. e certain parts of the data as needed A color opacity map can be saved Figure 15 15 Histogram tool specifies the opacity for different voxel intensities H Help ko e 7 i 3 18e4 Intensity 3 58e4 a Y Caliper Chapter 15 I 3D Multi Modality Tools 251 Specifying a Color Opacity Map 1 After the surface and volume data are loaded confirm that the Display Volume option is selected Figure 15 16 3D Multi Modality tools Tool Palette Performance Quality Color Opacity Map Grays 1 0 Opacity El Reverse Autofit air noise boundary Voxels below this threshold are not displayed The color table is mapped to voxels above threshold Opacity 0 00 Intensity E Logarithmic Histogram E Maximum Intensity Projection MIP El Gradient Illumination 2 To change the color table for the color opacity map make a selection from the Color table Opacity Map drop down list To apply the reverse color table select the Reverse option 3 Ifthe histogram intensity range appears narrow or suppressed choose the Logarithmic Histogram option Choosing this option enhances the histogram display by magnifying the smaller regions of interest in the histogram while keeping noise and air related intensity peaks high It helps bring out hidden regions visible in the histogram for easier identification of interesting intensity ranges
197. e orientation with good slice sampling spacing Total Slices Slice spacing Pixel spacing Parameters that determine the number and resolution of the slices to export Solid mesh If this option is chosen voxels generated inside the hollow mesh are assigned an intensity so that they are displayed as tissue when loaded into visualization software If no intensity is associated with the voxels they are considered noise or air and appear hollow Y Caliper ifeSciences Chapter 14 3D Reconstruction of Sources 219 Table 14 8 3D Scene Exporter dialog box continued Item Description Hollow mesh The intensity of pixels inside the surface is set to zero so that the exported surface appears as a hollow empty structure Viewing the DICOM Data The 3D scenes exported to DICOM can be viewed in the Living Image 3D Browser 1 Select File Browse 3D Volumetric Data on the menu bar 2 In the dialog box that appears select the DICOM data dcm or dc3 and click Open The 3D Browser window appears 3 To Figure 14 28 Living Image 3D Browser er IC Browse 3D Volumetric Data Seog ox Add tolist Browse Name Folder Path Slices Dimension Modality Pixel Spacing Slice Spacing Data Type Type BEM dcm 20110306103654 C Share Caliper LS Caliper Data 256 128 x 128 OT 0 289063 0 289063 10 3977 8 bit DICOM bsesssesesssssessssossssssvosssssssssssssssssssss ssssssssssssssssssosssosu
198. e Background ROI tab and puta check mark next to Use as BKG for future ROls in 3 Choose the image name or the Entire sequence option 8 7 Kinetic ROIs Kinetic ROIs help you track signal sources on an unanesthetized mobile subject The software automatically creates a separate ROI in each frame based on the user specified auto ROI settings As aresult kinetic ROIs are continuously displayed during kinetic data playback You can draw a kinetic ROI using any of the methods or shapes in Table 8 1 page 120 NOTE Large kinetic data sets may require more time to create plot and measure the ROIs because the software first applies corrections to a frame specified in the Corrections Filtering Tool Palette then draws the ROls in the frame The process can be aborted at any time These steps provide a quick guide on how to apply a measurement ROI to kinetic data For more details about measurement ROIs see page 125 1 Open the kinetic data and click ROI Tools in the Tool Palette 2 In the ROI tools select Measurement ROI from the Type drop down list 3 Click the Contour button and select Kinetic ROI The create tool appears on the image Y Caliper el os Chapter 8 ROI Tools 133 Figure 8 15 ROI create tool olea Tool Palette T m i ai gt Corrections Filtering HE Om YW Measure ROIs x Epi fluorescence Apply to Sequence ype Measurement ROI v Save ROIs Name ROI_3_KSA
199. e Clicks Browser Images Units Counts v E Use Saved Colors Options v Info 5 gt R oi a TLT20050624145507_001 JJH20050630142125 001 TLT20050624145507_002 JJH20050630142125_002 TLT20050624145507_003 JJH20050630142125_003 TLT20050624145507_004 lt lt Copy JJH20050630142125 004 TLT20050624145507_005 TLT20050624145507_006 Images in the Retired Images active SEQUENCE Min 2140 Images that have ee been removed from the active sequence Min 2949 if 586 Min 1122 Max 51182 95 Max 10348 96 0546 Y Ca i oh Chapter 7 Working With Optical Image Data 117 4 In the Edit Sequence box that appears choose the image s to add or remove retire from the sequence Figure 7 31 To add an image to the sequence select an image from the Browser Images and click Copy To remove an image from the sequence choose an image from Sequence Clicks and click Retire Nn Reactivate To restore a retired image to the sequence select the retired image and click 6 To reorder the sequence select an image and click Move Up or Move Down NOTE The Move Up and Move Down buttons are only available when the sequence view window displays images in the default sort order If the TimeStamp or UserID sort order is selected the images cannot be reordered 7 When you are finished editing the sequence click Close The updated image sequence is displayed 7 13 Creating a
200. e Kinetic Acquisition window click the Save button and select a save option Save Option Save Current Image Description Saves the currently displayed frame Save Accumulated Image Saves the accumulated signal for the selected frames tiff Note It is not necessary to select the Accumulate option to save an accumulated image Save Kinetic Data Saves the data photographic frames all luminescent or fluorescent frames and read bias in DICOM format dcm 2 In the Edit Image Labels box that appears enter information for the image label and click OK If you do not want to enter label information click Cancel 78 Living Image Software User s Manual Figure 6 7 The Edit Image Labels box Image window and Tool Palette appear when saving an image or kinetic stream e Gok Gs Unite Courter eet aa Kinetic Acquisition Fj Kinetic Argain PEET D ae gat rf You can edit and analyze kinetic data in the Image window 6 5 Playing Kinetic Data In the image window you can e Play kinetic data e Select and view a particular image e Select a range of images and extract as a separate kinetic data set YY Caliper 4 i Chapter 6 I Kinetic Imaging 79 Figure 6 8 Image window kinetic data fmn SNE TEAN E DA20081107162612 Tool Palette Units Radiance Photons v Display Overlay hd Options Info iw Image Adjust
201. e ROI e Drag a handle on the ROI e Edit the settings in the ROI Properties box NOTE You cannot change the size of an ROI that was created using the auto ROI or free draw tool To resize an ROI using a handle 140 Living Image Software User s Manual 1 Select the ROI and put the mouse pointer over a handle m on the ROI 2 When the pointer becomes a arrow drag the handle To resize an ROI using the ROI Properties box 1 Double click the ROI in the image The ROI Properties box appears and displays the positions and dimensions of the selected ROI Figure 8 22 ROI Properties dialog box Background ROI T Use as BKG for future ROIs in T1T20050624145507_005 only Entire sequence E Lock Position Xc pix 117 00218 Yc pix 97 93839 Angle deg 0 0000 F Lock Size Dimensions of the ROI Width pix 22 06161 selected in the image Height pix 20 43172 2 Enter a new width or height value in the ROI Properties box 3 To lock the current ROI size choose the Lock Size option NOTE The ROI size cannot be changed until the Lock Size option is cleared y Caliper A Bet Chapter 8 ROI Tools 141 Editing the ROI Line 1 Double click the ROI that you want to edit The ROI Properties box appears Figure 8 23 Figure 8 23 Editing ROI properties E ROI Properties sex ROL Label BKG 1 Shape Cirde Type Manual Background ROI
202. e fs arrow next to sea the browser contents in right click a row and ascending alpha numeric select Properties on order Click the column header the shortcut menu again to sort in descending aloha numeric order TL Living Image Browse CK20090729114835_SEQ Click Number EX Filter EM Filter amination Mode UserID User Group Experiment Commenti a HE CK20090729114835_SEQ i i XFM SN1007 irod7 srcA 1745 800 vevevevevevtevevevevevevereveverevevireveviverereveveveveveveveveviveviraevevivirerevevivevevevivevevevireveveverevrevirereveviver XFM SN1007 irod7 src A seee ee A Click No CK20090729114835_SEQ S Dataset Type Sequence Dataset No of clicks 16 coe Location C Share Caliper LS Caliper Data Sample Data FLIT CK20090729114835_SEQ Sequencelnfo txt ME CK20090729114835 nsilluminatio CK20100628141050_SEQ dea HX20070420121444 SEQ In vitro DecasgeecccccscccccucccccccscescesccsccsccusccccccccascesbocceseusesscecculsccscescceccccsceDecceccsceccnscecescesscesesccscecsleneccvccsccccscescescesabeccuceccsccecascessbecesesscusccccccscaecesscscessbssesccccscascasscscescessceceSscssescescesccusescsccscencescesacsccesescsccsscssescess IEG HX20070420122818 SEQ Invitro coll AFT00 AF 2colAF680 AF 3col QD700 AF700 QD800 g 3EQ TLT20050624145507_SEQ TLT a m Hide Browse View Close Preview Label Set All v Add to List
203. e or 3D sources The tools are organized by tabs 3D Tools Functions Page Surface Tools Adjust the appearance of the reconstructed animal See below surface and photon density maps Source Tools Adjust the appearance of reconstructed sources make 222 source measurements export voxel measurements Registration Tools Display organs on the reconstructed surface adjust the 225 location or scale of organs on the surface import an organ atlas Animate Tools Display preset animations of the 3D View scene Enables 231 you to create custom animations and record an animation to a movie file 14 10 3D Tools Surface Use these tools to adjust the appearance of the reconstructed animal surface and photon density maps Figure 14 29 Surface tools and example DLIT reconstruction with photon density maps Tool Palette Display Subject Surface 4 0 5 Opacity g Display Photon Density Map Apply Simulated Intensity asses E Color Table E F Reverse p T E Log Scale l 1 l i F i 20 i aos te Po aa Source Intensity SRE E epas YY Caliper Chapter 141 3D Reconstruction of Sources 221 Table 14 10 Surface tools Item Description Display Subject Choose this option to display the surface in the 3D View window Surface Drawing styles for the surface Wire frame amp i surface face ih Surface face Point cloud Wire frame i A
204. e present in the dataset Acquisition Background View Available Dark Charge Opens a dialog box that enables you to view the dark charge measurements for the system Acquisition gt Background Clear Available Dark Charge Clears all dark charge images from the system Acquisition Background Auto Background Setup Opens a dialog box that enables you to acquire background images or schedule or disable automatic background acquisition Acquisition gt Fluorescent Background Measure Fluorescent Background Starts a measurement of the instrument fluorescent background Acquisition Fluorescent Background Add or Replace Fluorescent Background Opens a dialog box that enables you to select an instrument fluorescent background measurement for the active image data If the Fluorescent Background Subtraction option is chosen in the Corrections Filtering tool palette the background measurement is subtracted from the image data y Caliper LifeSciences Appendix J Menu Commands Tool Bar amp Shortcuts 329 Table J 1 Menu bar commands and toolbar buttons continued Toolbar Menu Bar Command Button Acquisition Fluorescent Background Measure and Replace Fluorescent Background Description Measures fluorescent background under the same conditions as the currently selected image When the measurement is complete the newly acquired background ima
205. ect All Refresh Copies all rows in the table to the system clipboard Updates the ROI Measurements table for example after you draw new ROIs move an ROI and close or open image data Configure Displays the Configure Measurements box that enables you to specify and organize the data categories column headers for the table Export Displays the Save Measurements box so that the data can be saved to a txt or csv file Note Grid ROI measurements exported to a csv file can be opened in a spreadsheet application like Microsoft Excel Close Closes the ROI Measurements table Configuring the ROI Measurements Table You can customize the data and information column headers in the ROI Measurements table Several preset categories are available in the Measurement Types Click Attributes and ROI Dimensions drop down lists 1 To reorder the columns drag a column header left or right in the table 2 To change the measurement units make a selection from the Measurement Types drop down list Figure 8 30 ROI Measurements table 4 ROI Measurements olal ROI Measurements Grid ROI Measurements z Refresh Image Number ROI Image Laye Total Coun Avg Count Stdev Cour Min Count Max Count ROI Pixels Area Xc Yc Widt ccd Pixels pixels pixels pixe HX20070420121444 001 ROI1 Overlay 1 425e 06 4 167e 02 6 726e 02 8 462e 00 2 545e 03 3420 2189e 05 4
206. ections Window Size amp Position Most Recently Used Dataset History 5 gt Counts Total Count Radiance Photons Total Flux Effiency TotalEffidency Table B 1 General preferences Item Start Up Defaults Description Dock Tool Palette Choose this option to set the position of the Tool Palette in the application window Choose left or right Note To undock the Tool Palette click on the palette title bar and drag it a distance greater than its width Window Size Specifies the dimensions of the main application window Width Height Sets the dimensions of the image window Restore Defaults Click to apply the default settings Apply Individual Color Scale for Sequences Choose this option to apply a separate color scale to each thumbnail of a sequence If this option is not chosen all of the thumbnails are displayed using the same color scale Show Transillumination Locations Choose this option to display a cross hair at each transillumination location when you load transillumination data When you mouse over a cross hair a tool tip displays the transillumination coordinates If this option is not chosen you can choose the Transillumination Location option in the sequence view window to display the transillumination locations Show Advanced Options If this option is selected advanced features are available in the menu bar and Tool Palette including e Additional ROI
207. either of the following e Click Imaging Wizard and step through the wizard for more details on the Imaging Wizard see page 54 OR e Set up the sequence manually for more details see page 41 4 To set up the next sequence e If using the Imaging Wizard repeat step 3 Each sequence is dislayed in a separate tab e If setting up the sequence manually click the EA button in the sequence table to add a new tab then proceed with manual set up in the new tab NOTE Sequence tabs can be renamed Double click a tab name to edit it Alternatively right click the selected name to view a shortcut menu of edit commands for example Cut Copy Paste Figure 5 15 Multiple sequence tabs In this example three sequences are specified Adds a new tab use with Sequence tabs Removes the active tab and its sequence manual sequence setup oa a ia Subject D Mode Exposure Binning FStop Excitation Emission Structure FOW Height 1 FEZ auto Medium 1 Block 560 Yes c laso 2 BRS auto Medium 1 Block 580 No co 150 3 BS Auto Medium 1 Block 600 No co 150 4 ER Auto Medium 1 Block 620 No c i150 Ata Medam 1 Bib amp 140 No c liso Dnes E ra e 5 To remove a sequence click the sequence tab and then click the E button 6 Click Acquire when you are ready to capture the sequences Image acquisition proceeds with no intervening time delay between sequences NOTE If the check mark is removed next to t
208. el value the mean value of the nine pixels in the 3x3 neighborhood Y Ca i A ladias Appendix D Image Data Display amp Measurement Weegee Uae 6aeeeee GEE BRR RE ERD ORR EHR HEE OS BH 279 Quantifying Image Data ssa eee aR EERO EPEC RHEE EEE 281 PIAL ISIS ak KEE REE HHRERE DRE EER HRD EER DE HG BS 284 Cosmic Ray Corrections 6 4 i w 2iker wu kee ee beeee ne awed 284 D 1 Image Data Scientific Image Data Scientific image data is a two dimensional array of numbers Each element of the array pixel is associated with a number that is proportional to the light intensity on the element A charge coupled device CCD camera used for scientific imaging 1s essentially an array of photo sensitive pixels and each pixel collects photons during an image exposure The subsequent electronic readout provides a photon intensity number associated with each pixel In a bright area of the image more photons are detected and the photon intensity number is greater than the number in a dim area of the image The image data can be visualized in different ways including pseudocolor images generated by the Living Image software contour plots or isometric displays Graphic Image Data A graphic image is a two dimensional array of pixels with a color assigned to each pixel There are several schemes for digitally storing the images For example a common scheme assigns a red green blue RGB color code to each pixel The RGB code de
209. elect Remove Comment on the shortcut menu e To remove all comments right click the image and select Remove All Comments on the shortcut menu 96 Living Image Software User s Manual Insert Tag Insert Comment N Remove Tag Remove Comment Remove All Tags Remove All Comments Copy All ROIs Paste ROI Hide ROI Tags Delete All ROIs Sort ROIs Zoom Area Zoom In Zoom Out Reset Zoom Figure 7 12 Add a comment to an image C TEN r EENET EEEN E OAE EE e E E TLT20050624145507_006 E 11120050624145507_006 Units Counts v Display Overlay v Options vY Info wi Units Counts v Display Overlay e Options Y Info i Luminescence 15000 10000 5000 Counts Color Scale Min 1122 Max 19546 Luminescence 15000 10000 5000 Counts Color Scale Min 1122 Max 19546 Tagging an Image An image tag displays the x y pixel coordinates of the location and the pixel intensity z counts or photons You can apply a tag at a user selected location in an image To apply a tag 1 Right click a location in the image 2 Select Insert Tag on the short cut menu Figure 7 13 Insert a tag on an image left move the tag label right a Units Counts X Display Overlay v Options x Info g ig Luminescence 10000 Insert Tag Insert Comment Remove Tag
210. ematical function from the Result drop down list 5 To include a scaling factor k in the function enter a value for k 6 To view the new image click Display Result for Measuring To save the new image 1 Click the Save button fal Alternatively select File Save on the menu bar 2 In the dialog box that appears select a directory and click Save A folder of data is saved to the selected location AnalyzedClickInfo txt ClickInfo txt luminescent and photographic TIF images To export the image to a graphic file 1 Click the Export button Figure 9 2 2 In the dialog box that appears select a directory enter a file name and select the file type from the Save as type drop down list Click Save 09 y Caliper E per Chapter 9 Image Math 153 Table 9 1 Image Math window Item Description Color Ranges for A and B Full Choose this option to set the Max and Min values to the maximum and minimum data values in the image Auto When this option is chosen the software sets the Min and Max values to optimize image display and suppress background noise The Min and Max settings can be manually adjusted to further optimize the image display for your needs Note The color scale does not affect the image math result Color Ranges for Result Full See above Image Auto See above Min 0 Choose this option to set the minimum data value to zero Results Drop down list of mathematical
211. emiluminescent process associated with metabolic activity in living animals is the source of animal background This is supported by the observation that the level of background light drops significantly in euthanized animals In Figure E 2 the background light emission 1s clearly visible in the images of a control white furred mouse and a nude mouse The images are five minute high sensitivity high binning exposures The average emission from a white furred mouse and a nude mouse is approximately 1600 photons s cm2 sr and 1000 photons s cm2 sr respectively Since NP Caliper sven E I Luminescent Background Sources amp Corestons 289 these values are well above the lower limit of detection of the IVIS Imaging System 100 photons s cm2 sr the background light emission from the mouse determines the limit of detection An approximation of this background determined by making similar measurements on either control animals or regions of the subject animal that do not contain the primary signal can be subtracted from ROI measurements For more information on ROI measurements see Chapter 8 page 130 Note that the background light emission is not uniform over the entire animal In Figure E 2 images of control animals mice show a somewhat higher background component originating from the abdominal and thoracic regions Therefore care must be taken when selecting a representative background area Figure E 2 Background light emission
212. en beget eee ee uae ee ee ea ee Pa ee ee 119 e ADOUUROIS saesae ce cah et eke Giese eee Ge see ets ey oe eee eee eS 119 8 2 Quick Guide Drawing Measurement ROIs on an Image or Sequence 120 Oo ROLMOOIS Gaean th AP eee ee teenage ee ee eee ee eae ee ee eee he eee es 123 8 4 Measurement ROIS 4 422048 446688 hbo wee EEG HEE EERE oe eG 125 S3 PUDE ROIS ae eae ee eeeee bee So eR GG EER Re EES Eee he SG 129 8 6 Measuring Background Corrected Signal a n a a 2 2 ee 130 8 7 Kinetic ROL 2 25 24 444 24465 toh R bot ee eERH eae hdee Se Eee dakia 132 Oo Manasino ROIS akeveneadseeedekeee yes ee ee eke ee ees eee rangi 135 8 9 Managing the ROI Measurements Table 0 00 00 2 eee eee ees 145 9 IMAGE Malh 2 tae et be Pee ee eee eee eee eS EE eee 151 9 1 Creating a New Image Using Image Math 0 0 00 0 002 eee 151 9 2 Subtracting Tissue Autofluorescence ooon 153 10 Planar Spectral Image Analysis 2 0000 2 eee eee 157 10 1 Image Sequence Requirements 2 e a a a 157 10 2 Planar Spectral Image Analysis 2 2 2 ee 157 10 3 Viewing Graphical Results o a aaa 161 10 4 Managing Planar Spectral Imaging Results 2 2 2 0000 eee eee 162 11 Spectral UNMIXING 2 s ic ew be Saw eee ete ee HSS eee ES EE ew Es 165 11 1 Image Sequence Requirements 2 a a a a a 165 11 2 Performing Spectral Unmixing 2 2 ee 165 ILS Speciia WINdOW wines ese eet ee eee Ge
213. ents for all images click the arrow next to Current Frame and select All Frames then click the Refresh button 134 Living Image Software User s Manual Figure 8 16 ROI Measurements table Kinetic ROI measurements are displayed in a separate tab DA20081107162612 lo x A ROI Measurements Units Radiance Photc Display Options vY Info 4 iw ROI Measurements Kinetic ROI Measurements Plot Kinetic ROI Measurements Click DA20081107162612 v Rot Rota CurrentFrame Image Numb ROI Image Layer FrameTim Total Flux Avg Radiar Stdev Radiance Min Radiance Max Radiance p s p s cm s Epi fluorescence Frame132 ROIL Overlay 21 222 1898e 10 1 663e 09 9 955e 08 3 804e 08 4720e 09 40 9 x10 Customized Selections Measurements Types Image Attributes ROI Dimensions Radiance Photons y _none_ x _none_ A wo Configure Export ROI 1 8 1 898e 10 Radiance pisec cm2fsr Color Scale Min 4 51e8 Max 4 12e9 132 E gt Fa C Accumulate Plotting Kinetic ROI Measurements The kinetic ROI plot provides a convenient way to view and compare kinetic ROI measurements across user selected image frames from the same or different kinetic data sets 1 Open one or more kinetic data sets 2 Draw kinetic ROIs on the data sets in which you want to measure and compare ROIs 3 In the
214. es Otherwise the frames may not be properly animated 11 To view the animation click Play To stop the animation click Stop An animation setup series of key frames can be saved xkf or recorded to a movie mov mp4 avi mpg To save an animation setup 1 Click Save 2 In the dialog box that appears select a directory and enter a file name xkf To record the animation to a movie 1 Click Record 2 In the dialog box that appears choose a directory enter a file name mov mp4 avi and click Save 236 Living Image Software User s Manual To edit an animation setup 1 Open an image sequence and load a reconstruction 2 Open an animation setup To select a predefined setup make a selection from the Preset drop down list To select a saved user defined setup a Click Load b In the dialog box that appears select an animation setup xkf Figure 14 41 List of key frames in the selected animation E 3DAnimation Preset Animations Presets Spin CW on Y Axis Frame Factor Animation Setup Time Scale a p Key Frame 1 Key Frame 2 Key Frame 3 Key Frame 4 Key Frame 5 m Play Frames Per Second 10 Total Duration secs 5 Load 3 To add a key frame a Adjust the position of the reconstruction in the 3D view using an image tool for example or 4 For more details on the image tools see page 197 b Click the button 4 To reorder a key
215. es each pixel by a predetermined scale factor The scale factor for each pixel depends on its distance from the center of the image The scale factor near the center of the field of view is one but can be up to two or three near the corners on the IVIS Imaging Systems and Spectrum You may notice an increase in noise near the edges and corners of the FOV when flat field correction is applied this is normal D 4 Cosmic Ray Corrections Cosmic rays are extraterrestrial high energy particles that register a false signal on a CCD detector Cosmic rays as well as other sources of ionizing radiation cause infrequent interactions a few per minute on the CCD These interactions result in large signals that are usually isolated to a single pixel making them easy to correct The Living Image software searches for isolated high amplitude hot pixels and replaces them with a collective average of surrounding pixels The Cosmic Correction option should always be selected for in vivo image data because hot pixels can significantly affect an ROI measurement Cosmic ray correction is not recommended when imaging very small objects such as individual cells An individual cell may only light up one or two pixels and can sometimes be misinterpreted as a cosmic ray In this case clear the Cosmic Correction option in the Corrections Filtering tools to avoid filtering out single cell images Y Caliper Appendix E Luminescent Background Sources amp Correction
216. es eee eee ee ee es 171 11 4 Spectral Unmixing Parameters 2 2 173 11 5 Spectral Unmixing Options 2 2 sesse torraste trta ar teanas ia 174 Tr Y Caliper Contents iii 12 Reconstructing a 3D Surface 0 0 002 2 eee ee es 177 12 1 Generaune d Suae 2voees4 eue Geeta ede sdsta neeeve ee Rew ey eae eh ws 178 12 2 Managing SUuraces s s seasea be GH ede Oe wee REO ESS ERE HHS BGS ES 182 12 3 Export or Import a Surface 2 aa 183 13 3D Quantification Database 2 2 a es 185 13 1 Preparing amp Imaging the Samples aoaaa 185 13 2 Creating a Quantification Database 2 186 13 3 Managing Quantification Results a aoa a aa 189 14 3D Reconstruction of Sources a aaao es 191 14 1 Reconstructing Luminescent Sources oaoa 193 14 2 Reconstructing Fluorescent Sources ooo 201 14 3 3D Reconstruction Results a aoaaa 204 14 4 Checking the Reconstruction Quality a a a aa 207 14 5 Meas u ino SOUFCES s s s saca ta ae hah RR ee MRE Pee eee ee ee 209 14 6 Displaying Luminescent and Fluorescent Sources on One Surface 213 14 7 Comparing Luminescent and or Fluorescent Sources 2 2 2 2000 eee eee 213 14 8 Exporting a 3D Scene as DICOM 2 2 aa 217 Pe SD Tool OVCIVIOW 4 e oo 6 ek BOS eee GAS Gee HAS HE eS eS 220 14 10 3D Tools Surface 2 2 2 ee 220 l4ir SD Toob SONCE e wu aa eee ae Re eee ee ote Ree eee eee Gee ee BY 222 14 12 3D Tools Registratio
217. esult Results saved with the sequence selected in the upper box Select results to display from this box k Load V Display Voxels B i E Reverse F Log Scale V MIP V Display Surface Opacity i 50 fy Display Photon Density Map 214 Living Image Software User s Manual NOTE After the Longitudinal Study window is open more sequences can be added to the Longitudinal Study window by clicking the Open button amp and selecting sequenceinfo txt files found in the sequence data folder 2 To show particular results a Select a sequence in the upper box b Select one or more analysis results in the lower box To choose multiple adjacent results press and hold the Shift key while you click the first and last result To choose non adjacent results press and hold the Ctrl key while you click the results c Click Load 3 To show more results repeat step 2 4 To remove results from the Longitudinal Study window right click a surface and select Remove on the shortcut menu Alternatively select a surface click the Remove button and choose Selected Result To remove all results click the Remove button and choose All Results 5 To view a particular image in a sequence a Click the surface b For DLIT results make a selection from the Wavelength drop down list For FLIT results make a selection from the Image drop down list Figure 14 23 DLIT and FLIT results in the Lo
218. eters and any image label information entered at acquisition Figure 7 8 Figure 7 8 Image window displaying image information Click Info to display the image label and acquisition information D fie Edt View Toots Acquistion Window Heip aie amp AS R mins Acoly to of cone cain My um Bara ox _ Image Adjust gt Corrections Filtering Image informatie ROL Tools Planer Spectral lmegng Surlace Topography gt OLIT 20 Reconstruction JD Multi Modatty Toots Image label Another way to view information about images is available in the View menu 1 Open an image or sequence 2 Select View Image Information on the menu bar The Image Information window appears 3 Choose an image by making a selection from the Sequences drop down list and the Images drop down list Figure 7 9 94 Living Image Software User s Manual Figure 7 9 Viewing image information Drop down list of open sequences Drop down list of images in the selected sequence Choose Individual Images from the Or a list of single images if Individual Images is list to show the open single images in selected in the Sequences drop down list the Images drop down list Image Information na Sequences TLT20050624145507_SEQ Images TLT20050624145507_001 F Show All Sections Section Label luminescent image luminescent TIF hot hi
219. f View Horizontal Profile KAES ESSE NEEEEM ioue A e S T photons mm a Wleasured a Situated a Weasured Simulated ae 40 Photon density photons run 3 5 3 0 ah 2 0 1 5 1 0 0 5 0 0 40 20 Position irnn Position 4 To view the photon density profile at another location on the animal surface drag the cross hairs or click a point on the photon density map Table 14 6 Photon Density Maps window Item Image sources Description A list of images used in the reconstruction Select all images or a particular image number to display Angle of View The thumb wheel position Turn the thumb wheel to rotate the surface on the vertical axis Log Scale Choose this option to display the photon density using a log scale Simulated The photon density computed from DLIT or FLIT source solutions which best fit the measured photon density Measured The photon density determined from the image measurements of surface radiance Horizontal Profile The photon density line profile at the horizontal plane through the subject at the crosshairs location Vertical Profile The photon density line profile at the vertical plane through the subject at the crosshairs location Position mm Horizontal Profile The y axis position of the crosshairs horizontal line Vertical Profile The x axis position of the crosshairs vertical line feSciences i See Y Caliper Chap
220. f images that are grouped together in a single folder Figure 2 3 Table 2 1 IVIS Imaging Systems amp imaging modes Imaging Mode IVIS Imaging System Lumina Il Lumina XR 100 Series 200 Series Spectrum Kinetic A Photograph V V V V V V Luminescent v V V V V V m Fluorescent V V V V V V fe Structure as X ray V Kinetics v NOTE For details on your IVIS Imaging System please see the imaging system hardware manual Figure 2 3 Example image sequence overlay images luminescent image on photograph Double click an image in the Sequence View to open it in a separate window Ss Em a aay Se ee E Tries 20 sla s D Triem m Lint Counts Deciay etay EL Sequence View Spectra Lire Counts X Line faced Coker Mira cT Pa ort 8 Living Image Software User s Manual Table 2 2 Image types optical data Imaging Mode Description Example F Photograph A short exposure of the subject illuminated by the lights located in the ceiling of the imaging chamber The photographic image is displayed as a grayscale image Luminescent A longer exposure of the subject taken in darkness to capture low level luminescence emission The luminescent image is displayed in oseudocolor that represents intensity For more details on luminescent image data see Appendix D page 279 Luminescent image Overlay Luminescent
221. f the window and release Figure B 3 Main application window D Fle Edt View Tools Acquistion Window Help 2 x sIn ad Ag R utian ej Aooty to at T Sequence View Samta Too Palette ax Image Adjust ROI Toots o ii Q Measure ROts x i Type Measurement RO x Save RULS Neme Urvts Counts Use Saved Colors Ato ROL Peremeters g Type Description 3 Information Living Image 4 2 Mae 7 2011 111622 Version420 12 3 Information MIS configuration file fownd and loaded Information gt gt KSA Logged IN lt Aghi Wed X ES 268 Living Image Software User s Manual B 2 User Preferences Figure B 4 User preferences cE Preferences Optical Properties User Setting s Preferences Defaults Label Set Living Image Universal Edit Label Choices Table B 2 User preferences Item Description User s Settings Existing User ID The user ID displayed in the log on dialog box at startup New User ID Opens the Add New User box A new user is added to the Existing User ID drop down list Delete User ID Deletes the user selected from the Existing User ID drop down list Preferences Defaults Edit User label Choices Opens a dialog box that enables you to edit the Living Image Universal label set Default Units Choose counts or radiance photons for image display Y Caliper B 3 Acquisition Appendix B Preferences 269 Figu
222. fines how much of each color to apply in order to create the final pixel color Color photographs or color screenshots are examples of RGB images An RBG image is also a two dimensional array of numbers but unlike a scientific image the numbers are only color codes and are not related to light intensity A graphic image can be exported to a graphic display application Pseudocolor Images An image can be generated from scientific image data by assigning a color to each numerical value and plotting the array so that each pixel is filled with the color that corresponds to its numerical value A color table defines the relationship between the numerical data and the displayed color For example a grayscale color table assigns black to the smallest number in the array white to the largest number and shades of gray to the values in between Figure D 1 The resulting image is equivalent to a black and white photograph An illuminated photographic image acquired on an IVIS Imaging System is an example of a grayscale pseudoimage The reverse rainbow color table is also commonly used and assigns violet to the smallest number on the array red to the largest number and all of the spectral colors of the rainbow to the values in between Figure D 1 280 Living Image Software User s Manual Overlays Figure D 1 Example pseudoimages EF reranssoszaness07_005 sley trsta Counts Dagar Ponga Ooo 7 3 info Mrtoy ect Color t
223. functions that can be used to generate the new image including A B k A B k A B k A B if Counts B gt k Useful for fluorescence tomography A B k k Image Math window A user specified scaling factor applied in the results function Compute k from ROI This option is useful for subtracting fluorescence background Draw one ROI in an image on an area considered background In the Compute k from ROI drop down list select the this ROI with Photo from Choose this option to display the new image in overlay mode using the selected photographic image This option is only available if one of the selected images is an overlay Display Result for Opens the image generated by image math in an image window Measuring 9 2 Subtracting Tissue Autofluorescence To remove tissue autofluorescence from image data the IVIS Imaging System implements a subtraction method using blue shifted background filters that emit light at a shorter wavelength The objective of using a background filter is to excite the tissue autofluorescence without exciting the fluorophore To reduce autofluorescence signal in the primary image data use the image math tool to subtract the background filter image from the primary excitation filter image For more details on tissue autofluorescence see Appendix F page 305 The software computes the signal corrected for background A B x k where A primary image acquired using the ex
224. g Binning 2 smoothing 5x5 YY Caliper per Chapter 7 Working With Optical Image Data 101 7 7 Viewing Intensity Data amp Making Measurements The Image Information tools enable you to view intensity data and measure distance on an image You can view pixel data in different formats Image Information Description See Page x y coordinates and The x y pixel coordinates of the mouse pointer location in the 102 associated intensity image and the Intensity counts or photons at that location Histogram Histogram of pixel intensities in an image 103 Line profile Plots a line graph of intensity data at each pixel along a user 104 specified horizontal or vertical line in the image Figure 7 17 Tool Palette Image Information tools Tool Palette A gt Image Aadiust dS Corrections Filtering lan E eS an a E H Units cm r Image Binning amp Width 12 6 cem Height 12 6 cm Image X Y 6 843 2 047 on Image Data coum s Table 7 5 Tool palette Image Information tools Tool Description Click this button to display a histogram of pixel intensity For more details see page 103 Click this button to display a line profile For more details see page 104 Click this button to display a 3D representation of signal itensity For mre details see page 106 Click this button to display the distance measurement cursor in the image ca window For more details see page
225. g Image Software User s Manual Figure 14 17 Select and measure source voxels in the 3D View window ifthe surface includes voxels pasted from other Click the Measure Source button results select a source from the drop down list then draw a box around the source 4 o m Opacity _ 100 H Display Voxels V Maximum Intensity Projection 2 8510 photons sec 10 29 mm 3 3 4831 mm Center of Mass 1 5 10 3 19 7 Host Organ Unknown Click Center of Mass to obtain the measured source information Measure Source Depth 1 Select the source by drawing a box around it see Figure 14 17 2 Click the button The distance from the center of mass to the surface is measured in the three planes Figure 14 18 e Coronal and transaxial planes display the shortest distance from the center of mass to the surface e The sagittal plane displays the distance from the center of mass to the bottom of the subject 3 To display slice planes through the center of mass click the afr button For more details on planes see page 212 YF Caliper Chapter 141 3D Reconstruction of Sources 211 Figure 14 18 Slice planes This example shows slice planes through a selected source center of mass and distance measurements from the source center of mass to the surface k DM sr 7 Viewing Coordinates 1 In the Coronal Sagittal or Transaxial windowpane click a location
226. ge 279 Data Preview window 198 200 detection efficiency 275 detection sensitivity adjusting the lens aperture 275 exposure time 276 field of view 276 DICOM file size limit 77 DICOM Viewer 219 258 diffusion model 310 display units counts 281 efficiency 283 photons 282 DLIT sequence requirements 193 DLIT results 204 205 manage 205 DLIT FLIT troubleshooting 237 drift correction 285 E edit image label 94 edit image sequence 116 117 efficiency 283 293 298 299 electronic background dark charge 286 dark current 285 drift 285 read bias 285 EM gain 72 export image data 47 surface 183 export image data 47 export images 114 116 exposure time 276 F f stop 275 276 fluorescent imaging 297 fiducial registration 245 247 249 field of view 276 filter bandpass 294 fluorescent 296 filter spectra 294 flat field correction 100 flat fielding 284 FLIT sequence requirements 201 FLIT results 204 205 manage 205 fluorescence adaptive background subtraction 305 reconstruct 3D sources 201 203 fluorescence efficiency 298 299 fluorescence imaging components 291 294 fluorescent filters 296 fluorescent imaging efficiency 293 f stop 297 normalization 293 single image epi illumination 25 28 focus manually 264 G gradient illumination 255 graphic image data 279 H help 20 High Reflectance Hemisphere 287 high resolution imaging kinetic imaging 74 75 luminescent or fluorescent imaging 45 46 X ra
227. ge sequence acquisition The DLIT algorithm currently assumes a flat luciferin kinetic profile Therefore to optimize the signal for DLIT 3D reconstruction carefully plan the start and finish of image acquisition and ration the exposure time at each emission filter so that the sequence is acquired during the flattest region of the luciferin kinetic profile Image Sequence Requirements Use the Imaging Wizard to setup the image sequence required for DLIT analysis For more details on the Imaging Wizard see page 33 If you plan to manually set up the sequence Table 14 2 shows the recommended image sequence Analyzing more images usually produces more accurate results At a minimum the sequence must include data from at least two different emission filters 560 660 nm e Emission filter 1 Photographic luminescent e Emission filter 2 Luminescent image e One structured light image Table 14 2 Recommended image sequence for DLIT analysis Image Type Emission Filter Options 560 580 600 620 640 660 Photograph J Select the Reuse option in the control panel Structured light J Luminescent A J J J J J It is recommended that the binning level be the same for all of the luminescent images For more information on the DLIT algorithm and user modifiable parameters see Appendix H page 317 194 Living Image Software User s Manual Steps to Reconstruct Luminescent Sources Using DLIT 1 Loa
228. ge will be included in the data set of the current image replacing any existing background image that may be present In the data set Acquisition Fluorescent Background View Available Fluorescent Background Opens a dialog box that displays the fluorescent background measurements for the system If a fluorescent background is selected the Fluorescent Background Subtraction option appears in the Corrections Filtering tool palette Choose the Fluorescent Background Subtraction option to subtract the user specified background measurement from the image data Acquisition Fluorescent Background Clear Available Fluorescent Background Opens a dialog box that enables you to remove the fluorescent background measurements from the system Acquisition gt Auto Save If Auto Save is selected all images are automatically saved to a user selected folder Acquisition gt Auto Save To Opens a dialog box that enables you to select a folder where images will be saved to automatically Window gt Close Closes the active image window Window gt Close All Closes all image windows Window gt Cascade Organizes the open image windows in a cascade arrangement see page 88 Window gt Tile Organizes the open image windows in a tiled arrangement see page 88 Window 1 lt Image or Sequence name gt Window 2 lt Image or Sequence name gt Window g
229. ges for export to DICOM format includes photographs intensity signal and read bias or to a movie 1 In the image window click the 34 button Figure 6 9 page 80 2 If you want to select a particular range of data for export use the frame range selection to select the data Use the left slider to select the start image and the right slider to select the end image in the data range of interest The top slider automatically moves to denote the location of the current image with respect to the selected data range 3 To export the selected data to a movie a Click Extract and choose Save as a Movie b In the dialog box that appears select a folder enter a name for the movie and choose the file format for example mpg4 82 Living Image Software User s Manual Exporting an Image from a Kinetic Data Set 1 To select an image move the frame slider or enter a frame number in the spin box Figure 6 11 Image window selecting an image for export Units Radiance Photc Display Overlay Options Y 7 Info wj Export Graphics button Epi fluorescence Radiance ipisecicm isr Color Scale Min 4 51e8 Max 4 6969 Spin box Frame slider 2 Click Extract and choose Extract Current Image A new image window appears and displays the selected image 3 To save a snapshot of the current image click the Export Graphics button in the image window In the dialog box that appear
230. ges in the browser Note The Load as Group option is only available when two or more images non kinetic are selected in the browser Load Opens the selected image or image sequence Remove Removes a user selected image sequence s from the browser Close Closes the Living Image Browser Opening Data from the Menu or Toolbar NOTE To open a recently viewed file select File gt Recent Files on the menu bar 1 Click the Open button amp on the toolbar Alternatively select File gt Open on the menu bar 2 In the box that appears choose a file type filter from the drop down list Figure 7 4 Figure 7 4 Opening data from the toolbar or menu bar File Edit View Window Help Organize v WY Favorites E Desktop T Libraries a Music E Pictures E Videos j Computer we H a R R GOTE mee o eee a OC 5 7 i Downloads Documents a Homegroup Bre Libraries Open a library to see your files and arrange them by folder date and othe Documents amp ly Library Music a Library N Pictures A Library Videos Library amp Local Disk C E DVD RW Drive D Audio CD me o File name FE a ee ee TIFF Image Files tif tiff All Files Select the file type s 88 Living Image Software User s Manual Living Image files Click txt an image Living Image file format Sequence txt an image seque
231. gt _SEQ on the menu bar The overview appears YY Caliper per Chapter 7 Working With Optical Image Data 109 Figure 7 24 Transillumination overview D cedits seo ae Lit ae Beker a Se S z Fink uy m a D Qvervane_ CHODINDE IRE EDOSD 001 er cae ry 3 i Spee tees Linin Aadant Fick ingi Check gd Piscrescens 0 P iona F Trensiumrabeoiocbes Coton 7 5 d 110 Living Image Software User s Manual 7 9 Overlaying Multiple Images The image overlay tool provides a convenient way to view multiple reporters in one image You can use the image overlay tool to display multiple luminescence or fluorescence images on one photographic image To coregister multiple images 1 Acquire an image sequence using the appropriate filters for each reporter Alternatively create a sequence from images acquired during different sessions For more details see page 117 2 Load the image sequence Figure 7 25 Image sequence TF anwaosoaren2e002 s0 C Sequence view Lines Pliers EP fackericy Lise Saved Colors 3 Open one of the images and optimize the image display using the color scale Min and Max sliders in the Image Adjust tools To view all images in the sequence click the Display All button to open each image overlay mode in a separate image window 4 Select Tools Image Overlay for lt sequence name gt _SEQ on the menu bar
232. gth range of 450 850 nm Both epHllumination iluminationfrom above and trans illumination from below modes are available __concet __new__ s min Apply to All x Remover Al Update Insert Adc Imaging Wizard If this screen does not appear when the wizard Starts click Restart Wizard on the wizard screen to restart the wizard Step through the rest of the wizard In the wizard choose bioluminescence or fluorescence imaging Figure 5 7 In the next wizard screen choose the type of image sequence that you want to acquire To include an X ray image or photograph in the sequence put a check mark next to the X ray or Photograph option when you set the imaging parameters in the wizard Figure 5 8 56 Living Image Software User s Manual Figure 5 8 Imaging Wizard imaging parameters Imaging Subject Mouse x Exposure Parameters Auto Settings Luminescent Fq v Photograph BAT X Ray Field of View Cc 10cm X Focus Subject Height 1 50 2 cm Options Time Series Study C Imaging Wizard Bioluminescence Open Filter Manual Settings Focus use subject height x Restart Wizard Cancel Bak Next Table 5 3 Imaging Wizard Item Imaging Subject Description Choose the type of subject from this drop down list Exposure Parameters The Auto Settings exposure option is the default To manually
233. h Mode In batch mode you can set up multiple separate sequences which will be automatically acquired one after another without manual intervention To setup and acquire sequences in batch mode 1 In the Control Panel click Sequence Setup 2 Choose the Batch Sequences option Imaging Mode IVIS Acquisition Control Panel Exposure Time Binning F Stop 100 E eec gt CD CD Co CS Field of View E Mouse Imaging Shuttle Service 13 4 cm Figure 4 21 Control Panel GEI Excitation Filter Emission Filter Acquire Subject height 1 50 suicition Coni Ip Sam seg Exposure Time inni F Stop Field of View E Mouse Imaging Shuttle Excitation Filter Emission Filter o0 E eec v meam ea ea ono o Medum vls viy System Status cm Subject height 1 50 a Focus use subjectheight v Temperature M Batch Sequences option Number of Segments 1 amp 5 Delay 0 0 min XX Remover 4 Update Insert Gada 40 Living Image Software User s Manual 3 To set up the first sequence do either of the following e Click Imaging Wizard and step through the wizard for more details on the Imaging Wizard see page 33 OR e Set up the sequence manually for more details see page 41 4 To set up the next sequence e If using the Imaging Wizard repeat step 3 Each sequence is dislayed in a separ
234. h adaptive software correction works as effectively as the traditional method except the following cases e The subject is dark making it is difficult to mask the subject using the photo for example experiments that use black well plates e The subject occupies most of the FOV for example high magnification or multiple mice in the FOV As a result there is not enough information outside the subject that can be used to help fit the background F 8 Subtracting Tissue Autofluorescence Using Background Filters High levels of tissue autofluorescence can limit the sensitivity of detection of exogenous fluorophores particularly in the visible wavelength range from 400 to 700 nm Even in the near infrared range there is still a low level of autofluorescence Therefore it is desirable to be able to subtract the tissue autofluorescence from a fluorescent measurement The VIS Imaging Systems implement a subtraction method based on the use of blue shifted background filters that emit light at a shorter wavelength The objective of the background filters is to excite the tissue autofluorescence without exciting the 306 Living Image Software User s Manual fluorophore The background filter image is subtracted from the primary excitation filter image using the Image Math tool and the appropriate scale factor thus reducing the autofluorescence signal in the primary image data For more details see Chapter 9 page 153 The assumption here is
235. hat must be determined for quantitative image analysis e Dark current Electronic background generated by the thermal production of charge in the CCD To minimize dark current the CCD is cooled during use Read Bias amp Drift Prior to a luminescent image exposure the Living Image software initiates a series of zero time exposures image readout to determine a read bias measurement If a dark charge background is available for the luminescent image the average bias offset for the read bias image stored with the dark charge measurement is compared to the average bias offset determined with the read bias measurement made prior to the image The difference or drift correction 1s stored with the luminescent image data and is later used to correct minor drift typically less than two counts pixel that may occur in the bias offset since measuring the dark charge background If a dark charge background is not available at the time of the luminescent image exposure the software checks to see if the selected image parameters warrant a dark charge measurement large binning and long exposure time If a dark charge image is not required the read bias will be used If a dark charge is recommended the software provides the option of using the read bias measurement instead Since the read bias is by far the largest component of background using a read bias measurement instead of a dark charge measurement is often acceptable If read bias is used
236. hboring metastasis Figure G 3 After the ROI is defined start the planar spectral analysis for more details see page 157 The software e Measures the total flux inside the ROI on each filtered image e Normalizes the data to the luciferase spectrum Plot of Intensity vs Lambda Figure G 4 e Fits the normalized data to the analytical expression in Equation 1 page 310 where S absolute total photon flux emitted by the luminescence source and d source depth Plot of Linear Fit Results Figure G 4 Y Ca i esmi Appendix G I Planar Spectral Imaging 313 Figure G 3 Metastatic site femoral bone of the animal ROI includes the signal of the right kidney and separates it from other metastatic sites The signal coming from the lower back area is spread out due to the presence of two bright spots The dimmer signal in the lower bottom right of the image likely originates from the jw CK20031215150449 008 Units Photons vi Display Overlay Sew Image Min 1 26e3 Max 9 76e5 ROI 1 3 091e 06 Jae 10 p sec em 2 sr Color Bar Min 6 97e4 Max 6 44e5 aE Figure G 4 Planar spectral analysis results Fie Edit view Tools Window Help a gA H R amp R Units Photons C Apply to all gt Corrections Filtering gt Image Information gt ROI Tools 7 Planar Spectral Imaging Analyze Properties Results Spectral Results ee ROI Depth mm
237. he Batch Sequences option Figure 5 14 only the sequence in the active tab will be acquired feSciences Y a Iper Chapter 5 X Ray Imaging 63 5 4 High Resolution Imaging With the Optical Zoom Lens Attachment The optional Optical Zoom Lens attachment enables close up and high resolution imaging on the IVIS Lumina VIS Lumina XR and IVIS Kinetic Imaging Systems When the Zoom lens attachment is installed only the Z field of view setting 2 6 cm is available for single image or sequence acquisition The imaging system is set to the Z field of view position until the Zoom lens attachment is removed If the Zoom lens attachment is installed when the Living Image software is closed the stage will move to the Z position when the system is initialized NOTE When installing or removing the Optical Zoom Lens attachment avoid touching the optical glass Installing the Zoom Lens Attachment 1 Choose the Zoom option in the control panel Figure 5 16 You are prompted to insert the Zoom Lens attachment Figure 5 16 Control panel Zoom option selected Imaging Mode Sequence Setup Carefully insert the zoom lens in its position in the top center of the imaging chamber Close the door and click OK when done 2 After you install the Zoom Lens attachment in the imaging chamber click OK in the prompt The stage moves to the Z field of view position 64 Living Image Software User s Manual
238. he Imaging Wizard see page 33 If you plan to manually set up the sequence Figure 14 9 shows the an example image sequence Acquire the images using transillumination on the VIS Spectrum Imaging System using the same excitation and emission filters from at least four source locations that form a rectangle Figure 14 9 Example sequence setup for FLIT Imaging Wizard L Display Photographic Settings Subject Mone v Mode Exposure Binning FStop Excitation Emission Lamp Lewel Structure FOV Height Transilumination De Acquire a fluorescent image Ema Medium 2 640 700 High Yes c 1 50 15x23 2 13 structured light image and 2 Auta Medium 2 640 700 High No a 1 50 15x23 2 12 photograph at the first 3 i Auto Medium 2 640 700 High No E 1 50 15x23 2 12 transillumination location 4 Auto Medium 2 640 700 High Mo C 1 50 15x23 2 11 is E Auto Medium 2 640 700 High No C 1 50 15x23 2 11 Acquire a fluorescent image e i Auto Medium 2 640 700 High No a 1 50 15x23 2 10 at the remaining 7 E Auto Medium 2 640 700 High No C 1 50 15x23 2 10 aie e B Auta Medium 2 640 700 High No a 1 50 15x23 2 9 2 transillumination locations 3 i Number of Segments F Delay 10 0 min Apply to All X Remove Update Insert Add Steps to Reconstruct Fluorescent Sources Load an image sequence 2 Generate or load a surface For details on generating the surface see page 181 In the
239. he autofluorescent signal of tissue also increases at wavelengths less than 600 nm Table F 1 Standard filter sets and fluorescent dyes and proteins used with IVIS Imaging Systems Name GFP DsRed Cy5 5 ICG GFP Background Excitation Passband Emission Passband Dyes amp Passband nm nm 445 490 515 575 GFP EGFP FITC 500 550 575 650 DsRed2 1 PKH26 CellTracker Orange 615 665w 695 770 Cy5 5 Alexa Fluor 660 Alexa Fluor 680 710 760 810 875 Indocyanine green ICG 410 440 Uses same as GFP GFP EGFP FITC DsRed Background 460 490 Uses same as DsRed DsRed2 1 PKH26 CellTracker Orange YY Caliper per Chapter F Fluorescent Imaging 297 Table F 1 Standard filter sets and fluorescent dyes and proteins used with IVIS Imaging Systems Nama Excitation Passband Emission Passband Dyes amp Passband nm nm Cy5 5 Background 580 610 Uses same as Cy5 5 Cy5 5 Alexa Fluor 660 Alexa Fluor 680 ICG Background 665 695 Uses same as ICG Indocyanine green ICG F 3 Working with Fluorescent Samples There are a number of issues to consider when working with fluorescent samples including the position of the subject on the stage leakage and autofluorescence background signals and appropriate signal levels and f stop settings Tissue Optics Effects In in vivo fluorescence imaging the excitation light must be delivered to the fluorophore inside the animal for the fluorescent process to begin Once the excitatio
240. he line over the image The blue part of the line indicates the pixel intensities that are plotted in the line profile graph Y Ca i a Chapter 7 Working With Optical Image Data 105 The line profile is updated as you move the line move over the image Table 7 7 Line Profile window ltem Line Orientation Description Choose Vertical Horizontal or Free Hand from the drop down list to set the orientation of the line in the image window The Free Hand orientation enables you to drag each line segment endpoint to a user selected position Width Sets the line width Position Line position pixels Enables you to choose the grid line pattern to display in the line profile window Exports the line profile data to a csv or txt file Copies the line profile graph to the system clipboard Opens the Print dialog box a Displays the minimum and maximum value of the x axis Use the arrows to change the x axis min or max If photons is selected in the image window the x axis units pixels If counts is selected in the image window the x axis units cm To display the range available for the Min or Max place the mouse pointer over the Min or Max edit box Y Min Y Max a Displays the minimum and maximum value of the y axis Use the arrows to change the y axis min or max To display the range available for the Y Min or Y Max place the mouse pointer over the Min or M
241. he shortcut menu All of the results are copied to the system clipboard To export results 1 In the results tab right click the results table and select Export Results from the shortcut menu that appears 2 In the dialog box that appears choose a folder for the results enter a file name and click Save The exported results are saved in csv file format To delete results 1 In the Results tab make a selection from the Name drop down list 2 Click Delete The results are deleted from the system Y Caliper Chapter 14 3D Reconstruction of Sources 207 14 4 Checking the Reconstruction Quality To check the quality of a 3D reconstruction it is useful to compare the measured and simulated photon density plots The photon density is closely related to the measured radiance Photon density is the steady state measure of the number of photons in a cubic millimeter Light sources inside the tissue contribute to photon density in other portions of the tissue The reconstruction algorithms first convert the luminescent image of surface radiance to photon density just below the animal surface because this is what can be observed Then the algorithm solves for point source locations inside the tissue which would produce the observed photon density near the surface To view photon density maps 1 After the reconstruction is finished or results are loaded click Photon Density Maps in the Results tab The photon density maps
242. his option to apply the selected ROI to all images in a sequence 124 Living Image Software User s Manual Table 8 2 ROI tools continued Item Description Type Choose the ROI type from the drop down list Measurement Measures the signal intensity in an area of an image Average Bkg Measures the average signal intensity in a user specified area of the image that is considered background Subject ROI Identifies a subject animal in an image The software automatically associates a measurement and an average bkg ROI that are included in the same Subject ROI Using this type of ROI is optional Save ROIs Name Delete Load Save Creates a file that includes the ROI parameters for example the X Y coordinates type of ROI color shape width height ROIs that have been saved to file can be recalled and applied for another image at any time The name of the selected ROI set or the default name for a new ROI set Deletes the selected ROI set from the system Note This permanently removes the ROI from the system Applies the ROI set selected from the Name drop down list to the active image Saves the ROI set in the active image Note This is a global save the ROI is saved to the system and the ROI set can be loaded onto any image If you use the File Save commands to save an image that includes an ROI the ROI is saved with the image only not a global Save and is not available for
243. hosphoresce Materials should be screened so you can identify and eliminate problematic materials If a background source is phosphorescent and the phosphorescent lifetime is relatively short you can try keeping the sample in the dark for a long period before imaging to reduce background light emission Occasionally there is no way to eliminate the natural light emission of the sample The natural light emission associated with living animals autoluminescence is a major area of interest in in vivo luminescent imaging Most animals exhibit a low level of autoluminescence Usually this is only a problem when looking for very low signals at the highest levels of sensitivity Caliper has conducted tests to try to minimize the source of the background light emission in mice Test Description Observation Test 1 Subject animals were housed in Background emission levels were not reduced A the dark 12 hours prior to imaging phosphorescent component in mouse fur or skin is not the source of light emission Test 2 White furred animals were No increase or decrease in background emission levels shaved prior to imaging Test 3 Alfalfa known to be An altfalfa free diet reduced background emission phosphorescent was eliminated from slightly but not significantly the animal diet The sources of autoluminescence are not yet fully understood No external sources have been proven to cause natural light emissions so it is possible that a ch
244. ick Yes in the prompt and choose a folder in the dialog box that appears If autosave is enabled all images acquired during the session are automatically saved to a user selected folder You can choose a different folder at any time select Acquisition Auto Save on the menu bar Image acquisition proceeds During acquisition the control panel Acquire button becomes a Stop button To cancel the acquisition click Stop in the control panel The image window appears when acquisition is completed Figure 4 8 28 Living Image Software User s Manual Figure 4 8 Overlay fluorescent image on photograph in the image window For more details on the items in the image window see Table 4 1 page 24 D Fin Eda Views Too Acquvtion Wireiow Heip SAH tt AG R unsestecticeny O apo Units Redert Ciicerncy Cisplay Overlay s Info Image CK20100113162242 Mod Jun 13 2010 16 23 10 Group evel High Em 720 Ex 675 Epiiamination Image label Experiment MB FOV 12 9 12 3 Camera SO70QN4088 Andor ikon eee Commert 13013 molecules A620 dye Commert epi vectra Lor fluorescence 75 Check the image min and max in the color scale to determine whether the signal of interest is above the noise level and below CCD saturation 4 3 Fluorescent Optical Imaging With Transillumination This section explains how to acquire a fluorescent optical image with transillumination Transillumination provides a
245. iew Enables you to view a different area of a magnified image To view another area of the image choose this option then click and hold the pointer while you move the mouse over the image Crop Area To crop the image draw a rectangle over the area of interest in the image then right click the area in the box and select Crop Area on the shortcut menu Draw Grid Displays a grid over the frame Display Color Bar Choose this option to display the color scale Display Color Min Max Choose this option to display the color scale minimum and maximum Display Image Min Max Choose this option to display the minimum and maximum signal Y Ca i A i 6 4 Saving Kinetic Data Chapter 6 I Kinetic Imaging 77 The IVIS Kinetic instrument enables you to acquire a real time data stream which can generate very large files The file size limit for DICOM data is 2GB Kinetic data acquisition automatically stops when this file size limit is reached Table 6 3 shows how binning conditions affect the total number of frames that can be collected in overlay or luminescent fluorescent only mode Table 6 3 Frames collected per 1 GB DICOM file Luminescent or Binning Level Frame Size Overlay Mode Fluorescent Only DICOM File Size Total Frames Collected Bin 1 2 MB 250 500 Bin 2 512 KB 975 1950 1 GB Bin 4 128 KB 3900 7800 Bin 8 32 KB 15600 31250 Bin 16 8 KB 62500 125000 To save data 1 In th
246. igure 2 2 Steps to acquire an image sequence Start the Living Image software and initialize the IVIS Imaging System IVIS Acquisition Control Panel m A IVIS Acquisition Control Panel Calz Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter Eyes aS CHT CS Co CS Leet mi ppi for grey bount oF meam e crtreet nnn a en h bee fea ced or LaTe bates flsemcarce Pid fees map Click Imaging Wizard in the control panel Inthe Imaging Wizard double click Bioluminescence EJ or Fluorescence and step T through the wizard inthe control panel click Acquire Sequence ine i Lente age airtel faved Lebeey Lams Zl hack ap 0 i ar dipi 7 i i T Gm Enter image label information optional 2 Comet 7 nts The image window opens and displays the images as they are acquired The Tool Palette is displayed y7 Caliper por Chapter 2 Overview of Imaging amp Image Analysis 7 2 2 Imaging Modes Table 2 1 shows the imaging modes that are available on IVIS Imaging Systems Table 2 2 shows examples of the different types of images optical data You can acquire e Single images for example a luminescent image and a photograph After acquisition the Living Image software automatically coregisters images to generate an overlay image e An image sequence a collection o
247. igure 5 1 Example overlay fluorescence image on X ray image Units Radiant Efficiency Display _ Options z Info m Epi fluorescence 6 0 1 0 Radiant Efficiency yiicm Color Scale Min 9 56e8 Max 6 45e9 5 1 Acquire an X Ray Image This section explains how to acquire an X ray image on the Lumina XR Imaging System For information on including a luminescent or fluorescent image in the acquisition see Chapter 4 page 21 1 Start the Living Image software double click theFZ icon on the desktop 2 Initialize the Lumina XR Imaging System and confirm or wait for the CCD temperature to lock For more details see page 17 3 In the control panel put a check mark next to X Ray Figure 5 2 NOTE To enable X ray acquisition verify that the X ray enabling key on the front of the Lumina XR is set to ON and the orange X ray enable button has been depressed and is illuminated 50 Living Image Software User s Manual Figure 5 2 Lumina XR control panel id ACU ition Lo ntrol Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter Auto Medium jie Hion Res vla gt arma X ray acquisition settings Field of View C ha System Status X Rays will be produced rodu E xFov 24 E Zoom idle Acquire T Babecthaght L50 Ear Focus use subject height Temperature ees P Select the Auto expos
248. image Parallel laser lines are projected onto the subject to produce a structured light image Figure H 1 lf the Structure option is chosen in the Control panel a structured light image is automatically acquired 318 Living Image Software User s Manual The surface topography of the subject is determined by analyzing the displacement Ax or bending of the laser lines as they pass over the subject The displacement is defined as the difference between where the line should fall on the stage in the absence of the subject and where it appears in the image due to occlusion by the subject Figure H 1 Parallel laser lines projected onto a subject Given knowledge of the angle 9 the height of the subject h can be determined by analyzing the displacement Ax of the laser lines as they pass over the object AX k i The parallel lines are projected onto the surface of the subject at an angle 0 The angle is known by instrument calibrations of the distance between the structured light projector and the optical axis D and the distance between the stage and the structured light projector Figure H 2 Figure H 2 Structured light projector and subject SL projector A XEN ae A s l i il 4 p gt D and l form two perpendicular sides of a triangle giving tan D l Together Ax and h comprise a smaller version of this triangle The height A can be determined from h Ax tan
249. imately linear relationship between the source strength in each voxel S and the photon density or NTF Efficiency p at each surface element described by a Green s function G The photon density at the jt surface element is the sum of the contributions from all the voxels P 5G S 1 The Green s function contains information about the transport of photons through the tissue and the effects of the tissue air boundary By using a planar boundary approximation the Green s function can be calculated analytically as a solution to the diffusion equation Having an analytic expression for G allows Equation 1 to be computed very rapidly H 4 Determining the Best Approximate Solution to the Linear System Once the Green s functions Gi are known the goal is to solve Equation 1 for the source strength S in each voxel The DLIT and FLIT algorithms attempt to minimize Equation 2 while requiring that the source strength in each voxel is positive Equation 3 2 1 2 X gt Pj Lows 2 j J j S 20 3 A Non Negative Least Squares algorithm is used to find the approximate solution which minimizes 2 In order to reduce the number of variables in the problem the code only uses surface elements with signal above a certain threshold and only keeps the voxels that contribute significantly to these surface elements H 5 Source amp Tissue Properties DLIT analysis of spectrally filtered images requires knowledge of the spectral depe
250. ing Measurements To measure distance with the measurement cursor 1 Open an image and click the Distance Measurement Cursor button in the Image Information tools A measurement cursor 4_____s appears on the image Figure 7 22 The Tool Palette shows the position and length of the cursor Figure 7 22 Measurement cursor The Tool Palette displays the measurement cursor position and length Tool Palette an gt Image Adjust 5 Units Counts v Display Overlay 7 Options v Info itj i gt Corrections Fitering i Image Information w A Di TB units cm Y Image Binning 8 Width 12 6 cm Height 12 6 cm 3 Luminescence Image X Y 12 451 10 941 an 10000 Image Data 6 counts a ne 2000 6 90 7 28 1 76 0 00 Distance 1 76 am 6000 gt ROI Tools _ Planar Spectral Imaging gt Surface Topography gt DLIT 3D Reconstruction _ 3D Multi Modality Tools _ Spectral Unmixing 4000 2000 Counts Color Scale Min 586 Max 10348 2 To change the cursor position or size drag the A or B end of the cursor to a new location on the image The measurement information in the Tool Palette is updated 3 To hide the cursor click the pa button Table 7 9 Measurement cursor position amp length Item Description Jor Pixel x y coordinates of p
251. ing Shuttle ue Subjectheight 1 50 f cm 22 Living Image Software User s Manual 2 Puta check mark next to Photograph and select Auto exposure click the arrow 3 Make a selection from the Field of View drop down list For more details on the field of view see page 261 z Set the proper Focus by choosing one of the methods below The focal distance to the camera is set at stage z 0 for each field of view To focus at the top of the animal the stage will move down so that the top of the animal is at z 0 You can either enter the height of the animal using the use subject height option or let the software determine the animal height by choosing the scan mid range option e Select use subject height and use the e arrows or the keyboard arrows to specify a subject height cm e Select manual focus For more details on manual focusing see page 264 e Select scan mid range available on the IVIS 200 or Spectrum Imaging System only The imaging system determines the subject height using a laser scan This value is automatically entered in the subject height box and is used to set the focal length Nn To acquire an overlay image coregistered images for display after acquisition put a check mark next to Overlay NOTE To view the subject s inside the chamber before image acquisition take a photograph Uncheck the Luminescent option choose the Photograph and Auto options and click A
252. ing can be applied without loss of spatial resolution For example an image acquired at binning 4 and FOV 20 cm has the same spatial resolution as an image acquired at binning 8 and FOV 10 cm Due to the increase in binning the latter image has a four fold increase in sensitivity compared to the former A charge coupled device CCD is a photosensitive detector constructed in a two dimensional array of pixels After an image is acquired each pixel contains an electrical charge that is proportional to the amount of light that the pixel was exposed to The software measures the electrical charge of each CCD pixel and assigns a numerical value counts For more details on counts and other measurement units see Appendix D page 279 The resulting image data comprise a two dimensional array of numbers each pixel contains the counts associated with the amount of light detected IVIS Imaging Systems are equipped with a CCD that ranges from 102451024 to 2048x2048 pixels in size and thus have a high degree of spatial resolution At binning 1 YY Caliper REF nce Chapter C Detection Sensitivity 277 each pixel is read and the image size number of pixels is equal to the physical number of CCD pixels Figure C 3 Figure C 3 A small segment of the CCD At binning 2 4 pixels are summed together at binning 4 16 pixels are summed Binning 1 Binning 2 Binning 4 CCD pixel Signal 4 times larger Signal 16 times larger Sp
253. instead of a dark charge background the read bias image is stored with the image data rather than the usual background information 286 Living Image Software User s Manual Dark Charge If the amount of dark charge associated with an image is negligible read bias subtraction is an adequate substitute for dark charge background subtraction Dark charge increases with exposure time and is more significant at higher levels of binning A good rule of thumb is that dark charge is negligible if TB2 lt 1000 where T is the exposure time seconds and B is the binning factor Under these conditions dark charge contributes less than 0 1 counts pixel and may be ignored Dark charge refers to all types of electronic background including dark current and read bias Dark charge is a function of the exposure time binning level and camera temperature A dark charge measurement should be taken within 48 hours of image acquisition and the system should remain stable between dark charge measurement and image acquisition If the power to the system or camera controller a component of some IVIS Imaging Systems has been cycled or if the camera temperature has changed a new dark charge measurement should be taken The dark charge is measured with the camera shutter closed and is usually performed automatically overnight by the Living Image software The software acquires a series of zero time exposures to determine the bias offset and read noise foll
254. ion to optimize settings so that the photographic image luminescent or fluorescent signal is not saturated To perform a test acquisition 1 Confirm that the Accumulate option is selected Do not select the Auto color scale option WwW N Start the acquisition click the Record button Record If the photographic image is saturated stop the acquisition click the button and reduce the photograph light level CAUTION Extended acquisition of saturated images can shorten the life of the EMCCD and should be avoided 4 Restart the acquisition If necessary repeat step 3 and step 4 74 Living Image Software User s Manual 6 2 High Resolution Imaging With the Optical Zoom Lens Attachment The optional Optical Zoom Lens attachment enables close up and high resolution imaging on the IVIS Lumina VIS Lumina XR and IVIS Kinetic Imaging Systems When the Zoom lens attachment is installed only the Z field of view setting 2 6 cm is available for single image or sequence acquisition The imaging system is set to the Z field of view position until the Zoom lens attachment is removed If the Zoom lens attachment is installed when the Living Image software is closed the stage will move to the Z position when the system is initialized NOTE When installing or removing the Optical Zoom Lens attachment avoid touching the optical glass Installing the Zoom Lens Attachment 1 Choose the Zoom option in the cont
255. ions for the Image Layout window For Layout Style h example the 2x2 layout style provides 4 separate layout areas in the window A different image can be pasted into each layout area Anmalion oe To apply notes to an image enter text in the annotation box and press Enter aa Drag the text to the location of interest in the image PE Opens a dialog box that enables you to select a font or edit the font style and SIZe E Opens a color palette that enables you to select a font color or specify a custom font color Opens a text editor that enables you to edit the selected text 7 12 Editing an Image Sequence You can add or remove individual images from a sequence Only individual images not an image sequence can be added to a sequence 1 Open the image sequence that you want to edit 2 If you plan to add images to the sequence browse for the images that you want to add in the Living Image browser For more details on browsing see page 83 NOTE If you plan to add images to the sequence browse for the images that you want to add in the Living Image Browser For more details on the browser see page 83 3 In the image window click the Edit button Figure 7 31 Opening the Edit Sequence dialog box Single images in the Living Image Browser that can be added to the sequence Oe Deer CEA oo z E T2005062414550 SEQ co x EP Edit Sequence Sequence View Spectra Sequenc
256. is Is typically used to remove natural animal background luminescence and should not be confused with the dark charge and read bias background corrections that are applied by default to the raw CCD data to remove electronic noise before any measurements For more details see page 130 Replace ROIs If this option is chosen all auto ROIs are replaced when new ROI s are created Restore Defaults Restores the factory set defaults for the auto ROI parameters Save Load Click to display or hide the tools that enable you to save load or delete auto ROIs in the active data Note The save function saves parameters the not actual ROIs This means that when you load saved auto ROI parameters the software draws a new ROI using the saved values Threshold Lower Limit Minimum Size Y Caliper Ber Chapter 8 ROI Tools 125 8 4 Measurement ROIs To obtain the intensity signal in a user specified area of an image draw a measurement ROI on the image This section explains in detail the three ways to draw measurement ROIs NOTE For a quick guide to drawing measurement ROIs see page 120 Drawing Description cP Method p Page Manual Places one or more ROls circular square or grid shape on the image 120 Automatic The software automatically locates and draws an ROI s on the image 127 To do this the software locates the peak pixel intensities in the image and searches the neighborhood around a
257. is The ROI should include as much of the light emission from a single source as possible Figure 10 3 Selecting ROIs for planar spectral image analysis a I 11120050624145507_SEQ Oo Sequence View m Spectra CoE Toot Potete F Use Saved Colors Units Counts z ROI 1 50 ROI 2 50 ROI 5 50 ROI 7 50 ROI 6 50 wa a Options 7 zno S gt Q ROI 3 50 Min 2140 Max 36446 ROI 9 50 o J ROT 10 50 sP Min 1122 Max 19546 Image Adjust gt ROI Tools Planar Spectral Imaging Analyze Properties Results Sequence 72720050624145507_SEQ Tissue Mouse Tissue Source Firefly Select Filters Filters Imageset lt lt No active ROI selected gt gt l i TLT20050624145507_001 TLT20050624145507_002 TLT20050624145507_003 TLT20050624145507_005 b b b TLT20050624145507_004 gt b TLT20050624145507_006 gt All ROI1 Y Caliper Chapter 10 Planar Spectral Image Analysis 159 5 Choose the tissue properties a In the Properties tab choose Mouse Tissue or XPM 2 XFM 2 mouse phantom from the Tissue Properties drop down list The software automatically sets the internal medium index of refraction based on the selection in the Tissue Properties list Figure 10 4 Planar spectral imaging tools Analyze tab Properties tab Results t
258. isible and near infrared part of the spectrum Figure C 1 shows detection efficiencies for several commonly used photon detectors The back illuminated CCD has the highest efficiency particularly in the 600 800 nm region of the spectrum the area of greatest interest for in vivo imaging Figure C 1 Quantum efficiencies Jom Back kma ted 30 E Front Whaniriated i amp maga Intensifier 80 n Oo 70 Bialkali photocathode A a Back illuminated CCD i oe Front illuminated CCD W Li gi E 4i IVIS systems use back illuminated CCDs 4 30 Y 200 40 600 BOG oo Wavelength nm IVIS Imaging Systems are equipped with a high light collection f 1 lens The sensitivity of the IVIS Imaging System can be adjusted by changing the f stop setting that controls the lens aperture The detected signal scales approximately as 1 f stop For maximum sensitivity select f 1 the largest aperture setting on the IVIS Imaging System Figure C 2 This provides the greatest light collection efficiency but results in the minimum depth of field for the image The depth of field refers to the depth over which the image appears to be in focus and is determined by the f stop and the field of view FOV At f 1 the depth of field ranges from 0 2 cm at FOV 3 9 cm IVIS Imaging System 200 Series only to 2 cm at FOV 25 cm You can use the manual focus option on the Control 276 Living Image Software User s Manual panel to easily
259. l To apply the new value to all of the cells in the same column click Apply to all 3 Click outside the cell to lose focus To edit a parameter in the control panel 1 In the sequence table select the row that you want to modify 2 In the control panel choose new parameter values and or imaging mode 3 Click in the sequence table Inserting Images in a Sequence Method 1 1 In the sequence table select the row next to where you want to insert the image 2 Set the imaging mode and parameters in the control panel 3 To insert the new image above the selected row click insert Method 2 Select the row s of interest and right click the sequence table to view a shortcut menu of edit commands Figure 4 27 Sequence table edit commands in the shortcut menu ela I E Display Photographic Settings Subject Mouse Probes E seqo 5eg 0 o Mode Exposure Binning FStop Excitation Emission Structure FOV Height 1 DEED Auto Medium 1 Block 1 50 Medium la Block 580 ae Auto Medium i Block C150 Copy rent Select All Auto Medium 1 Block 620 Mo C 1 50 Delete row Auto Medium 1 Block 640 No co 1 50 soe Replace Rowls Paste Rows E Number of Segments 1 gt Delay 0 0 min Apply to All Remover Update Insert Add Table 4 6 Sequence table shortcut menu edit commands Command Description Copy row s Copies the selected row s to the system
260. l or lens option that are installed Table A 1 IVIS acquisition control panel Item Description Luminescent Choose this option to acquire a luminescent image Fluorescent Choose this option to acquire a fluorescent image If the Fluorescent option is selected on the IVIS Spectrum Imaging System the following options also appear in the control panel Transillumination Choose this option to acquire a fluorescent image using transillumination excitation light located below the stage Normalized This option is selected by default when the Fluorescent and Transillumination options are chosen so that NTF Efficiency images can be produced 260 Living Image Software User s Manual Table A 1 IVIS acquisition control panel continued item Exposure time Description The length of time that the shutter is open during acquisition of an image The luminescent or fluorescent signal level is directly proportional to the exposure time The goal is to adjust the exposure time to produce a signal that is well above the noise gt 600 counts recommended but less than the CCD camera saturation of 60 000 counts Luminescent exposure time is measured in seconds or minutes The minimum calibrated exposure time is 0 5 seconds The exposure time for fluorescent images is limited to 60 seconds to prevent saturation of the CCD There is no limit on the maximum exposure time for luminescent images however there is little benefit
261. lanar Spectral Imaging gt Surface Topography gt DUT 3D Reconstruction 3D Multi Modality Tools Volume Slice Results m pD H H tD TAE gt as GO Share gt CaliperLS Caliper Data RAW v 4 Search RAV p Organize v Open v Burn New folder v oil di Caliper LS a Name 3 Date modified Type Size d Caliper Data J ALI20110120171831_SEQ di AutoFluorBkgCorr J BkgSub di CoReg_Demo J dem 20110306103654 DLIT Datasets dJi emfilters J 1V20110114162356_SEQ P Katherine Quantum Ji Kidney KSA20110305104918 SEQ di LognitudinalData a Lung J MAT20090302190433 X aneurism_8bit_256x3 raw Date modified 9 26 2001 4 42 AM Date created 2 1 2011 5 11 PM OOO RAW File Size 16 0 MB Display Volume _ aneurism_8bit_256x3 raw 9 26 2001 4 42 AM RAW File 16 384 KB Level Of Detail Color Opacity Map 1 0 Opacity aw i Gi Reverse _ Spectral Unmixing 244 Living Image Software User s Manual 2 In the Volume Information dialog box that appears Figure 15 6 enter the e Data width height and the number of slices e Slice row column pixel size and the slice spacing in millimeters Figure 15 6 Volume information I Volume Information mE j Volume Information File C Share aneurism_8bit_256x3 raw E Signed pixels Height 1 pi Select a data type
262. larly on the IVIS Imaging System 200 Series For example it is possible for a large subject 10 cm ventral dorsal height to contact the top of the imaging chamber if you set the subject height 0 and choose a small FOV Focus Drop down list of focusing methods available Use subject height Choose this option to set the focal plane at the specified subject height Manual Choose this option to open the Focus Image window so that you can manually adjust the stage position For more details on manual focusing see page 264 Batch Sequences Choose this option if you want to specify multiple separate image sequences for batch acquisition multiple image sequences are automatically acquired one after another without user intervention For more details see page 39 Temperature The temperature box color indicates the temperature and status of the system White box System not initialized r Red box System initialized but the CCD temperature is out of range ea Green box System is initialized and the CCD temperature is at or within acceptable range of the demand temperature and locked The system Is ready for imaging Click the temperature box to display the actual and demand temperature of the CCD and stage For more details see page 18 Acquire Click to acquire an image using the settings and options selected in the control panel or to acquire an image sequence specified in the Sequential Setu
263. lative uncertainty in the optical properties An analysis of the dorsal and ventral views of the mouse left lung in Figure G 5 results in total flux values that are very similar The measured depth values are also close indicating that the cells are distributed about the same distance from the front and back of the animal Figure G 5 Planar spectral analysis results Top Dorsal view of the left lung bottom ventral view of the left lung O Fie Edit View Image Window Help C Image Information gt ROI Tools Planar Spectral Imaging Analyze Properties Results Spectral Results Unsaved ROI Depth mm Total Flux phot s ROI1 2 326 0 191 3 01e6 3 87e5 aa n a a A T TTAN T TATT Fie Edit view Tools Window Help SES SESEEESSSSSESSSESSSESSSESSEEESESESEESS SESS SESS SESE TEE es Units Photons Display Overlay v into image Adust dD Corrections Filtering B i gt Surface Topography DLIT we A a A A amp R i Units Photons jo Apply to all gt a We RO 1 1 250e 06 JEK Image Min 1 26e3 Max 9 76e5 p sec em 2 sr lt m w Save Results Color Bar Name E pl m2 v reed Delete Load JA XI S AH A amp R Units Photons w C Apply to all gt Corrections Filtering B gt ROI Tools Image z Min 1 09e3 Planar Spectral Imaging Max 6 57e5 A
264. lecules and generates a quantification database The software uses the quantification database to determine the number of cells in a DLIT source or the number of cells or dye molecules in a FLIT source Image Overlay Displays multiple luminescent or fluorescent images on one 110 photograph Colorize Renders luminescence or fluorescence data in color enabling you to 113 see both intensity and spectral information in a single view The tool provides a useful way to visualize multiple probes or scale probe signals that are not in the visible range Transillumination Available for FLIT image sequence Generates an overview image for 109 Overview for each filter pair that includes the data from all of the transillumination lt name gt _SEQ locations The overview image can be analyzed using the tools in the Tool Palette Image Math A method for mathematically combining two images add subtract 151 multiply or divide Use image math to remove autofluorescence from a fluorescent image 14 Living Image Software User s Manual This page intentionally blank Y Ca i iA 3 Getting Started Starting the Living Image Software 0 2 0 eee ee ee ee 15 Initializing the IVIS Imaging System a 0 0 2 ee eee ee ee 17 Checking the System Temperature e 2 ee ee ee ee es 18 About the IVIS Acquisition Control Panel amp Auto Exposure Feature 19 Tracking System and User Activity 2 2 00
265. lette Corrections Filtering tools continued Tool Description Flat Field Correction Select this check box to apply a lens correction factor to the image data For more details on flat field correction see Appendix D page 284 Note In photons mode flat field correction is a mandatory default In counts mode the check box can be cleared Cosmic Correction Select this check box to correct image data for cosmic rays or other ionizing radiation that interact with the CCD For more details on cosmic correction see Appendix D page 284 Adaptive FL Opens the Photo Mask Setup box that enables you to set the photo mask Background for adaptive fluorescent background subtraction For more details on Subtraction adaptive fluorescent background subtraction see Appendix F page 305 Binning Specifies the number of pixels in the image data that are grouped together to form a larger pixel called soft binning Binning changes the pixel size in the image Figure 7 16 For more details on binning see Appendix C page 2 6 Smoothing Computes the average signal of the specified number of pixels and replaces the original signal with the average signal Figure 7 16 Smoothing removes signal noise without changing pixel size Click this button to return the binning or smoothing to the previous setting and update the image Figure 7 16 Example of binning and smoothing image data Binning at acquisition 8 no smoothin
266. lly blank 7 Caliper _ Appendix G Planar Spectral Imaging Planar Spectral Imaging Theory 2 eee ee ee ee 309 Upisi Fronin 644 hob Kh Dee EER ER EE HEHE Hwee HG 311 Luciferase Spectrum naaa a a ee a 311 An Example of Planar Spectral Imaging aoaaa a a a a 312 Optimizing the Precision of Planar Spectral Analysis 315 The unique spectral signatures of the luciferase emission spectrum and the optical properties of tissue enable the Living Image software to determine the depth and intensity of light sources inside a living animal The planar spectral imaging algorithm relies on a diffusion model of light propagation in tissue and assumes a point source of light embedded in a flat surface approximation of the mouse The algorithm is designed to provide a fast and robust method to approximate source location and brightness The analysis requires two or more single view images at wavelengths between 560 and 660 nm The Diffuse Tomography DLIT algorithm is a more complete and accurate model It analyzes images of surface light emission to produce a three dimensional 3D reconstruction of the luminescent light sources in a subject For more details on DLIT analysis see Chapter 14 page 191 and Appendix H page 317 G 1 Planar Spectral Imaging Theory An image acquired on an IVIS Imaging System is a diffuse projection on the surface of the animal from the luminescent sources located deeper inside Information abou
267. lts to a text file Figure 13 8 Well plate quantification results a Well Plate Type Measurement Sample Wells 3D 3A V Background Wells 1D 1A Apply to Sequence IC Well Plate Quantification Window For Sequence EL20090414101005_SEQ Click EL20090414101005_001 v WellPlate Quantification Plots Results Database WPQUANT_1 Copy Select All Export Results Le A ET UY Fluorophore Type Dye molecules C Cells C Set 1 115e 08 4 821e 09 IRS 2 251 09 8 518e 10 Sequence WPQUANT_1 v Name Name Overwrite Overwrite Database WPQUANT_1 X Y Ca i a 1 4 3D Reconstruction of Sources Reconstructing Luminescent Sources 0 0 02 ee eee ees 193 Reconstructing Fluorescent Sources aaoo a a saaa 201 3D Reconstruction Results oaoa aa a a a a a 204 Checking the Reconstruction Quality 2 858228 eas 207 Measuring Sources 1 eee ee a 209 Displaying Luminescent and Fluorescent Sources on One Surface 213 Comparing Luminescent and or Fluorescent Sources 213 Exporting a 3D Scene as DICOM 2 00020 2 eae 217 3D Tools Overview 2 we a a a saaa 220 3D Tools SUrlaCS 6 be RETESET wR ee Oe Ye awe ES 220 Oe TOOL SOUC 24 ee eR ER RRR BLE RRR EHR ES a ee 3D Tools Registration 1 ee ee ikaw se 2eeo SU AMMANN a ss ee OH eee hh ERE Ee ee wee 23
268. m a straight line could indicate possible issues with the dilution series or errors when entering sample dilution values 16 To export the quantification plot values Tie e Y Caliper Chapter 13 3D Quantification Database 189 a Click the gy button b In the dialog box that appears select a folder for the file csv and click Save 17 To copy the quantification plot values to the system clipboard click the button Table 13 1 Quantification results Item Description Fluorescence Excitation nm The excitation and emission filter wavelengths for the image Emission nm Excitation and Emission filters will be specified for fluorescent images and the Open filter for Emission will be specified for bioluminescent images Extinction Coeff A measure of excitation photon absorption interaction with the well plate samples based on a base 10 logarithmic derivation The quantum efficiency factor of the conversion of the absorbed photon to the emission wavelength is also included Cross Section A measure of excitation photon absorption interaction with the well plate samples based on a natural logarithmic derivation The quantum efficiency factor of the conversion of the absorbed photon to the emission wavelength is also included Bioluminescence Total Flux cell A measure of total flux ohoton sec emitted from a single cell This number can be used to estimate the number of cells from the t
269. m tool button B To check the organ fit 1 Check the fit in the coronal sagittal and transaxial windowpanes 2 Click the Change view toolbar button The Top view is displayed me Y Caliper _ Chapter 14 3D Reconstruction of Sources 229 Figure 14 34 Skin pink fitted to surface gray Subject Height 26 3 mm _ Perspective 3 Press the V key or the button to display alternative views of the surface Figure 14 35 Alternate views of the surface Top Bottom Front Left Right 230 Living Image Software User s Manual Importing an Organ Atlas An organ atlas iv dxf or stl one organ per file consisting of segmented organ surfaces derived from an MRI or CT scan can be imported into the Living Image software for registration with the animal surfaces derived from IVIS data Organ files must be segmented from MRI or CT 3D volumetric data in third party medical imaging analysis software NOTE The imported atlas must include a surface skin file which delineates the animal surface The file name must include the word skin for example rat skin iv 1 Load a DLIT or FLIT image sequence that is associated with the mouse comprising the organ files in iv dxf or stl format Select File Import Organ Atlas on the menu bar In the dialog box that appears click Add Organ Files Figure 14 36 W Figure 14 36 Import Organ Atlas di
270. maging System and the imaging mode selected luminescent or fluorescent Image Setup or Sequence Setup mode For more details on the control panel see Appendix A page 259 The IVIS Imaging System is ready for imaging after the system is initialized and the operating demand temperature of the CCD camera is reached locked Y Caliper REF nce Chapter 3 Getting Started 19 3 4 About the IVIS Acquisition Control Panel amp Auto Exposure Feature The control panel Figure 3 5 provides the image acquisition functions For details on the imaging parameters in the control panel see Appendix A page 259 The auto exposure setting is useful in situations where the signal strength is unknown or varies widely for example during a time course study If auto exposure is chosen Figure 3 5 the system acquires an image at maximum sensitivity then calculates the required settings to achieve as closely as possible an image with a user specified target max count If the resulting image has too little signal or saturated pixels the software adjusts the parameters and takes another image In most cases the default auto exposure settings provide a good luminescent or fluorescent image However you can modify the auto exposure preferences to meet your needs For more details see page 269 Figure 3 5 IVIS Acquisition Control Panel To acquire an image using auto exposure click the gm arrow and select Auto Lu m m e
271. mapped back to the surface of the object to produce an absolute value of the surface radiance photon s cm2 steradian from each imaged surface element Figure H 3 Figure H 3 Light emission from a surface element passes through the lens entrance pupil and ts recorded in the image The imaging system collects the light emitted from the surface element at an angle 0 measured with respect to the normal to the surface element into the solid angle dQ subtended by the entrance pupil The value of the surface radiance L 0 is directly related to the photon density p photons mm just inside the surface of the element FLIT analysis uses NTF Efficiency data and takes into account the photon density of both the fluorescent image and transmission image 320 Living Image Software User s Manual H 3 Defining the Linear Relationship Between a Source and Photon Density or NTF Efficiency The software divides the interior of the subject into a solid mesh of volume elements voxels Each voxel is considered to contain a point light source at its center The index i enumerates the set of voxels S is the value of the strength of the point source inside the i voxel The solid mesh defines a collection of point sources that approximate the actual source distribution The accuracy of the approximation is improved by increasing the density of the solid mesh The reconstruction method is based on the principle that there is an approx
272. minescent calibration database see page 185 7 In the Analyze tab click Start The Data Preview window appears and displays the image data that will be included in the reconstruction Usually no data adjustment is required However it is possible to exclude or include user selected pixel data from the analysis For more details see page 198 Figure 14 4 Data Preview window right FENA E TLT20050624145507_SEQ Sequence View 4 3D View Spectra Data Preview E3 Tool Palette gt Q JO il Analyze Properties Results Sequence 71720050624145507_SEO Tissue Mouse Tissue Source Firefly Filter Threshold V Image Label V Median Filter Restore Threshold Data Adjustment E Select All Cancel Reconstruct 196 Living Image Software User s Manual 8 In the Data Preview window click Reconstruct The reconstruction normally requires less than one minute depending on the reconstruction volume parameter settings and computer performance When the analysis is finished e The 3D View window displays the animal surface and the reconstructed sources e In the Tool Palette the Results tab displays the results data and the algorithm parameter values e The 3D Tools appear after a reconstruction 1s generated or loaded For more details on the 3D
273. n VIS Imaging Systems and provides tools for optimizing image display and analyzing images 1 1 What s New In Living Image 4 2 Software The following table provides a brief description of new or improved features in the Living Image software New or Improved Features See Page Image Acquisition and Analysis Living Image acquisition and analysis sofware supports Windows 7 operating system Batch mode for sequence acquisition 39 Expanded selection of fluorescent probes in the Imaging Wizard including BoneProbe680 Integrin750 RJ 2 DG 750 Faster DLIT and FLIT algorithm speed Dynamic voxel display during the DLIT or FLIT reconstruction process shows voxel size refinement during analysis Working With Images Image Labels improvements e Edit comments directly on image sequence in the Info section 94 e New toolbar button k for quick access to the Image Label dialog box for editing image 95 labels e New Options Labels menu allows flexible selection of labels for display 95 Add comments to images 95 Export well plate quantification results 190 View a 3D representation of intensity signals 106 ROI Tools Apply measurement ROls to all images in the Sequence View window 120 ROI tag includes the auto ROI threshold 121 Sort auto ROI numbers 121 125 Select multiple ROIs and move them together 126 DLIT or FLIT 3D Reconstructions Longitudinal Study Browser quantitativel
274. n 1 Medium Photo Bin 4 Bin 2 Bin 2 Bin 2 1Some early IVIS 100 Systems with Spectral Instruments SITe cameras and all Roper and Princeton Instrument cameras are not supported in Windows 7 Living Image 4 2 software 2 Default setting You can also apply soft binning after an image is acquired Conceptually soft binning is the same as hardware binning groups of pixels are summed and a smaller lower resolution image is produced However in soft binning the summing is performed digitally on the stored image data not on the electronic charge before readout as in hardware binning Although soft binning does not improve the signal to noise ratio for read noise it may enhance the signal visibility because it reduces the statistical scatter of nearby pixel contents Usually hardware binning is preferred but if it is not possible to take another image applying soft binning to the data may provide a worthwhile solution C 3 Smoothing Smoothing is a filtering method that reduces noise in the image data To apply smoothing the software replaces the intensity of each pixel with the average intensity of a nearby pixel neighborhood that includes the pixel Figure C 4 shows a 3x3 pixel neighborhood Smoothing does not change the pixel size and helps e Eliminate outlier pixel values that are extremely high or low e Reduce noise fluctuations in the image to help reveal small signals Figure C 4 3x3 pixel neighborhood Center pix
275. n 2 4 ses be kee ee eee ee ee ee ee ee RE Se Des 225 14 13 3D Animation aoaaa 231 14 14 DLIT FLIT Troubleshooting o aoa a a 0 2 00 eee ee 237 15 3D Multi Modality Tools nonoo aa 239 15 1 3D Multi Modality Tools Requirements n aoaaa 2 02 eee ee ee 239 15 2 Loading Data for Registration ooa aa 241 15 3 Registering Multi Modal Data aoaaa 245 15 4 Classifying 3D Volumetric Data 2 250 15 5 Volume Display Options 2 0 00 254 15 6 Viewing a Slice 2 cn do eee REGO eRERS ERO HERE EEE RES EHS Gow a 256 15 7 Volume Information and Results 2 a aaa 257 S Voume Daa VIEWER sane eb eee ee ee Oe ee Eee ee eee eee ee ee 258 Appendix A IVIS Acquisition Control Panel 82 5808 259 Ant Control Panel 22 6eeeeenececteeeete eee eee eee eee es ee eee 259 A 2 Manually Setting the Focus 2 a 264 Appendix B Preferences 2 2 see ee ee a ee 265 B 1 General Preferences ooa ee 266 Deo Wiser PicictemCcs 2a eeu ea gehaet eke eh ete ee eh ee eee thee wets eee oes 268 Boo ACquUIsION 5 2 areeubena base beeuntdeteececetaeocuce be oe ho wa oa oe G 269 B 4 VWhie e 228564 056 58 eek eee SeS Ee See wee eee ee eee ee Se ee 270 iv Living Image Software User s Manual B5 Optical Properties 2228444255824 eS aoe eee See wwe Ee eee 273 Appendix C Detection Sensitivity 2 00 0 eee eee ee 275 CU CCD Detection EMiciency x s s S 44465444 2284 Y oe BoD Eee PAE EG oe HG
276. n Image Sequence from Individual Images You can create a sequence from images acquired during different sessions 1 In the Living Image Browser browse for the images of interest For more details on browsing see page 83 NOTE Browse for individual images which may or may not be part of a sequence not image sequences Figure 7 32 Living Image Browser Individual images highlighted blue in this example that may or may not be part of a sequence can be selected for grouping into a new sequence ME JJH20050630142125_003 i M JJH20050630142125 002 ME JJH20050630142125_001 b SE amp HX20070420122818 SEQ Gnana ae se In vitro In vitro nao strona vew AL Uxing rage Browse c C fa Click Number h EX Filter EM Filter Illumination Mode User ID User Group Experiment Commenti i M JJH20050630142125 004 620nm JH i coll AF700 AF680 QD700 QD800 AF750 2col AF680 AF700 AF750 3col QD700 AF70C i coll AF700 AF680 QD700 QD800 AF750 2col AF680 AF700 AF750 3col QD700 AF70 Feesececssuscsssessoseseossesscessessosscesosessesssesoesdsssssscessessoessessosssslsssssssosossesssseseessoesissscsssesssssssessssssessesossesossoesessseoissesssesessssessosssessolsssessesssessessseoissessosossessssseesssssssseessseseesosossessessssesossssssscsosessossoeisosssssessessossscessesesosscssssesesosoessesdusssssesssssseessesssessosssess 0
277. n excitation light source located below the stage FLIT reconstruction of fluorescent sources analyzes a transilluminated image sequence For more information about fluorescent imaging see page 291 If the fluorescent source is deep relative to the imaged side of the animal acquisition with transillumination is recommended By default acquisition with transillumination includes an Normalized Transmission Fluorescence NTF Efficiency image in which the fluorescent emission image is normalized by the transmission image measured with the same emission filter and open excitation filter Figure 4 9 Transillumination is only available on the IVIS Spectrum Imaging System Y caliper oa ter tcl maging 29 Figure 4 9 Fluorescent images acquired with transillumination In this example the NTF Efficiency image highlights the presence of fluorescence in the animal while the Radiant Efficiency image shows signal ambiguous with autofluorescence NTF Efficiency Image Radiant Efficiency Image Unie MP enep Dele eri fp e ce yah E inanan aa Unda Baai oeer e gi Diay E jfi oe fya e E raran ein aa To acquire a fluorescent image with transillumination NOTE Before setting the imaging parameters the IVIS Imaging System should be intialized and the temperature locked For more details see Chapter 3 page 15 1 In the control panel put a check mark next to Fluorescent and Transillumination Figure 4 10
278. n light is absorbed by the fluorophore the fluorescence is emitted However due to the optical characteristics of tissue the excitation light is scattered and absorbed before it reaches the fluorophore as well as after it leaves the fluorophore and is detected at the animal surface Figure F 9 The excitation light also causes the tissue to autofluoresce The amount of autofluorescence depends on the intensity and wavelength of the excitation source and the type of tissue Autofluorescence can occur throughout the animal but is strongest at the surface where the excitation light is strongest Figure F 9 Illustration of the in vivo fluorescence process p CCD Array gt Imaging Lens Tissue Autofluorescence Photon Scattering Mean free path 0 5 mm Bioluminescent or Fluorescent Source At 600 900 nm light transmission through tissue is highest and the generation of autofluorescence is lower Therefore it is important to select fluorophores that are active in the 600 900 nm range Fluorophores such as GFP that are active in the 450 600 nm range will still work but the depth of detection may be limited to within several millimeters of the surface Specifying Signal Levels and f stop Settings Fluorescent signals are usually brighter than luminescent signals so imaging times are shorter typically from one to 30 seconds The bright signal enables a lower binning level that produces better spatial resoluti
279. nalyze Properties Results Spectral Results lt af ROI Depth mm Total Flux phot s FRO 2 2 426 7 0 199 2 4206 3 2385 af 40 x10 3 0 20 cim gt 10 Plot Linear Fit p sec com 2 st Save Results Color Bar Name Splm_2 K v Min 6 32e4 Delete Load Max 6 4165 gt Surface Topography Z DLIT YY Caliper per Appendix G I Planar Spectral Imaging 315 G 5 Optimizing the Precision of Planar Spectral Analysis The accuracy of the planar spectral analysis is highly dependent on the quality of the e Measured data for the firefly luciferase spectrum and the tissue optical properties e Fit of the experimentally measured total flux at each wavelength to UL effective attentuation coefficient In general more experimental values produce a better fit of the data It is particularly important to be able to extract signals at all wavelengths to optimize the quality of the fit If the software detects no signal above the animal background level at 560 nm and 580 nm the wavelengths that absorb the most light the dynamic range of the optical properties is reduced and with it the precision of the fit If a luminescent signal is dim or buried deep in the tissue it may barely exceed the tissue autoluminescence at the shorter more absorbing wavelengths 560 and 580 nm In this case it is recommended that you subtract the tissue autoluminescence from the image data For more details on subtracti
280. name appears in the Name drop down list y7 Caliper 7 Der ces Chapter 10 Planar Spectral Image Analysis 161 10 3 Viewing Graphical Results 1 In the Results tab select an ROI 2 Click Plot Intensity or Plot Linear Fit Figure 10 5 Linear fit graph Plots the logarithm of the intensity normalized to the selected source spectrum and the filter transmission properties against the optical property of the tissue The slope of the line is the source depth If any of the measured points in red deviate significantly from the straight line fit then the analysis results may be suspect The horizontal error bars represent the uncertainty in the optical properties usually estimated at 10 The vertical error bars represent noise in the image Intensity graph Displays a graph of the measured intensity in the selected ROI at each wavelength in the analysis The intensity is normalized to the selected source spectrum and the filter transmission properties Figure 10 5 View the Linear Fit or Intensity graph 7 Plot of Linear Fit Results Tool Palette a Linear Fit Plot for ROI 1 Log Norm Flux K mueff crn LC Plot of Intensity Vs Lambda el Intensity vs Lambda Plot for ROI 1 Normalized Flux a u 600 Wavelength nr To export graph data 1 Click the Export Data button gj 2 In the dialog box that appears select a directory for the data and enter a file name
281. nce Living Image file format dcm kinetic data or an image that was exported to a DICOM file TIFF Image Files Graphic files tif tiff All Files All file types 3 Navigate to the file and click double click it Alternatively select the data and click Open Organizing Images When multiple image windows are open you can organize them in a cascade or tile arrangement Choose Window Cascade or Window Tile on the menu bar Figure 7 5 Image windows cascade top or tiled bottom Pis fdt View foh Window Hrip ae Qo oo AA E ulcer ety ea YY Caliper per Chapter 7 Working With Optical Image Data 89 7 2 About the Image Window amp Tool Palette Image Window An image image sequence or kinetic data set is displayed in an image window Multiple image windows can be open at the same time Figure 7 6 Image windows sequence view and single image The options available in the image window depend on the type of active image data File Egt View Tools Window Help 28am J BR amp as F dey to al Tool Palette gx Image Adjust EE Q Sequence View Specta Corrections Filtering ol Units Counts Use Saved Colors _ Image Information gt ROI Tools L gt Planar Spectral Imaging Surface Topography _ Orme 30 Mult Modality Tools E ti r200s0624145507 SEQ Color Scale Min SUG May 103448 Table 7 2 Image window
282. nce histogram shows the part of variance y axis that can be explained by a number of principal components x axis Figure 11 10 PCA explained variance histogram Ez TLT20060406164950_5EQ l Unmixed Images Composite PCA Variance I Sequence View Spectra oe ls a i a 100 gg J g5 g3 g3 0 ao at a6 a 176 Living Image Software User s Manual PCA Biplot The PCA biplot is a visualization tool for principal component analysis It shows a simultaneous display of n observations pixels and p variables wavelengths on a two dimensional diagram Figure 11 11 PCA biplot E TL120060406164950_SEQ alla 2 Lif Soames en soe Sequence View Spectra Unmixed Images nixed Images Comp Composite _ PCA Variance e PCA Biplot K Axis Componenti w Y Axis Component2 a 0 6 0 4 0200 02 04 06 O48 10 Y Y Caliper 1 2 Reconstructing a 3D Surface Generating a Surface 1 a 178 Managing SUITaCES bac ke tb reada dau HEH ee HES SS es 182 Export or Import a Surface 1 2 a a a a 183 A surface is a 3D reconstruction of the animal surface topography derived from structured light images The Living Image software requires a surface to perform some types of analyses Figure 12 1 You can e Save a surface and use it for any of the analyses shown below e Export a surface for viewing in other 3D viewer applications e Im
283. nce per incident excitation power p sec cm2 sr MW Fluorescent emission yield normalized to the incident excitation intensity radiance of the subject illumination intensity Fluorescent emission image normalized to the transmission image which is measured with the same emission filter and open excitation filter Y Ca i ermi Chapter 8 ROI Tools 147 Table 8 4 ROI Measurements table continued Item Description Image Attributes None All Possible Values All Populated Values Living Image Universal Make a selection from the drop down list to specify the click number image file information to include in the table Click attributes include label name settings and camera settings Excludes image attributes from the table Includes all of the image attributes for example label name settings and camera settings in the table Includes only the image attributes with values in the table Includes all Living Image Universal label name settings in the table ROI Dimensions None Pixels cm Make a selection from the drop down list to specify the ROI dimensions to include in the table Excludes the ROI area x y coordinates and dimensions from the table Includes ROI area x y coordinates and dimensions in pixels in the table Includes ROI area x y coordinates and dimensions in cm in the table Copy Copies the selected row s in the table to the system clipboard Sel
284. nces are acquired during the same session the generated surfaces are nearly identical 1 Load a DLIT reconstruction and a FLIT reconstruction 2 Choose one of the reconstructions click the gpa button and select Copy source voxels 3 In the other reconstruction click the ga button and choose Paste source voxels NOTE Pasted voxels can be measured For more details on measuring sources see page 209 14 7 Comparing Luminescent and or Fluorescent Sources Multiple DLIT and or FLIT reconstruction results can be viewed side by side in the Longitudinal Study window Voxel intensity within the entire surface or a user selected area can be measured in all results in the Longitudinal Study window The Longitudinal Study window provides a convenient way to compare different results for example results obtained at different time points or results from different types of reporters Viewing Results in the Longitudinal Study Window 1 Load the DLIT and or FLIT sequences with the results that you want to display Select Tools gt Longitudinal Study on the menu bar The Longitudinal Study window appears Figure 14 22 Longitudinal Study window p Longitudinal Study Window GEJ 2 woe Of ew ETT STS TS iT Select a Sequence 3D View Loaded DLIT or E pur EL20100601160926_SEQ FLIT sequences EE DUT EL20100611111118_SEQ EE DLI EL20100623110413_SEQ Wavelength 7 XE Remove Select Analysis R
285. nd volume deformation After fitting organs to the surface using the ii or A tool if necessary you can click this button to restore the defaumt fit i Display Organs Choose this option to display the organs on the surface Organs that are check marked will be displayed For more details see page 226 226 Living Image Software User s Manual Table 14 12 Registration tools continued Item Description Drawing styles for the organs see Display Organs PAN ie Ae Shading styles for the organs see Display Organs Ge E E i Opacity Adjusts the opacity of the organ display Organ Atlas Choose a type of organ atlas Click to select all organs in the database and display them on the surface Click to clear the selected organs and remove all organ diagrams from the surface Displaying Organs With the Reconstruction 1 Load reconstruction results and confirm that the surface is in the perspective view click the toolbar button in the 3D View window or press the R key 2 In the 3D registration tools choose the Display Organs option and select an organ atlas The organs in the selected atlas appear on the surface 3 To fit the organs to the surface click a registration tool fi Rigid registration Performs linear transformation but keeps the shape of the atlas surface A Full registration Performs linear transformation and volume deformation F
286. ndence of luminescent light emission Table H 2 shows the factory set source spectra provided by the software The source spectra is not an input to the 3D reconstruction of fluorescent sources ol is Y Caliper Appendix H DLIT amp FLIT Reconstruction of Sources 321 Figure H 4 DLIT 3D reconstruction tools Properties tab Tool Palette XPM 2 mouse phantom Choose Source Spectrum from the drop down list trom the Plot drop down list to display the selected spectrum This is required for DLIT Select a luminescent source spectrum Result not found E Browse for a 3D Quantification Database os AE l For more details on the database see page 185 Table H 2 Source spectra Source Spectrum Description Bacteria Bacterial luciferase CB Green Click beetle green luciferase CB Red Click beetle red luciferase Firefly Firefly luciferase hRenilla Sea pansy Renilla reniformis luciferase Tritium Bead 5 Phosphor coated glass bead containing tritium gas Spectrum for bead 5 XPM 2 LED LED in the XPM 2 mouse phantom NOTE The firefly luciferase spectrum is dependent on temperature and pH The data provided are valid only for measurements performed at 37 C and at pH 7 0 7 5 Selecting other temperature and pH conditions for a specific experiment requires the use of the associated spectral curve for the spectral analysis For more information about pH and temperature dependence
287. ndle W at a corner of the image 4 To reposition the image in the window drag the image YY Caliper per Chapter 7 Working With Optical Image Data 115 Figure 7 30 Image Layout window E rime E Image Layout Window a oe x D W x e oO oO le fv amp v X amp LayoutStyle Freestyle v TE w a o r Annotation Table 7 13 Image Layout window Item Description i Clears the Image Layout window Note If you do not clear the layout click the button before you close the Image Layout window the same window contents are displayed the next time the window is opened Opens a dialog box that enables you to save the Image Layout window contents to a graphic Tile Pastes an image of the active data in the Image Layout window Copies the contents of the Image Layout window to the system clipboard Pastes the contents of the system clipboard to the Image Layout window Rectangle drawing tool Ellipse drawing tool Pointer tool OG LU gh a a 1 Arrow and line drawing tool x A 2 z Select an the item in the Image Layout window To move the item to the front EZEN or back in the window choose an option from the drop down list VY Send backward W Send to back wE Deletes the selected image 116 Living Image Software User s Manual Table 7 13 Image Layout window continued Item Description A drop down list of formatting opt
288. ng tissue autoluminescence see Appendix E page 288 It is also recommended that you inspect all images in the sequence to confirm that the luminescent signal is greater than the tissue autoluminescence If the luminescent signal does not exceed the tissue autoluminescence at a particular wavelength do not include that wavelength in the analysis 316 Living Image Software User s Manual This page intentionally blank y Caliper LifeSciences Appendix H DLIT amp FLIT Reconstruction of Sources Determining Surface Topography 2 0 6 ee eee ee es 317 Converting Light Emission to a Photon Density Map 319 Defining the Linear Relationship Between a Source and Photon Density or NTF EIOS Y 2 hee he REEDS RPE REED SHEDS SH OS HEH 320 Determining the Best Approximate Solution to the Linear System 320 Source amp Tissue Properties 1 ee a a Diffuse Luminescence Imaging Tomography DLIT is a technique that analyzes images of the surface light emission from a living subject to generate a three dimensional 3D reconstruction of luminescent light source distribution inside the subject Fluorescence Imaging Tomography FLIT analyzes images of surface light emission to generate a 3D reconstruction of fluorescent light source distribution inside the subject To reconstruct luminescent sources the Living Image software requires a photograph a structured light image and luminescent images obtaine
289. nge between luminescent and fluorescent imaging applications For more details see the appropriate hardware manual e IVIS Spectrum System Manual e IVIS Imaging System 200 Series System Manual e IVIS Lumina System Manual e XFO 6 or XFO 12 Fluorescence Option Manual Figure F 1 IVIS Spectrum CCD Camera oo e Scanning Custom Lens ee and Emission Assembly Filter Wheel Imaging Chamber Heated Shef o AER Anesthesia Manifold Transillumination manifold Compartment Soundproof for CCD Portable Camera Water Cart with a Chiller and 25x34 inch Camera Footprint Controller 292 Living Image Software User s Manual Figure F 2 IVIS Imaging System 200 Series Integrated Emission Figs filter wheel uorescent imaging eaBIles Excitation filter wheel Light source Figure F 3 IVIS Imaging System Lumina Lumina 100 Series and Lumina 50 Series Excitation Emission Filter Wheel Filter Wheel Assembly not visible A 150 watt quartz tungsten halogen QTH lamp with a dichroic reflector provides light for fluorescence excitation The relative spectral radiance output of the lamp reflector combination provides high emission throughout the 400 950 nm wavelength range Figure F 4 The dichroic reflector reduces infrared coupling gt 700 nm to prevent overheating of the fiber optic bundles but allows sufficient infrared light throughput to enable imaging a
290. ngitudinal Study window Choose an image to display Use the thumb wheel from the selected results to rotate the surfaces i Angle Of view PEE E E a 1 14 a Select a Sequence 3D View Plots BD pur EL20100611111118_SEQ E DLI EL20100601160926_SEQ araale l DLIT EL20100623110413_SEQ Voxel color scale If both DLIT and FLIT results are loaded two color scales are displayed Reverse Log Scale V MIP Display Surface z Opacity i a photons sec Display Photon Density Map DLIT Results For more details on Click a surface to select it these display controls see Table 14 7 page 217 v i ge Y Caliper Chapter 14 3D Reconstruction of Sources 215 Measuring Intensity 1 After results are loaded click the button By default a selection box appears around each surface Figure 14 24 This means that measurements for the entire surface will be computed 2 To select a particular region of the surface for measurements draw a box by clicking and dragging the mouse around the area The same box is applied to the other surfaces in the Longitudinal Study window 3 To clear boxes click the button again Figure 14 24 Selection boxes around each surface photons sec DLIT Results Viewing Plots In the Plots tab make a selection from the Analysis Type and Plot drop down lists Figure 14 25 The following graphs are available in the Plots tab e Quantificati
291. ni tet Mice Browse View Preview picture of the data selected in the browser blue row Color Scale Provides a reference for the pixel intensities in a luminescent or fluorescent image Pixels less than the color scale minimum do not appear in the image Pixels greater than the color scale maximum are displayed in the maximum color 4 2 Fluorescent Optical Imaging With Epi lIllumination This section explains how to acquire a single fluorescent optical image Epi illumination provides an excitation light source located above the stage For more details about fluorescent imaging see page 291 For information on acquiring an image sequence see Acquire a Sequence Using the Imaging Wizard page 33 NOTE Before setting the imaging parameters the IVIS Imaging System should be intialized and the temperature locked For more details see Chapter 3 page 15 1 In the control panel put a check mark next to Fluorescent and select Auto exposure click the arrow When you select Auto exposure the software automatically determines the binning and F Stop settings Alternatively you can manually set the exposure binning and F Stop For more details on these control panel settings see page 259 Figure 4 5 Acquisition control panel F Stop Excitation Filter Emission Filter Field of View E Mouse Imaging Shuttle Subject height 1 50 l m Focus use subject height Temperature M Locked 2
292. nits Photograph Choose the type of units for displaying the fluorescent or luminescent image For more details on measurement units see page 281 Fluorescent or A drop down list of the photographs in the image sequence The sequence images Luminescent Images 112 Living Image Software User s Manual Table 7 11 Image Overlay window Item B Description Copies the overlay to the system clipboard Click to export the overlay to a graphic file C4 Click to include all fluorescent or luminescent images in the overlay Click to remove all fluorescent or luminescent images from the photograph Image Adjust Tools for adjusting the appearance of the highlighted fluorescent or luminescent image Adjustments can only be made on one image at a time Min The minimum pixel intensity associated with the color scale for an image Pixels less than the minimum value are not displayed Max The maximum pixel intensity associated with the color scale for an image Pixels greater than the maximum value are displayed in the maximum color Opacity Controls the opacity of the fluorescent or luminescent image Color Table Tools selecting and modifying the color scale associated with an image Color Scale Type Choose BlackLevel to show black at the low end of the color scale Choose WhiteLevel to show white at the low end of the color scale Red Click the drop do
293. ns In the spectral unmixing tools the Options tab shows the user modifiable parameters in the spectral unmixing algorithm Figure 11 9 It is recommended that you first perform spectral unmixing using the default settings Then if necessary change the option settings and reanalyze the data Figure 11 9 Spectral unmixing options Tool Palette PCA Explained Variance NA me NifA Sort T Force Denoise by PCA Medium Default N A 7 N A 7 Bipi Table 11 6 Spectral unmixing options Option Constraints Description The constraints for unmixing the components Reset Values Returns all constraint settings to the default values Init The method for generating the initial guess of the spectrum for the selected component Auto means this is automatically determined by the software Alternatively you can used a loaded spectrum as the initial guess Fix This option determines whether the spectrum is allowed to change If this option is chosen the spectrum of that component is not updated during unmixing HP Sets a high pass filter for the spectrum Signal below the HP cut off frequency is forced to zero Choose N A to turn off the high pass filter Otherwise the value represents the high pass cut off frequency This constraint can help isolate components that are physically mixed and difficult to distinguish Y Ca i i a Chapter 11 Spectral Unmixing 175
294. nt 2 After you remove the Zoom Lens attachment click OK in the prompt Always store the lens wrapped in its protective container Figure 6 5 Control panel remove the check mark next to Zoom we vis Acquisition Control Panel k Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter 1 d Field of view ey System Status C xeoy 24 T Acquire Sn Subject height 15 cm Sequence Setup Please remove the zoom lens by pulling down firmly from tts magnetic attachment Click OK when done Canes 6 3 Viewing amp Editing Data in the Kinetic Acquisition Window After stopping acquisition you can view the data in the Kinetic Acquisition window 1 To start the playback click the Play button ra After playback starts the button changes to a Stop button so _ 2 To stop the playback click the Stop button m st 3 To view a particular frame do either of the following e Move the top frame slider or enter a frame number in the box next to the frame slider e Click a location in the Maximum vs Time graph 4 To select a particular range of kinetic data move the start and end frames selection handles Alternatively enter a frame number in the box next to each slider Only the selected frames will be played back or saved NOTE Kinetic data dcm can also be edited in the Image window For more details see page 109 76 Living Image Software User s Manual
295. nt food bottom Images were taken using the GFP DsRed Cy5 5 or ICG filter set The data is plotted in efficiency on the same log scale Regular Rodent Food Alfalfa free Rodent Food GFP DsRed ICG Figure F 18 shows a comparison of fluorescence and luminescence emission in vivo In this example 3x10 PC3M luc DsRed prostate tumor cells were injected subcutaneously into the lower back region of the animal The cell line is stably transfected with the firefly luciferase gene and the DsRed2 1 protein enabling luminescent and fluorescent expression The fluorescence signal level is 110 times brighter than the luminescence signal However the autofluorescent tissue emission is five orders of magnitude higher In this example fluorescent imaging requires at least 3 8x105 cells to obtain a signal above tissue autofluorescence while luminescent imaging requires only 400 cells 304 Living Image Software User s Manual Figure F 18 Images of stably transfected dual tagged PC3M luc DsRed cells The images show the signal from a subcutaneous Injection of 3x106 cells in an 11 week old male Nu nu mouse Fluorescent Bioluminescent 8 gt 9 5x10 4 wW 7 1x10 2 photons s cm sr 1 0x10 olx 7 8x10 Signal Background 7 8 Signal Background 7500 Min detectable cells 3 8x10 Min detectable cells 400 NOTE When you make ROI measurements on fluorescent images it is important to
296. ntrol cells are always recommended in any experiment to assess the autofluorescence of the native cell Miscellaneous Material Autofluorescence It is recommended that you place a black Lexan sheet Caliper part no 60104 on the imaging stage to prevent illumination reflections and to help keep the stage clean If you are working in transillumination mode do not use the black Lexan sheet it will block the signal The black paper recommended for luminescent imaging Swathmore Artagain Black 9 x12 Caliper part no 445 109 has a measurable autofluorescent signal particularly with the Cy5 5 filter set Figure F 14 shows a fluorescent image of a sheet of black Lexan on the sample stage as seen through a GFP filter set The image includes optical autofluorescence light leakage and low level autofluorescence from inside the IVIS System imaging chamber The ring like structure is a typical background autofluorescence leakage pattern The image represents the minimum background level that a fluorophore signal of interest must exceed in order to be detected Figure F 14 Light from black Lexan This image shows the typical ring like structure of light from a sheet of black Lexan a low autofluorescent material that may be placed on the imaging stage to prevent illumination reflections Imaging parameters GFP filter set Fluorescence level High Binning 16 FOV 18 6 f 2 Exp 5sec Other laboratory accessories may exhi
297. ntrol panel and sequence table Exposure Time Binning F Stop Excitation Filter Emission Filter F Display Photographic Settings CEE Auto sec_y Medium open M E seqo Mode X Ray Exposure X Ray Binning X Ray FStop X Ray Energy Reuse X Ray Exposure Binning Medium _v i16 gt 1 EAS Auto Animal 2 Animal Yes Auto Medium Medium v 2 amp Animal O 2 gg Auto Medium 2 Animal Yes Auto Medium a Medium Animal Yes Auto Medium Field of View System Status E XFov 24 E Zoom 10 om Subject height 1 50 cm rons leesiectheght Terpener M ced _ aaie o E umber ofsegmenss oey 0 0 mn Caspi toat O lt Remover unite C imeen Cras X ray reuse option is selected by default One X ray image is acquired for the sequence 58 Living Image Software User s Manual Acquire the Image Sequence 1 Confirm that the VIS Imaging System is initialized and the CCD temperature is locked For more details see page 17 2 When you are ready to acquire the images click Acquire Sequence in the control panel The Edit Image Labels box appears Figure 5 11 Edit Image Labels box 7 Edit Image Labels sz UserID KSA Living Image Universal Saved Labels LABELS 1 ia Check any 5 fields for display v User v 7 Group The information entered here appears T Experiment in the image label Figure 5 13 v Commenti V Comment2 Time Point
298. ntum FX uCT instrument The subject must be contained in the Mouse Imaging Shuttle during both optical and CT imaging and the CT data must be exported to DICOM format After registration classify the 3D volumetric data to help you identify and separate objects see page 250 About the Mouse Imaging Shuttle The Mouse Imaging Shuttle Caliper part no 127744 contains the subject during imaging and enables the subject to be transferred between an IVIS Imaging System and the Quantum FX uCT instrument without disrupting the subject s position The Mouse Imaging Shuttle must be correctly docked to the docking station in the IVIS Imaging System and the Quantum FX uCT instrument The docking station in the Quantum FX uCT system is marked with a triangle shaped fiducial pattern under the plane where the Mouse Imaging Shuttle docks Automatic fiducial registration is available if both sides of the triangle fiducial pattern are included in the CT images For more details on using the Mouse Imaging Shuttle see the Mouse Imaging Shuttle Instructions Caliper part no 127820 _RevA 246 Living Image Software User s Manual To perform automatic fiducial registration 1 Load the data that you want to register see page 241 2 Click the Fiducial Registration button B The multi modal data are automatically registered and cropped Figure 15 9 3 To undo the registration click the Reset Registration button I Figure 15 9 Registered 3D
299. o help you fine tune the registration Figure 15 11 Example surfaces before and after registration 3D volumetric surface 3D structured light surface Surfaces before registration Registered surfaces To manually register data 1 Load the data that you want to register for more details see page 241 The software determines a default air noise boundary for the 3D volumetric data Figure 15 12 2 If you need to remove noise from the 3D volumetric data move the air noise boundary to the right in the histogram tool 248 Living Image Software User s Manual Figure 15 12 Adjusting the air noise boundary in the histogram tool olor Opacity Map Grays 1 0 Opacity m x Reverse Example noise in the 3D volumetric data Default air noise boundary E Reverse Adjust the air noise boundary to reduce noise in the 3D volumetric data Reduced noise 3 Ifthe volumetric data needs cropping for example to remove structures such as the stage from the CT view follow step a to step c below If cropping is not needed proceed to step 4 To crop the data a Click the crop tool button H The crop tool appears and has six control points crops the data along the x axis Crops data along the y axis crops data along the z axis Figure 15 13 Crop data along the x y or z axis Une Tale berg te erable tripi feat Cae oop hai iia idii No crop
300. o a 0 25 inch fused silica fiber optic bundle inside the imaging chamber Fused silica fibers core and clad unlike ordinary glass fibers prevent the generation of autofluorescence The fused silica fiber bundle splits into four separate bundles that deliver filtered light to four reflectors in the ceiling of the imaging chamber Figure F 1 The reflectors provide a diffuse and relatively uniform illumination of the sample stage Analyzing image data in terms of efficiency corrects for nonuniformity in the illumination profile When the efficiency mode is selected the measured fluorescent image is normalized to a reference illumination image For more details on efficiency see page 283 The IVIS Spectrum provides both transmission and epi illumination Emitted light from the excitation filter wheel feeds through a fiber optic bundle to illuminate the specimen 294 Living Image Software User s Manual from either the top in epi illumination reflectance mode or from underneath the stage by means of an automated bundle switch Transilluminating the subject from below at precise x y locations allows for transmission imaging enabling more sensitive detection and accurate quantification of deep sources Transmission fluorescence imaging also reduces the effects of autofluorescence A computer controlled imaging switch allows you to change between the two imaging modes using IVIS Acquisition Control Panel or the Imaging Wizard The emissi
301. o carry 1 To start the Living Image software click the icon on he desktop z out a procedure the p Document names are italicized Living Image Software User s Guide Note information NOTE A note presents pertinent details on a topic or Note Notes may also appear in this format Caution information CAUTION A caution note warns you that your actions may have nonreversible consequences or may cause loss of data Important information 1 IMPORTANT ALERT Important information advises you of actions that are essential to the correct performance of the instrument or software YY Caliper Y DET nce Chapter 11 Welcome 3 Living Image Help There are several ways to obtain help on the software features e To view a tooltip about a button function put the mouse cursor over the button e To view a brief description about an item in the user interface click the K toolbar button then click the item e Press F1 or select Help User Guide on the menu bar to display the Living Image Software User s Manual pdf 1 3 Contacting Caliper Technical Support If you need technical support please contact Caliper at Telephone 1 877 522 2447 Toll Free in the United States 1 508 435 9761 E mail tech support caliperLS com Fax 1 508 435 0950 Address Caliper Life Sciences 68 Elm Street Hopkinton MA 01748 USA 4 Living Image Software User s Manual This page intentionally blank
302. o register multi modal data aj x i Load the optical data e Bioluminescence or fluorescence image sequence and structured light surface e 3D source reconstruction DLIT or FLIT results page 206 Load 3D volumetric data CT or MRI page 241 Register the 3D source reconstruction and the 3D volumetric data by performing either e Automatic fiducial registration Available for data acquired on the Quantum FX UCT instrument using the Mouse Imaging Shuttle page 245 Or e Manual registration Match animal surface representations using the Manual Registration tool page 247 oe Classify the 3D volumetric data to help identify and separate objects page 250 Save the color opacity map optional Save the registered 3D multi modality results page 257 s i Ir a_j Y Caliper Chapter 15 3D Multi Modality Tools 241 15 2 Loading Data for Registration 1 Load a DLIT or FLIT image sequence and the 3D reconstruction results NOTE The 3D Multi Modality tools appear in the Tool Palette after you load optical image data If the 3D Multi Modality tools do not appear in the Tool Palette confirm that the 3D Multi Modality Tools license is installed and that the workstation graphics card meets the specifications in Table 15 1 page 239 2 To browse for DICOM or TIFF volumetric data select File Browse 3D Volumetric Data on the menu bar 3 In the Browse For
303. o save the selected data Save As a Movie Saves the kinetic data set as a movie mp4 mov avi mpg Y Ca i a Chapter 6 Kinetic Imaging 81 Viewing Kinetic Data 1 Open the kinetic data 2 To start playing the kinetic data click the Play button gt If you want to start the playback at a particular image first move the top slider to the starting image then click the Play button gt To stop playing data click the Stop button 4 To view the cumulative signal during playback choose the Accumulate option If the accumulated image maximum exceeds the current color scale range use the image adjust tools to adjust the color scale Kinetic Plot The Kinetic Plot is a graph of the maximum signal versus time To view the Kinetic Plot click the button in the Image Information tools Figure 6 10 Kinetic Plot ss I Kinetic Plot Window o E fmt Tool Palette 5 _ Image Adj gt a 5 Adit gt Corrections Filtering gt x10 Maximum Vs Time DA20081107162612 Image Information Maxim 1 len c B r f E units m Binning 4 Width 12 5cm Height 12 5 cm Image X Y 12 276 2 136 an Image Data 7 039768 photons sec an2 sr Crop Distance TA 0 00 0 00 B i 0 00 0 00 Time secs 0 00 0 00 Distance 0 00 E ROI Tools a Exporting Kinetic Data You can select a range of ima
304. ociated with these points can be changed v ie 7 Y Caliper Chapter 15 I 3D Multi Modality Tools 253 Figure 15 18 Histogram tool Color Opacity Map Each control point specifies a particular opacity intensity color Double click a control point to open the color palette Perspective TT Titi ie SSennn TILL HTT E i sgg D 4 Adi to Custam Cetera 3 To select a color for particular data double click a control point In the color palette that appears choose a color and click OK The software interpolates the color range between adjacent control points 4 To delete a control point right click the point To delete all control points click the button The and control points cannot be deleted from the histogram Saving a Color Opacity Map A color opacity map can be saved and applied to any volumetric data set 1 Click the Save button l Figure 15 19 2 In the dialog box that appears select a folder for the file tfn and enter a file name 3 Click Save Loading a Color Opacity Map 1 Click the Open button Ga Figure 15 19 2 In the dialog box that appears navigate to the map file tfn and click Open 254 Living Image Software User s Manual Figure 15 19 Save or load a color opacity map gt Se Tool Palette gt ROI Tools gt Planar Spectral Imaging _ Surface Topography gt DUT 3D 3D Multi Modality Tools slice S
305. of the luciferase spectrum please contact Caliper Life Sciences technical support You can view tissue optical property values Uefe U s Ha in the Tissue Properties drop down list The tissue properties are plotted as a function of wavelength Select the optical property descriptor most representative of the imaged subject Mouse Tissue is a good choice for general reconstructions in vivo NOTE Default tissue optical properties and source spectra are specified in the Preferences box For more details see Appendix B page 273 322 Living Image Software User s Manual This page intentionally blank YY Caliper Appendix IVIS Syringe Injection System Controlling the Infusion Pump 0 0 ee ee ee ee 323 Tracking Infusion in the Maximum vs Time Graph 2 2 2 4 325 Closing the Infusion Pump Control Panel 00 325 The IVIS Syringe Injection system is designed for use with the IVIS Kinetic Imaging System You can control the infusion pump in the Living Image software or manually For more details on the setup and manual control of the infusion pump see the V S Syringe Injection System instructions from Caliper or the PHD 22 2000 Syringe Pump Series User s manual from Harvard Apparatus Both are included on the Living Image installation CDROM The IVIS Syringe Injection system can be used during kinetic or still image acquisition however subjects must remain immobile 1 1 Cont
306. ol appears Figure 14 32 Displaying the transform tool Tool Palette Dasme 7 amp Ge ig BB liver lungs I ovaries E pancreas Wi rectum N is Use Tab key to switch between transformation tools Use X Y or Z keys to restrict scaling to only one axis us F y Scaling On XYZ photonsjsec Source Intensity 4 5 To adjust the x y or z position of the organ drag the transform tool Press the Tab key to put the transform tool in scale mode A red cube W appears at each corner of the transform tool 228 Living Image Software User s Manual 6 To increase or decrease scale the size of the organ drag a red cube at a corner of the transform tool To restrict scaling to a particular axis press the X Y or Z key then drag a red cube 7 Press the Tab key again to put the transform tool in rotate mode A red green and blue circle appear around the surface Figure 14 33 Rotating the organ on the surface pasties ee ee x ms A Use Tab key to switch between transformation tools Perspective Perspective The selected circle line is thicker 8 To rotate the organ on the x y or z axis click the blue green or red circle and drag the mouse arrow in the direction of interest To return the digital organ to the default position and size click the Reset button then g button 9 To turn off the transform tool click the Transfor
307. on Units Select the measurement units for the image display from this drop down list The available units depend on the type of image data For more details on measurement units see page 281 Display Select the image type for example X ray that you want to display from this drop down list For more details on the different types of image displays see Table 2 2 page 8 Note If the acquisition included more than two imaging modes for example luminescent X ray and photograph additional drop down lists appear so you can conveniently choose any two images to overlay Display E Fluorescent on E Ray z Info Click to display or hide the image label information Opens a dialog box that enables you to export the active view as a graphic file 54 Living Image Software User s Manual Table 5 2 Image window continued Item Description Takes a snapshot that is displayed with the data in the Living Image Browser A Living Image Browser anme EE Click Number EX Filter EM Filter Illumination Mode User ID Experiment Commenti 460 CK20100628141050 SEQ eee o ck CFT50 dye in pillows 0 25 pmol ul 2 ul bead 12 ee E E E EEA EAEE m PAT E oer tuner Doa cUdisassuntuveccensssessuns cae SAONE AANEEN ES EOIN TEENAA EAN 29 moas O Living Image O yO EEEE Close Preview Label Set Al v V Add to List Browse View Default v Configure Load as Group Load Remove
308. on Further the f stop can often be set to higher values f 2 or f 4 is 298 Living Image Software User s Manual recommended for fluorescence imaging A higher f stop improves the depth of field yielding a sharper image For more details on the f stop see Lens Aperture page 275 F 4 Image Data Display Fluorescent image data can be displayed in e Counts e Radiance photons Radiant efficiency Efficiency Illumination Power Efficiency calibrated normalized For more details see Quantifying Image Data page 281 If the image is displayed in any units other than counts you can compare images with different exposure times f stop setting or binning level When an image is displayed in terms of efficiency the fluorescent image is normalized against a stored reference image of the excitation light intensity Efficiency image data is without units and represents the ratio of emitted light to incident light For more details on efficiency see page 283 Fluorescent Efficiency and Radiant Efficiency The detected fluorescent signal depends on the amount of fluorophore present in the sample and the intensity of the incident excitation light At the sample stage the incident excitation light is not uniform over the FOV It peaks at the center of the FOV and drops of slowly toward the edges Figure F 10 To eliminate the excitation light as a variable from the measurement the data can be displayed in terms of efficiency Figure
309. on Profile Plots the measured intensity within the user selected area on the surface If no box was drawn on the surface measures the total intensity for the entire surface e Reduced Chi Squared Profile A measure of the difference between the computed and measured photon density maps at the optimum solution A smaller y value indicates a better quality of fit e Voxel Size Plots the voxel size at the start of the 3D reconstruction and at the end of the 3D reconstruction 216 Living Image Software User s Manual Figure 14 25 Example Quantification plot Quantification aC Reduce quared Voxel Size es Quantification photor sec 0 First surface 1 Second surface 2 Third surface and so on te E AE h ve Y Ca i BS or Chapter 14 3D Reconstruction of Sources 217 Table 14 7 Longitudinal Study window Item Display Voxels DLIT Red E Reverse T Log Scale MIP Description Voxel display controls Display Voxels Choose this option to show voxels with the surface From the drop down list select a color scheme for the color scale Move the sliders to adjust the color scale minimum and maximum values Reverse Choose this option to apply the colors of the selected color table in reverse order to the photon density scale For example the Red color table represents the source intensity photons sec from low to high using a color scale from transparent to red If
310. on filter wheel at the top of the imaging chamber collects the fluorescent emission from the target fluorophore and focuses it into the CCD camera All IVIS Imaging Systems require that one filter position on each wheel always be open for luminescent imaging IVIS Imaging System Number of Emission Filter Wheel Number of Available Positions Fluorescence Filters Spectrum 24 two levels each with 12 positions 22 60 mm diameter Lumina 8 7 4 sets of 7 high resolution filter wheels or a wheel with 4 standard filters 100 or 50 6 5 75 mm diameter F 2 Filter Spectra High quality filters are essential for obtaining good signal to background levels contrast in fluorescence measurements particularly in highly sensitive instruments such as the IVIS Imaging Systems Figure F 6 shows typical excitation and emission fluorophore spectra along with idealized excitation and emission filter transmission curves The excitation and emission filters are called bandpass filters Ideally bandpass filters transmit all of the wavelengths within the bandpass region and block absorb or reflect all wavelengths outside the bandpass region This spectral band is like a window characterized by its central wavelength and its width at 50 peak transmission or full width half maximum Figure F 7 shows filter transmission curves of a more realistic nature Because the filters are not ideal some leakage undesirable light not blocked by the filter
311. on preferences Camera Settings E7 Preferences Acquisition Auto Exposure Camera Settings Default Image Exposure Photographic Auto Luminescent 1 00 Fluorescent 1 00 Auto Save Optical Properties Folder C Users Katherine Documents Temp docs Default Image Binning Photographic Luminescent 4 7 5 Manual a Standard Restore Defaults Table B 4 Camera settings Item Default Image Exposure Description Sets the default exposure settings that appear in the IVIS acquisition control panel Default Image Binning Standard Binning choices include Small Medium and Large These are predetermined factory loaded binning values that depend on the imaging system camera Manual Allows the user to choose a binning value 1 2 4 or 16 Auto Save Specifies the folder where images are automatically saved Click the L J button to select a folder Restore Defaults Click to apply the default settings B 4 Theme Figure B 7 Image view preferences Ez Preferences Accson Image View Luminescent Fluorescent Background amp Text Color Background Color Text Color Text Size YellowHot ka E Reverse E Use saved color palette while loading datasets L a Color Palette ROI Color Luminescent B E Fluorescent Table B 5 Image view preferences Item Color Palette Description Use these controls to
312. opography Auto ROI Parameters DLIT 3D z Threshold o Reconstruction Lower Limit Lo E These Auto ROI 3D Multi Modality Tools A parameters are gt Spectral Unmixing i M E aE available if Show Preview Use Bkg Offset _ Replace ROIs Advanced O pti ons Restore Defaults Save Load is selected in the General Preferences For more details on setting Preferences see Appendix B page 265 Table 8 2 ROI tools Item Description oO Click to select the number of circle ROIs to add to the active image a C Click to select the number of square ROIs to add to the active image 2 H Click to specify the grid pattern for a measurement ROI that you want to add to a the active image This tool is useful for an image of a multi well culture plate or microplate Ch Click and select Auto All to automatically draw ROls in the image using the auto ROI parameters Click and select Auto 1 to automatically draw one ROI at a user selected location using the auto ROI parameters For more details on using the auto ROI features see page 127 wd Measure ROIs Click to display the ROI Measurements table or compute intensity signal in an ROI x Click to display a drop down list of options to delete an ROI s in the active image For more details see page 143 Note These commands do not delete the ROIs that are saved to the system listed in the Menu Name drop down list Apply to Sequence Choose t
313. optical and 3D volumetric data E 120101119144617_SEQ Calta i Sequence View LA 30 View Spectra k v E a e Bey G4 amp fev ig Sagittal x 0 7 Transaxialfy 8 0 Subject Height 15 0 mm Perspective To save the registration information 1 In the Results tab confirm the default name or enter a name for the results 2 Click Save Figure 15 10 3D Multi Modality Tools Results 3D Multi Modality Tools Volume Slice Results Volume Information MULTIMODALITY_6 Loaded Key Value StudyDate 20101119 NameOfVolume Mouse3_3_Day 28_145522 00 PatientsName Mouse3 Modality CT Manufacturer Rigaku BitsAllocated 16 SamplesPerPixel 1 Rows 256 Columns 256 NumberOfFram 512 PixelSpacing 0 236 0 236 SliceThickness 0 236 ImagePositionP 30 208000 30 208000 60 4160 ImageOrientati 1 0 0 0 1 0 PatientsBirthDate 20101018 4 wm p Save Results Name MULTIMODALITY_7 v Delete Load Save Y Ca i oe Chapter 15 3D Multi Modality Tools 247 Registration information is saved with the results for the volumetric data and is specific for a particular optical data set Manual Registration To manually register data use the 3D Multi Modality tools to translate scale or rotate the 3D volumetric surface so that features common to both surfaces are matched and aligned in the x y and z planes Examine the matched surfaces in the 3D slice views t
314. option is not chosen the data selected in the Browse for Folder box replaces the contents of the 3D Volumetric Data Browser except for loaded data Browse Opens the Browse For Folder box Load in a new window If this option is chosen multiple data sets can be loaded each in a separate window If this option is not chosen only one data set can loaded ata time Load Click to open the data selected in the 3D Volumetric Data Browser Chapter 15 3D Multi Modality Tools 243 Y Caliper feSciences Figure 15 4 3D optical and 3D volumetric data loaded but not registered Teci Palette REL Tena Planar Spectral Inugig Surface Topography DUT J0 Reconstruction 20 Melt Hedality Took Em zk Pues he tom hae E eLo seq D Sequence View e 30 view Soecin et Danay ime Ravel CHF etm Perfomance Chitty Cak Goudy Map Gayi 0 padty well Rrepa 1 E E a mni pagg inirmsihy 5 Eig J Legpertthee Halny am Maus ipes Prmeraan MIM mo Peete Gredent During JD Took Sera omiaa Histogram of voxel intensities Loading RAW Volumetric Data 1 Drag a single RAW file raw or vox from Windows Explorer to the 3D Multi Modality tools Figure 15 5 Only single raw or vox files consisting of multiple slices of a 3D volume can be loaded into Living Image Figure 15 5 Opening RAW volumetric data Tool Palette _ ROI Tools _ P
315. or examples of the views see Figure 12 7 ka Select this tool from the drop down list to display the perspective view E Click to show or hide measurement cursors in the coronal sagittal or transaxial views 7 7 After you perform DLIT or FLIT analysis click a voxel in the 3D reconstruction then click this button to display measurements for the voxel in the 3D tools source voxel measurements x Enables you to save the 3D view to a graphic file for example jpg x Y Caliper Chapter 12 Reconstructing a 3D Surface 181 Changing the View Perspective Figure 12 7 shows examples of the available views You can view the surface from different perspectives by doing one of the following e Select w to change the view Figure 12 6 e Alternatively click the surface in the 3D View window then press the V key to cycle through the different views of the surface Figure 12 6 Surface perspective view E TLT20050624122348_SEQ BAE b 3D View Spectra Sequence View Gronal z 11 0 Sagittal x 1 0 i Transaxial y 14 0 Subject Height 22 9 mm Perspective a View name 182 Living Image Software User s Manual Figure 12 7 Alternate views of a surface Bottom Right Back l Left Right 12 2 Managing Surfaces After the surface is saved it can be shared by the DLIT or FLIT tools Figure 12 8 Tool palette Surface to
316. or more details on measurement units see page 281 Display A list of image types available for display for example overlay For more details on the different types of image displays see Table 2 2 page 8 Note If the acquisition included more than two imaging modes for example luminescent x ray and photograph additional drop down lists appear so you can conveniently choose any two images to overlay Display E Luminescent on EA Photograph hi Info Click to display or hide the image label The image label includes information you enter in the Edit Image Labels dialog box Figure 4 2 and other image information automatically recorded by the software ig Opens a dialog box that enables you to export the active view as a graphic file y Caliper ie Chapter 4 Optical Imaging 25 Table 4 1 Image window continued Item Description wj Creates a preview picture snapshot of the image or thumbnails that the Living Image Browser displays when the data are selected in the browser For more details on the browser see page 83 ee rerem ak i Lit Ba Pen z ai ey Fehrs fe gepint gt ME CRBODSLIORIONSS SEQ ra Tina LMQ03TL1 DUIT subjects phantom gt Cy TLIA Seq TLT l i ISCO TITAH EO iope Preview Label Set all add to List Brose View Defaut coniare Levee ss crue Load Remove Di Location Chane Caliper L5 jipe Data Scrape Data TW1S200 data irarria LT 0457 err i
317. orescent images only Spectral Unmixing page 165 Extracts the signal of one or more fluorophores from the tissue autofluorescence Distinguishes the spectral signatures of different fluorescent or luminescent reporters when more than one reporter is used In the same animal model NOTE The 3D Multi Modality tools require a separate license YY Caliper DOr ces Chapter 2 Overview of Imaging amp Image Analysis 13 Additional tools are available in the menu bar after data are loaded Figure 2 6 Tools accessible from the menu bar Living Image 4 2 ie File Edit View Window Help ay LW amp i 3D Animation ily to all Longitudinal Study Well Plate Quantification for TLT20050624145507_SEQ Image Overlay for TLT20050624145507_SEQ Colorize Image Math for TLT20050624145507 SEQ Tool Description See Page 3D Animation Tools for creating an animation from an image sequence For example 231 an animation can depict a rotating 3D scene The animation can be recorded to a movie file mov mp4 or avi Longitudinal Enables you to view multiple DLIT and or FLIT reconstruction results 213 Study side by side provides a convenient way to compare longitudinal study results Voxel intensity within the entire surface or a user selected area can be measured in all results Well Plate Analyzes images of known serial dilutions of luminescent cells or 185 Quantification fluorescent dye mo
318. osition A on the cursor Pixel x y coordinates of position B on the cursor Length of the cursor from A to B number of pixels vertical distance from A to B number of pixels Distance Length of the cursor from A to B number of pixels 108 Living Image Software User s Manual To measure distance using the crop box 1 Open an image and in the Image Information tools click the Image Crop button i_i Figure 7 23 Using a crop box to make measurements Crop Distance 1 60 1 42 Sectaeageeey eeepc Sane RT Tool Palette E E 71120050624145507_005 ka a gt Image Adjust O O OOOO O O 2 Units Counts Display Overlay 7 Options v Info _ itj gt Corrections Filtering le Image Information a w A D E f BB Units cm v Image Binning amp Width 12 6 cm Height 12 6 cm Image X Y 0 497 11 311 am Image Data 7 counts Luminescence 10000 8000 Prrvesceseees 6 81 8 04 Distance 2 14am 6000 gt ROI Tools _ Planar Spectral Imaging J 4000 Surface Topography gt DLIT 3D Reconstruction 2000 gt 3D Multi Modality Tools _ Spectral Unmixing a Counts Color Scale Min 586 Max 10348 2 When the mouse pointer changes to a draw a rectangle on the area of interest To change the size or position
319. otal flux in the 3D quantification 13 3 Managing Quantification Results The quantification results can be saved with the image sequence and as a calibration database that is made available in the DLIT or FLIT 3D reconstruction tools in the Properties tab When you define the properties for performing a 3D reconstruction and a calibration database is specified the 3D reconstruction results will be displayed in calibrated units for cell numbers or molecule quantities in picomole units Figure 13 7 Save the quantification results 7 Well Plate Quantification Window Col E Jm Tool Palette For Sequence EL20090414101005_SEQ Click EL20090414101005_001 _ Image Adjust gt Fluorophore Type Corrections Filteri gt ering H Well Plate Type jl Dye molecules Cells _ ROI Tools A Measurement Sample Wells JD 3A C Set _ Surface Topography 7 Background Wells 6D 6A C setie FLIT 3D Reconstruction V Apply to Sequence Analyze Properties Results E Weli Piate Quantification Plots Results Tissue Properties Mouse Tissue Well Plate Quantification Results Unsaved Fluorescent Quantification Excitation Emission Extinction Coeff Cross Section WPQUANT_1 mm nm nm Qe M cm 1000 Qoa mm N cs 7 Cy Tissue Properties Plot 1 465 520 2 919e 07 1 115e 08 2 465 540 1 2
320. oto Adjustment Brightness Click and move the slider left or right to adjust the brightness of an image displayed in overlay or photograph mode Alternatively enter a brightness value Contrast Click and move the slider left or right to adjust the gamma of an image displayed in overlay mode Alternatively enter a gamma value Gamma is related to image contrast Opacity Click and move the slider left or right to adjust the opacity of the pseudocolor luminescent data of an image displayed in overlay mode Alternatively enter an opacity value Color Scale Min The minimum pixel intensity associated with the color scale for an image Pixels less than the minimum value are not displayed Max The maximum pixel intensity associated with the color scale for an image Pixels greater than the maximum value are displayed in the maximum color Color Scale Limits Auto If this option is chosen the software sets the Min and Max values to optimize image display and suppress background noise The Min and Max settings can be manually adjusted to further optimize the image display for your needs Full Choose this option to set the Max and Min values to the maximum and minimum data values in the image Manual Choose this option to enter Max and Min values for the image display Individual Applies a separate color table to each image in a sequence Note This option is only available when an image sequence
321. ound Corrected Signal 2 05825 eas 130 RIC Ae se wee bk eGR EEO EEE ORR EEE REE eS 132 Manado AUIS 4 kt tek OEP RE RHAEEEECKESERKEGHRER ERE EGS OS 135 Managing the ROI Measurements Table 2 005 ewes 145 8 1 About ROIs A region of interest ROI is a user specified area in an optical image Figure 8 1 The ROI tools enable you to create three types of ROIs measurement average background or subject ROI Table 8 1 During a session the Living Image software records information about the ROIs you create and computes statistical data for the ROI measurements The ROI Measurements table displays the data and provides a convenient way to review or export ROI information Figure 8 1 For more details see Managing the ROI Measurements Table page 145 Figure 8 1 Example measurement ROIs and ROI measurements table EF T1120050624145507 005 _ 7 Oeoiay Overa gt un Ga Image Number Image Layer Total Counts Avg Count Stdev Counts Min Count Max Count TLT200506241 45507_005 Overlay 6 Die 05 S5We Dd Jate O 2920 02 117le 0t TLT20050624145507_005 012 Overlay 3087e 05 1 319e 03 S4l3e 02 SAS3e 02 26556203 120 Living Image Software User s Manual Table 8 1 Types of ROIs Measurement ROI Type of ROI Average Background ROI Subject ROI Measures the signal intensity in an area of an image Measures the average signal intensity in a user specified area of the image that is considere
322. owed by three dark exposures The dark charge measurement usually takes more than three times as long to complete as the equivalent luminescent exposure E 2 Background Light On the Sample An underlying assumption for in vivo imaging is that all of the light detected during a luminescent image exposure is emitted by the sample This is not accurate if there is an external light source illuminating the sample Any reflected light will be detected and is indistinguishable from emission from the sample The best way to deal with external light is to physically eliminate it There are two potential sources of external light a light leak through a crack or other mechanical imperfection in the imaging chamber or a source of external illumination IVIS Imaging Systems are designed to be extremely light tight and are thoroughly checked for light leaks before and after installation Light leaks are unlikely unless mechanical damage has occurred To ensure that there are no light leaks in the imaging chamber conduct an imaging test using the High Reflectance Hemisphere Figure E 1 A more subtle source of external illumination is the possible presence of light emitting materials inside the imaging chamber In addition to obvious sources such as the light emitting diodes LEDs of electronic equipment some materials contain phosphorescent compounds Do not place equipment that contains LEDs in the imaging chamber Phosphorescence is a physical proce
323. p table Sequence Setup Click to display the sequence table so that you can specify and manage sequence acquisition parameters or open sequence acquisition parameters xsq For more details on setting up an image sequence see page 33 Image Setup Click to close the sequence table Initialize Click to initialize the IVIS Imaging System For more details on initializing the system see page 1 7 YW Caliper Appendix A I IVIS Acquisition Control Panel 263 Table A 2 Additional controls for the IVIS Imaging System 200 Series or IVIS Spectrum Item Alignment grid Description Choose this option to activate a laser generated alignment grid on the stage when the imaging chamber door is opened The alignment grid is set to the size of the selected FOV The grid automatically turns off after two minutes If subject alignment is not completed in two minutes place a check mark next to Enable Alignment Grid to turn on the grid Note The horizontal cross hair of the alignment grid is offset appropriately to take into account the height entered in the Subject height box Focus Scan Mid Image Choose this option in the Focus drop down list to set the focal plane at the maximum dorso ventral height of the subject at the middle of the animal This focusing method uses the laser to scan horizontally across the middle of the subject to determine the maximum subject height along this line This option is well
324. peak pixel A pixel is included in the ROI if the pixel intensity is greater than the threshold a user specified percentage of the peak pixel intensity Free draw Draw line segments that define the ROI 129 Manually Drawing a Measurement ROI 1 Open an image or image sequence and in the ROI tools select Measurement ROI from the Type drop down list 2 Select the ROI shape a Click the Circle OJ Square GJ or Grid button J The grid shape is useful for drawing a grid of ROIs on an image of a well plate b On the drop down list that appears select the number of ROIs that you want to add to the image or the grid ROI dimensions The ROIs and intensity measurements appear on the image NOTE Manual ROIs are numbered in the order they are created You may want to arrange the ROIs in a known order for easier comparison between images To renumber the ROIs ascending order from right to left right click the image and select Sort ROIs on the shortcut menu If the Apply to Sequence option is selected in the ROI tools choose Sort ROIs in Sequence to sort all of the ROIs in the sequence The sort options are only available if the ROIs have not been sorted 126 Living Image Software User s Manual Figure 8 7 Placing two circular ROIs on the image i E 11720050628145507_006 Ce ss s me m Luminescence 15000 Auto All Auto 1 Free Draw 20 g 10000 I Me Bkg Offset Replace RO
325. pecially when only part of the emission excitation spectrum is sampled Use Constraints Choose this option to apply the recommended constraints when performing spectral unmixing For more details on the constraints see page 174 To disable the constraints uncheck this option Alternatively in the Options tab of the Spectral Unmixing tools click Reset Values and then click Update Number of components to unmix The total number of components background and probe signals selected for unmixing Caliper Y p Chapter 11 Spectral Unmixing 169 ifeSciences 6 Click Finish when you are done choosing the components to unmix The unmixed images and results are displayed Figure 11 4 The results include a signal distribution map of each unmixed result and a composite image that includes all of the fluorescent signals each displayed in a different color Figure 11 4 Spectral unmixing results oS tr fee Tool Palette g E TLT20060406164950_ SEQ Spectral Unmixing ii Sequence View Spectra Unmixed Images EE og Analyze Options Results 7 Show Labels V Individual Scale amp sere e omens Item Tissue AF UmMX 2 Number of Iterations Number of Components Number of Wavelengths Number of Samples Lack of Fit PCA 3 56937 Lack of Fit EXP 3 84347 Divergence counter 0 Denoise PCA Medium Save Results Min 0 00 Min 0 00 Name
326. pectrum calculated for a user selected ROI ba Deletes the spectrum selected in the spectrum list from the plot Type The type of spectrum UMX A spectrum generated by the spectral unmixing algorithm LIB A user selected library spectrum The library includes spectra obtained of different sources obtained using excitation and emission filters ROI A spectrum calculated for a user selected ROI Name The spectrum identifier used by the unmixing algorithm The name cannot be modified Label The spectrum name in the spectrum plot key Double click the label to edit it Color The plot line color for a particular soectrum For the UMX type spectrum it is D also the color in the composite image Double click a color swatch to open the D color palette that enables you to change the plot line color for the spectrum 172 Living Image Software User s Manual Table 11 4 Spectra window continued Item Description Normalized Normalizes ROI spectra to library spectra Legend Click to show or hide the spectra plot legend Red Shift Adjusts library and ROI spectra to compensate for tissue absorption simulates the red spectral shift of a spectrum produced by a signal that is located at a depth of 5 mm in tissue Adding Spectra to the Plot You can add library spectra or a spectrum calculated for an ROI to the plot To display library spectra 1 Click the button arrow and select From Library A ne
327. pography tools Tool Palette Surface name F Y Caliper Chapter 12 Reconstructing a 3D Surface 183 Item in the Surface Description Topography Tools Name Name of the selected surface Delete Removes the selected surface from the system Load Opens the selected surface Save Saves a surface to the selected name Overwrite Saves the surface and overwrites the previous surface results 12 3 Export or Import a Surface A surface can be shared with other users or viewed in other 3D viewer applications Surface import capability is only available if Show Advanced Options is selected in the general preferences see page 266 1 Load a surface 2 Select File Export or Import 3D Surface on the menu bar In the dialog box that appears select a folder enter a file name and select a file type see Table 12 2 Importing a surface by this method is for viewing purposes only not for registration with optical reconstructions in Living Image software To import a surface or other organs for registration purposes import an organ atlas For more details see page 230 Table 12 2 Surface file types Export Option Description Export Import Surface mesh A native file format of the Living Image software that is yes yes xmh used to exchange 3D surface information between Living Image software and other third party analysis tools It is based on a basic indexed fa
328. port a surface For more details on how the software generates a surface see Appendix H page 317 Figure 12 1 Example surface and analyses that require a surface Perspective Generate a surface for one m o DLIT Analysis FLIT Analysis 3D reconstruction of luminescent sources 3D reconstruction of fluorescent source displayed as voxels page 193 displayed as voxels page 201 178 Living Image Software User s Manual 12 1 Generating a Surface P Load the image sequence for the reconstruction For example a sequence that was acquired for DLIT analysis In the surface topography tools make a selection from the Object drop down list nude mouse fur mouse or phantom Select an orientation dorsal or ventral Select a smoothing level Click Reconstruct The Tomography Analysis box appears By default the entire subject is selected for the reconstruction Figure 12 2 Surface topography tools and tomography analysis box Tool Palette i Image adiust JEE O E single view surface Topography analysis anar Spectralimegng crop image Surface Topography Surface Reconstruction Crop image Subject Nude Mouse w Orientation Dors v Draw a region of interest Surface Smoothing rectangle to proceed ia Snooth Save Results Name SURFACE_22348 TLT20050624122348 001 Valid crop region 6 If you want to reconstruct only a particular region of the subject resize
329. r 5 I X Ray Imaging 57 Table 5 3 Imaging Wizard Item Description Focus Drop down list of focusing methods available Use subject height Choose this option to set the focal plane at the specified subject height Manual Choose this option to open the Focus Image window so that you can manually adjust the stage position For more details on manual focusing see 264 Options Time Series Study Choose this option to specify the number of segments to acquire and a time delay between segments This option is usetul for acquiring data for kinetic analysis Note The sequence specified in the sequence table is called a segment Restart Wizard Returns the wizard to the starting screen Figure 5 9 Control panel and sequence table j B ee ng a y A ica tion Contro ani E Ti Binni F c ission Fi i i j xposure Time ing Stop Excitation Filter Emission Filter E Display Photographic Settings CH eee Medium rhs y Medium i2 I a Field of View E xFov 24 E Zoom w m Subjectheight 1 50 om Focus use subject height Temperature I Locked itiali E Number of Segments 1 Delay 0 0 min Apply to All X Remover Zf Update Insert Add Reuse photograph amp Reuse X ray options 7 Complete the rest of the Imaging Wizard When you complete the wizard the sequence information appears in the sequence table Figure 5 10 Figure 5 10 Co
330. r Measunng 3 In the Image Math window that appears select an image from box A and from box B The Image Math window shows a thumbnail of image A image B and the new image 152 Living Image Software User s Manual Figure 9 2 Image Math window and new image Click to export the image to a graphic Tile 7 E Image Math Window o amp se f tm20060510114512_005 BECEJ x Sequence TLT20060510114512_SEQ Units Radiant Efficiency Display Options z l Info a ig TLT20060510114512_001 a 71720060510114512_002 a ia TLT20060510114512_005 TLT20060510114512_001 1 00 TLT20060510114512_003 ana O TLT20060510114512_004 E TLT20060510114512_005 TLT20060510114512_006 i p TITINNENCINIVACIT ANT Epi fluorescence T1T20060510114512_001 a TLT20060510114512 002 E Radiant Efficiency TLT20060510114512_003 TLT20060510114512_004 W 6 0 TLT20060510114512_005 TLT20060510114512_006 TITIANGNS1N114517 ANT z 8 Color Scale Limits for A and B 4 9 x10 Full Auto ams Result Color Scale Limits 7 2 0 Radiant Efficiency Full Auto E Min 0 Result A B k z 0 Radiant Efficiency k 1 00 pisecicm aisr Wicm2 Compute k from ROI Y k Color Scale V with Photo from A X Min 3 98e7 Max 7 77e8 l Display Result For Measuring NOTE For more details on items in the Image Math window see Table 9 1 page 153 4 Select a math
331. r of the figure legend for the image selected in the image list Click the color swatch to open a color palette that enables you to select a new color for the figure legend Label The name of the image selected in the image list To edit the name double click the name in this box Right click the label name to show a short cut menu of edit commands for example Cut Copy Paste Copies the composite image to the system clipboard oS wy Click to export the composite image to a graphic file for example jpg Opens the Print dialog box YY Caliper i Chapter 11 Spectral Unmixing 171 11 3 Spectra Window The Spectra window plots the normalized spectra of the unmixed results Figure 11 6 Spectra window E TLT20060406164950_SEQ Lalaj z La Sequence View Spectra Unmixed Images Composite V Normalized V Legend Red Shift ee Spectrum List UMX 2 r y a TissueAF Loo Normalized Amplitude On Type Name Label Color V UMX 1 TissueAF W UMX 2 UMX 2 0 0 720 740 760 800 20 840 780 Wavelength nm Table 11 4 Spectra window Item Description ya Enables you to select a spectrum to add to the graph From Library Choose this option to select a probe from the Living Image database The library includes spectra of different sources obtained using excitation and emission filters From ROI Choose this option to display a s
332. ransillumination fluorescence Description Make a selection from the drop down list to specify the type of ROI measurements to include in the table Excludes ROI measurements from the table Includes Total Counts Avg Counts Stdev Counts Min Counts and Max Counts in the table Total Counts the sum of all counts for all pixels inside the ROI Avg Counts Total Counts Number of pixels or super pixels Stdev Counts standard deviation of the pixel counts inside the ROI Min Counts lowest number of counts in a pixel inside the ROI Max counts highest number of counts in a pixel inside the ROI For more details on count units see page 281 Note These numbers are displayed if the units selected in the ROI Measurements table and the image are the same Otherwise N A appears in each column Total Flux the radiance photons sec in each pixel summed or integrated over the ROI area cm x 4 Average Radiance the sum of the radiance from each pixel inside the RO number of pixels or super pixels photons sec cm2 sr Stdev Radiance standard deviation of the pixel radiance inside the ROI Min Radiance lowest radiance for a pixel inside the ROI Max Radiance highest radiance for a pixel inside the ROI For more details on photon units see page 282 Epi fluorescence Fluorescence emission radiance per incident excitation irradiance p sec cm2 sr HW cm 2 Transillumination fluorescence Fluorescence emission radia
333. re 3 4 After the system is initialized the temperature box turns green when the temperature is locked at the demand temperature 90 C or 105 C for IVIS Systems cooled by a Cryotiger unit indicating the instrument is ready for operation and image acquisition The demand temperature for the CCD camera is fixed Electronic feedback control maintains the CCD camera temperature to within a few degrees of the demand temperature The default temperature of the stage in the imaging chamber is 37 C but may be set to a temperature from 20 40 C Figure 3 4 IVIS Acquisition Control Panel IVIS Lumina XR Imaging Mode aT Time Binning F Stop Excitation Filter Emission Filter eee ok auto Emad fishes ee gt Field of View System Status X Rays will be produced when energized E xFov 24 E Zoom ide Acquire m e A Subject height 1 50 a cm a Sequence Setup Setup Focus use subjectheight v Temperature flocked maie Click the temperature box to Temperature box color indicates view the demand and oo measured temperatures of system not initialized the CCD camera and stage hit eae l J System is initialized but CCD camera temperature is out of range and not ready for imaging System is initialized and CCD camera is at or within acceptable range of the demand temperature and locked The system is ready for imaging NOTE The items in the control panel depend on the particular IVIS I
334. re B 5 Acquisition preferences Auto Exposure Ez Preferences Auto Exposure First Preference Second Preference Range Values Exp Time sec Binning Min 0 50 Max 60 Luminescent Fluorescent Auto Exposure Preferences Third Preference Min Target Count Minimum Luminescent 3000 Exposure Time F Stop Epi fluorescent 6000 Trans fluorescent 10000 F Stop Restore Defaults Table B 3 Auto exposure settings Item Luminescent Fluorescent Auto Exposure Preferences First Preference Second Preference Third Preference Target Count Minimum Description During auto exposure the software acquires a luminescent or fluorescent image so that the brightest pixel is approximately equal to the user specified Target Count Minimum If the target minimum count cannot be closely approximated by adjusting the first preference for example exposure time the software uses the first and second or first second and third preferences to attempt to reach the target max count during image acquisition A user specified intensity Range Values Exp Time sec Binning F Stop The minimum and maximum values define the range of values for exposure time F Stop or binning that the software can use to attempt to reach the target max count during image acquisition Restore Defaults Click to apply default settings 270 Living Image Software User s Manual Figure B 6 Acquisiti
335. rescence incident excitation intensity radiance of the subject illumination intensity NTF Efficiency Fluorescent emission image normalized Transillumination fluorescent to the transmission image which is measurements measured with the same emission filter and open excitation filter When image data is displayed in counts the image pixel contents are displayed as the numerical output of the charge digitizer on the charge coupled device CCD Figure D 2 The counts measurement also known as analog digitizer units ADU or relative luminescence units RLU is proportional to the number of photons detected in a pixel Counts are uncalibrated units that represent the raw amplitude of the signal detected by the CCD camera A signal measured in counts is related to the photons incident on the CCD camera The signal varies depending on the camera settings for example integration time binning f stop or field of view setting All IVIS Imaging Systems include a CCD digitizer that is a 16 bit device which means that the signal count range is from zero to 65 535 Sometimes the displayed signal count may appear outside of this range due to corrections applied to the image data for example background corrections 282 Living Image Software User s Manual Figure D 2 ROI measurements counts mode Select Counts Photons or Efficiency for the image data E 1172 050624145507_005 fete is wes
336. rol panel Figure 6 4 You are prompted to insert the Zoom Lens attachment Figure 6 4 Control panel Zoom option selected IVIS Acquisition Control Panel mE Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter T d Field of view E System Status C xFov zoom ide Acquire Pe em Subject height 15 g cm Sequence Setup 9 Da a re Carefully insert the zoom lens in its position in the top center of the imaging chamber Close the door and click OK when done 2 After you install the Zoom Lens attachment in the imaging chamber click OK in the prompt The stage moves to the Z field of view position NOTE When the Zoom Lens attachment is installed single images or sequential images can only be acquired at the Z field of view setting During sequence setup if you attempt to change away from the Z setting the sequence table will be cleared If you attempt to change from another field of view setting to the Z setting the sequence table is cleared of all previous settings before the camera settings for the Z position are added Y Caliper ie Chapter 6 I Kinetic Imaging 75 Removing the Zoom Len Attachment The imaging system is set to the Z field of view until the Zoom Lens attachment is removed 1 Remove the check mark next to Zoom in the control panel Figure 6 5 The stage moves to position C then you are prompted to remove the lens attachme
337. rolling the Infusion Pump After the IVIS Kinetic Imaging System is initialized and locked you can access the infusion pump controls 1 Select Acquisition gt Infusion Pump Setup on the menu bar The Infusion Pump control panel appears above the IVIS acquisition control panel Figure I 1 Display the Infusion Pump control panel m o e IE we te 288 amp m d me Ficar Pista Syringe Tesi Dier wu Etdin Site mirma i NOTE If you are going to acquire kinetic data open the infusion pump control panel before you open the kinetic acquisition control panel When the kinetic control panel is open the Acquisition menu is unavailable 2 Set the volume and flow rate 324 Living Image Software User s Manual Figure 1 2 Volume amp flow rate settings Infusion Pump Control Infusion Pump Control Panel Volume Flow Rake Syringe Type Diameter C Auto Start After Stark Mow 1OuLimin B D icc M 4 099 mr C Auto Stop After Acquisition Stop Mow 3 Make a selection from the Syringe Type drop down list the associated syringe diameter is automatically entered To enter a custom syringe a Select Custom from the drop down list b Click OK in the dialog box that appears Figure I 3 Confirmation message for a custom syringe Pl Information SHARING s Diameter not known For syringe type Custom 1 Please enter a valid diameter for the syringe selected
338. rs NOTE You can view multiple animations sequentially For example if you select Spin CW on X Axis and Spin CW on Y axis from the Presets drop down list the animation shows the 3D reconstruction spinning clockwise on the x axis then spinning clockwise on the y axis 5 To view the animation click Play Figure 14 38 3D Animation tools For more details on the animation tools see Table 14 13 page 232 Presets Spin CW on Z Axis k A A ES Select a preset animation Animation Setup Time Scale 100 Key Frame 1 Key Frame 2 Key Frame 3 Key Frame 4 Key Frame 5 Key frames box Key Frame 6 Key Frame 7 Key Frame 8 Key Frame 9 Key Frame 10 Frames Per Second 10 234 Living Image Software User s Manual Creating a Custom Animation To create an animation specify a custom animation setup or edit an existing setup 1 Open an image sequence and load 3D reconstruction results 2 Select properties to display in the 3D View window for example organs sources surface or photon density maps 3 Select View 3D Animation on the menu bar The 3D Animation tools that appear Clear the key frame box if necessary click the button and select Delete All Figure 14 39 3D Animation tools 3D Tools Surface Source Registration Animate Preset Animations Presets Spin CW on Axis Frame Factor 1 4 Animation Setup Time Scale o ol a
339. s Elg tronic BAGKOICUNG s sa 2be52c eee wR disnas ES Re EE Eo SOS 285 Background Light On the Sample 2 0 0 0 eee ee eee 286 Background Light From the Sample 2 05828 ee eee 288 The background sources of light from luminescent images are inherently very low This appendix discusses sources of background and how to manage them Due to the extreme sensitivity of the IVIS Imaging System residual electronic background dark current and luminescent emission from live animals autoluminescence are measurable and must be taken into account For information on fluorescent background see Appendix F page 299 E 1 Electronic Background The cooled CCD camera used in an IVIS Imaging System has electronic background that must be accurately measured and subtracted from the image data before the light intensity is quantified Raw data that is not corrected for electronic background results in erroneous ROI measurements Incorrect background subtraction may also result in serious errors However it is not necessary to subtract the electronic background when making a simple visual inspection of an image The types of electronic background include e Read bias An electronic offset that exists on every pixel This means that the zero photon level in the readout is not actually zero but is typically a few hundred counts per pixel The read bias offset is reproducible within errors defined by the read noise another quantity t
340. s select a destination folder enter a file name select a file type and click Save YW Caliper _ 7 Working With Optical Image Data Opening Optical Image Data 2 ee ee a 83 About the Image Window amp Tool Palette 0 2 558248 89 Viewing Image Information 2 ee ee a 93 Adding Comments or Tagstoanlmage 2 05082 8 ee eee 95 Adjusting Image Appearance 2 eee ee ee a 97 Correcting Optical Image Data 2 0 0 eee ee ee ee 99 Viewing Intensity Data amp Making Measurements 26 101 Creating a Transillumination Overview a e ee ee ee ee es 109 Overlaying Multiple Images eee ee ee 110 Rendering Intensity Data in Color 0 0 0 ee ee ee es 113 Exporting or Printing Images 1 25 sc ee cet Pee wei bebee dade s 114 Editing an Image Sequence 0 eee eee a 116 Creating an Image Sequence from Individual lmages 117 This chapter explains how to open Joad view and perform other operations on the optical image data obtained on an IVIS Imaging System 7 1 Opening Optical Image Data You can load optical images from the e Living Image Browser see below e Toolbar or menu bar page 87 Multiple data sets can be open at the same time NOTE To open a recently viewed file select File gt Recent Files on the menu bar Loading Optical Images From the Living Image Browser The Living Image Browser provides a con
341. s Acquisition Window Help se A amp R cnits Radiant Efficiency v E Apply to all ie EL20090623142613_ SEQ epee Tool Palette Sequence View gt Image Adjust Corrections Filtering Units Radiant Effic Use Saved Cc Options Info a R _ Image Information L gt ROI Tools 7 120090623142613 002 vse t saa Units Radiant Efficien v Display Overlay Options igi Image EL20090623142613_002 User Tue Jun 23 2009 14 27 33 Group E Level High Em Cy5 5 Ex Cy5 5 Epi llumination Bin 4 FOV 7 5 f2 1s OE Camera IVIS 11209 Andor iKon Commenti Animal Num Epi fluorescence Radiant Efficiency sec cm2 sr Es a Min 4 98e ywicm Color Scale Min 4 05e7 Max 1 82e8 Check the image min and max in the color scale to determine whether the signal of interest is above the noise level and below CCD saturation Table 5 4 Image window Sequence view Item Description Units Select the measurement units for the image display from this drop down list The available units depend on the type of image data For more details on measurement units see page 281 Use Saved Choose this option to display the image data using the color table that was specified Colors in the Preferences at the time of acquisition If this option is not selected image data are displayed using the color table currently spe
342. s a more complex expression that is derived from the model and includes terms that describe the effect of the tissue air boundary on the light propagation Both ueg and the function K are dependent on the wavelength Equation 1 shows that if the total integrated intensity ROI measurement is measured at several wavelengths it is proportional to an exponential function of the product of the depth and the optical property 4 4 Therefore the steps to planar spectral image analysis include e Acquire two or more images at different wavelengths e Measure the total integrated intensity on each image e Fit the measured values to the exponential function of Equation 1 The results of the fit are the total flux of the luminescence source S and the source depth d Y Caliper per Appendix G I Planar Spectral Imaging 311 G 2 Optical Properties Planar spectral image analysis requires prior knowledge of the tissue optical properties at the wavelength used at image acquisition The two main optical parameters are the e Absorption coefficient u that defines the inverse of the mean path before photons are absorbed by the tissue e Reduced scattering coefficient u that defines the inverse of the mean path before photons are scattered isotropically in the tissue The effective attenuation coefficient uep is a function of the absorption and reduced scattering coefficients Her Sua us OY 2 Calculation of the function K in
343. s table click Configure The Configure Measurements box appears Figure 8 31 Configure Measurements dialog box L Configure Measurements User Lists Name Counts Update A Sort Available Items tsai Analysis Comment Total Counts Analysis User ID Avg Counts Angle Stdev Counts Animal Model Add Min Counts Move Down Animal Number Max Counts Animal Strain Area ced Pixels Area cm7 Avg Dark Charge Counts Avg Efficiency Avg Fluorescent Bkg Counts Avg Radiance p s cm sr Avg Radiant Efficiency p s crm Binning Cell Line 4 mT Move Up Column headers in the active ROI table Select a configuration from the User Lists drop down list and click Customize To add column header to the ROI table make a selection from the Available Item list and click Add To remove column header from the ROI table select the item that you want to remove in the Selected Items list and click Remove To reorder an item in the Selected Items list select the item and click Move Up or Move Down The columns in the ROI Measurements table are updated Enter a name for the custom configuration in the Name box and click Save Y Caliper Chapter 8 ROI Tools 149 To delete a custom table configuration 1 Select the configuration from the User Lists drop down list and click Delete NOTE Preset table configurations cannot be deleted Copying or Exporting the ROI Measuremen
344. s the cumulative signal in each frame YW Caliper Chapter 6 Kinetic Imaging 73 Table 6 1 Kinetic acquisition settings continued ltem Color Scale Description Auto If this option is chosen the software chooses the color scale minimum and maximum Note Do not choose this option if the Accumulate option is selected Minimum A user specified threshold for the color scale minimum that is applied to the data if the Auto option is not selected Intensity signals less than the minimum are not displayed Maximum A user specified threshold for the color scale maximum that is applied to the data if the Auto option is not selected File Size Displays the Tile size of the kinetic stream dcm being acquired The file size display is only available in the Kinetic Acquisition panel Save Click to select an option for saving the data Save Current Image Saves the currently selected frame single image photograph and read bias Save Accumulated Image Saves the accumulated signal for the selected frames tiff Save Kinetic Data Saves all selected photographic luminescent or fluorescent images frames and the read bias image dcm The signal is not accumulated Done Closes the Kinetic Acquisition window Accumulated Signal The Accumulate option enables you to view increasing signals in real time If you plan to accumulate signals it is recommended that you perform a test acquisit
345. sce Contact Caliper technical support for a list of tested and approved cleaning compounds If it is necessary to introduce suspect materials into the imaging chamber screen the materials by imaging them Acquire an image of the material alone using the same settings for example FOV and exposure time that will be used to image the sample to determine if the material is visible in the luminescent image Microplates white black or clear plastic can be screened this way Screen all three types with a test image White plates appear extremely bright by IVIS Imaging System standards and interfere with measurements Black or clear plastic microplates do not phosphoresce making them better choices The High Reflectance Hemisphere provides a more definitive way to determine the presence of an undesirable light source Figure E 1 It is a small white hemisphere that is coated with a non phosphorescent material A long exposure image of the hemisphere should produce a luminescent image in which the hemisphere is not visible Figure E 1 High Reflectance Hemisphere and a plastic marker pen Left Photographic image Right Photograph with luminescent overlay The hemisphere is illuminated by phosphorescence emitted from the pen If any part of the hemisphere exhibits what appears to be luminescent emission it is actually the light reflected from a source illuminating the hemisphere Observe the side of the hemisphere that is
346. sce NEE F IYIS Acquisition Control Panel Imaging settings Imaging Mode Exposure Time Binning Fstop Excitation Filter Emission Filter a boie e h ee oen I Fluorescence E imaging settings puto Qo zips I Photographic a futo S ho afe Animal imaging settings Iv E i Field of View B System Stat Structured light Se X Rays will be produced imaging settings 4 when energized Service fio crn Acquire X ray imagin rere y ging a H Sequence Setup Subject height 0 00 cm settings Focus Juse subject height Temperature I locked Initialize NOTE The options available in the control panel depend on the selected imaging mode the imaging system and the installed filter wheel or lens option 3 5 Tracking System and User Activity Activity Window The Activity window shows the imaging system activities The software creates and saves a log of the system activities related to data acquisition for example the type and number of acquired images fluorescent lamp usage X ray tube accumulated usage and kinetic camera usage This information may be useful for Caliper field service engineers to understand the imaging system behavior over time or for troubleshooting The activity log is located at C Program Files Caliper Life Sciences Living Image 20 Living Image Software User s Manual Figure 3 6 Activity window View Tools QATools Acquisition Window Help Fhe Ww
347. scheme for voxel display Use the left and right sliders to set the minimum and maximum colors Reverse Choose this option to apply the colors of the selected color table in reverse order to the photon density scale For example the Red color table represents the source intensity photons sec from low to high using a color scale from transparent to red If Reverse is chosen the source intensity photons sec from low to high is represented using the color scale from red to transparent Log scale Choose this option to apply a logarithmic scale to the color table Measured Sources Quantification DLIT For uncalibrated sources the total flux measured for the sources selected using the Measure Source tool pod For calibrated sources this unit will be in cell units For details on using this tool see page 209 Quantification FLIT For uncalibrated sources the fluorescence yield measured for the voxels selected using the Measure Source tool fae Fluorescence yield is expressed in units of pmol M cm here for uncalibrated sources For calibrated sources this unit will be in either cells or pmol For details using this tool see page 209 Volume Volume of the selected source mm 3 Center of Mass DLIT or FLIT The weighted average x y and z coordinates of the selected voxels where the weights are the flux of each highlighted voxel Host Organ The location of the selected source can be referen
348. se the Photograph and Auto options and click Acquire 7 Click Acquire when you are ready to capture the image NOTE If necessary click mage setup jin the control panel to operate in single image mode In single image mode the Sequence Setup button appears in the control panel Click this button to set up sequence acquisition For details on sequence setup see page 33 YY Caliper Chapter 41 Optical Imaging 27 Figure 4 6 Edit Image Labels box bda mage Usbels z UserID KSA Living Image Universal Saved Labels LABELS_1 A E X Check any 5 fields for display J User X 4 4 4 4 4 4 4 4 4 4 4 i Apply To Sequence x cane The information entered here appears in ee the image label Figure 4 8 page 28 8 Inthe Edit Image Labels box that appears enter information about the image and click OK NOTE You can enter image label information at any time during or after acquisition If you do not want to enter image information click Cancel 9 If this is the first image of the session you are prompted to enable the autosave function Figure 4 7 Autosave prompt E Living Image 4 2 This can be changed anytime from the Acquisition menu 3 Do you want to enable auto saving of acquired data for this session 10 To enable autosave cl
349. se the Photograph option and click Acquire 10 When you are ready to capture the image click Acquire 32 Living Image Software User s Manual Figure 4 12 Edit Image Labels box eG Edit Image Labels ss UserID KSA v Living Image Universal Saved Labels LABELS_1 A B Xx Check any 5 fields for display User v cams The information entered here appears in 7 Experiment the image label Figure 4 8 page 28 J Commenti Treatment Luc Injection Time IACUC Number E 4 4 4 4 4 4 4 Apply To Sequence ox canei 11 In the Edit Image Labels box enter information about the image and click OK NOTE You can enter image label information at any time during or after acquisition If you do not want to enter image information click Cancel 12 If this is the first image of the session you are prompted to enable the autosave function Figure 4 13 Autosave prompt Living Image 4 2 eal Do you want to enable auto saving of acquired data for this session This can be changed anytime from the Acquisition menu 13 To enable autosave click Yes in the prompt and choose a folder in the dialog box that appears If autosave is enabled all images acquired during the session are automatically saved to a user selected folder
350. set the exposure parameters select the Manual Settings option For more details on the exposure parameters see page 259 E Luminescent To include a photograph or X ray image in the EG Photograph acquisition put a check mark next to the an Photograph or X ray option a y X Ray grap y Op In the control panel the Photograph and X ray Reuse option is selected by default Figure 5 9 This means the same X ray image and photograph will be used if camera conditions do not change for example binning or F Stop If you do not want to reuse the X ray image or photograph you can manually edit the image sequence in the sequence table for more details see page 44 Alternatively remove the check mark next to Reuse in the control panel before you begin the Imaging Wizard Field of View Sets the size of the stage area to be imaged by adjusting the position of the stage and lens The FOV is the width of the square area cm to be imaged A smaller FOV gives a higher sensitivity measurement so it is best to set the FOV no larger than necessary to accommodate the subject or area of interest The FOV also affects the depth of field range in which the subject is in focus A smaller FOV results in a narrower depth of field but gives a higher resolution image Select the FOV by choosing a setting A B or C from the drop down list For more details on the calibrated FOV positions see Table A 3 page 263 Y Caliper feSciences Chapte
351. software from the application folder The main window appears Figure 3 1 16 Living Image Software User s Manual Figure 3 1 Living Image main window Fle Eiti Toos Window SAUSAS R 2 Select a user ID from the drop down list or enter a new User ID up to three letters and click OK The control panel appears if the workstation controls the IVIS Imaging System Figure 3 2 For more details on the control panel see Appendix A page 259 Figure 3 2 Living Image main window and IVIS Acquisition Control Panel Menu bar for more details see Appendix J page 327 o DRAN Toolbar F Stop Excitation Filter Emission Filter Field of View C System Status _ Mouse Imaging Shuttle System needs initialization Ba ln onmia buono Subjectheight 1 50 om i Sequence Setup Activity Focus use subjectheight v Temperature ___ Unlocked OO niie Oe window a Description i Living Image 4 2 Mar 7 2011 11 16 22 Version 4 2 0 13104 i IVIS configuration file found and loaded gt gt KSA Logged IN lt lt NOTE The Living Image software on the PC workstation that controls the IVIS Imaging System includes both the acquisition and analysis features The Living Image software on other workstations includes only the analysis features Macintosh users have access to the analysis features only 3 2 Initializing the IVIS Imaging System The imaging
352. source measurements page 209 e Export voxel measurements csv Figure 14 30 Source tools and example DLIT reconstruction Tool Palette Display Source Surface madi Adi Opacity Display Voxels Maximum Intensity Projection Threshold 101 E Gradation fe 5 a Voxel Size 0 31 Display Voxels As snooting 5x5 gt Color Scale min E 2 83e6 gt Color Table E Reverse n n T E Log Scale Measured Sources P Value Subiect Height 263mm Perspective ification 0 00 photons sec 3 61e 10 mm 3 0 00 mm Center of Mass 0 00 0 00 0 00 Host Organ Unknown Export Voxels Center of Mass Table 14 11 Source tools Item Description Select Source A drop down list of available sources Original Results saved with the data lt sequence name SourceVoxels gt Pasted voxels Click the a button to remove pasted voxels from the surface Display Source Choose this option to display the source surfaces reconstructed using Surface DLIT or FLIT A surface will be wrapped around the currently displayed voxels Adjust the voxel display by moving the Threshold slider YW Caliper Chapter 14 3D Reconstruction of Sources 223 Table 14 11 Source tools continued ltem A ih te Description Drawing styles for the source surface see Display Source Surface Ge E E i Shading styles for the source surface see Display Sour
353. ss similar to fluorescence but the light emission persists for a longer period Phosphorescent materials absorb light from an external source for example room lights and then re emit it Some phosphorescent materials may re v iw Y Caliper Appendix E Luminescent Background Sources amp Corrections 287 emit light for many hours If this type of material is introduced into the imaging chamber it produces background light even after the chamber door is closed If the light emitted from the phosphorescent material illuminates the sample from outside of the field of view during imaging it may be extremely difficult to distinguish from the light emitted by the sample IVIS Imaging Systems are designed to eliminate background interference from these types of materials Each system is put through a rigorous quality control process to ensure that background levels are acceptably low However if you introduce such materials inadvertently problems may arise Problematic materials include plastics paints organic compounds plastic tape and plastic containers Contaminants such as animal urine can be phosphorescent To help maintain a clean imaging chamber place animal subjects on black paper for example Artagain black paper Strathmore cat no 445 109 and change the paper frequently Cleaning the imaging chamber frequently 1s also helpful OLN ALERT Use only cleaning agents approved by Caliper Many cleaning compounds phosphore
354. ssssssosssssssssssossssssssssessstssssssessssosssosssostssssssssssesesssssesessssseses sssssssssososososssssssosso sssssesssesessevosssssssessssossssotosssssssssosssesssesssesesesesesosotsssossssssssosssosssossossssetsssesssessssssesoss dcm 20110306103654 094 dcm a dcm 20110306103654 095 dcm dcm 20110306103654 096 dcm dcm 20110306103654 097 dcm dcm 20110306103654 098 dcm dcm 20110306103654 099 dcm dem 20110306103654 100 dem dcm 20110306103654 101 dcm dcem 20110306103654 102 dem dcm 20110306103654 103 dcm _dcm 20110306103654 104 dcm Ei Start Index 1 M Auto Preview bI EndIndex 256 oar H ond la 100 Data will be loaded in 7LT20050624145507_SEQ Move the slider to select a particular slice for viewing or click an image Table 14 9 Living Image 3D Browser DICOM viewing controls Item Description Start Index Specifies the first image slice for viewing Auto Preview Select this option to automatically play back the images End Index Specifies the last image slice for viewing Load Opens the DICOM data in a 3D View window Load data in If this option is selected DICOM data are opened in a new 3D View new window window when you click Load If this option is not selected DICOM data are loaded in the active 3D View window 220 Living Image Software User s Manual 14 9 3D Tools Overview The Tool Palette includes the 3D Tools after you reconstruct or load a surfac
355. subtract the autofluorescence background For more details see Subtracting Tissue Autofluorescence page 153 F 6 Subtracting Instrument Fluorescent Background The fluorescence instrumentation on an IVIS Imaging System is carefully designed to minimize autofluorescence and background caused by instrumentation However a residual background may be detected by the highly sensitive CCD camera Autofluorescence of the system optics or the experimental setup or residual light leakage through the filters can contribute to autofluorescence background The Living Image software can measure and subtract the background from a fluorescence image Fluorescent background subtraction is similar to the dark charge bias subtraction that is implemented in luminescent mode However fluorescent background changes day to day depending on the experimental setup Therefore fluorescent background is not measured during the night like dark charge background 1s After you acquire a fluorescent image inspect the signal to determine if a fluorescent background should be subtracted Figure F 19 If background subtraction is needed remove the fluorescent subject from the imaging chamber and measure the fluorescent background select Acquisition Fluorescent Background Measure Fluorescent Background on the menu bar In the Living Image software the Sub Fluor Bkg check box appears on the Control panel after a background has been acquired You can toggle the
356. subtract tissue autofluorescence background from signal Living Image Tool Use This Tool To See Page Image Math Mathematically combine add multiply subtract or divide two 151 user specified images Image Math Remove autofluorescence from a fluorescent image Sa To perform image math open an image sequence or a group of images For more details on creating a sequence from individual images see page 117 9 1 Creating a New Image Using Image Math 1 Load an image sequence 2 Select Tools Image Math for lt name gt _SEQ on the menu bar Figure 9 1 Opening the Image Math window File Ect View Window Help CEE E yea Lorsqituchmal Study Well Plate Quantification for TLT20050510114512 SEQ image Overlay for TLT20080510114512_SEQ Colonte Image Math for TLT20060510114512 SEQ Memory Tracker E tirma SEQ Sequence TL120060510114512 SLQ C Sequence View spectra FLE20060510114512_001 Urita Radantificency TLT20060510114512_002 TIT200605101 1455 2 003 TiTa0060S1011455 7 004 TLr2cce0s10114512 905 TLr20060510114512_008 TIT ORNS 10114589 ANT v TL20060510114512 00 TI2060510114512_002 TIT2OOOS101 14517008 TITA00605101 14517004 TIT200G0S10114512 005 TLP200G0S10114512 006 TIT WWENSINI ASS iT Color Seale Limits for A and D or Auto Rest Color Scale Liewts Radan Elficerny w ru Auto Renit dress k 1 00 Compute K from RO F Z wh Photo from A l Deolay Resut Fo
357. suited for animal imaging because the peak height is clearly identified as the maximum height on the dorsal side along the mid plane of the animal Note This focusing method is not recommended for microplates or when using a high magnification field of view FOV A 4 0 cm In these situations Manual or Subject Size focus methods are recommended Transillumination Setup IVIS Spectrum only Choose this option to display the transillumination setup window that enables you to select the locations for image acquisition using bottom illumination that originates beneath the stage Table A 3 Typical field of view FOV settings e IVIS Imaging System Lumina Lumina XR 100 Series 200 Series Spectrum Kinetic FOV cm A 5 5 10 4 4 4 B 7 5 Ta 15 6 5 6 5 7 C 10 10 20 13 13 10 D 12 5 12 58 25 22 5 19 5 22 5 19 5 12 E 246 N A N A 22 5 26 22 5 26 245 Zd 2 6 2 6 N A N A N A 2 6 a Position D is not available for X ray imaging on the Lumina XR b Available with removable lens option XFOV 24 Not available on the IVIS Lumina XR c Some imaging systems may have the FOV in parentheses FOV 19 5 and 26 were replaced by FOV 22 5 d Position Z is available when the Zoom lens is installed on the IVIS Lumina IVIS Lumina XR or IVIS Kinetic Imaging System 264 Living Image Software User s Manual A 2 Manually Setting the Focus The IVIS Imaging System automatically focuses
358. surements table E ROI Measurements ROI Measurements Image Number TLT20050624145507_001 TLT20050624145507_002 TLT20050624145507_003 TLT20050624145507_003 TLT20050624145507_004 Customized Selections Configure Export Image Laye Total Coun Avg Count Stdew Cour Min Count Max Count Overlay Overlay Overlay Overlay 6 536e 03 2 653e 04 290e 05 2 687e 05 1 621e 06 2 334e 02 6 982e 02 2 961e 04 4 405e 03 4 053e 04 4395e 01 1 402e 02 5 80 7e 03 9302e 02 599e 05 1 f23e 02 4 730e 02 2 209e 04 3 042e 03 2576e 04 amp lls 3 301e 02 9 393e 02 4280e 04 6 048e 05 5 699e 04 Measurements Types Image Attributes ROI Dimensions Counts Y _none_ ha none _ ka Overlay LT20050624145507 004 ROLIG Overlay 7273e 05 8 456e 05 1 784e 03 6007e 03 119 Copy Select All Close The ROI Measurements table displays data for all ROIs created in images or sequences during a session one ROI per row The table provides a convenient way to review and export ROI data For more details on the table see Managing the ROI Measurements Table page 145 To automatically draw an ROI at a user specified location 1 Open an image 2 Click an ROI shape button Circle OJ Square O or Contour and select Auto 1 from the drop down list The create tool appears on the image Figure 8 10 ROI create tool r PSS eS SS E TLT20050624
359. system must be initialized each time the Living Image software is started or if the power has been cycled to the imaging chamber or the camera controller a component of some IVIS Imaging Systems The initialization procedure moves every motor driven component in the system for example stage and lens to a home position resets all electronics and controllers and restores all software variables to the default settings Initialization may be useful in error situations For further details on instrument operation see your IVIS Imaging System hardware manual To initialize the IVIS Imaging System 3 Start the Living Image software double click the f4 icon on the desktop 4 Inthe control panel that appears click Initialize You will hear the motors move Figure 3 3 IVIS Acquisition Control Panel F Stop Exgtation Filter Emission Filter Field of View Mouse Imaging Shuttle fsa m Subjectheight 1 50 2 om ran ervoor ND ies ee NOTE The control panel is only available on the workstation that controls the imaging system The items available in the control panel depend on the particular IVIS Imaging System and the imaging mode selected luminescent or fluorescent Image Setup or Sequence Setup mode 18 Living Image Software User s Manual 3 3 Checking the System Temperature The IVIS acquisition control panel indicates the temperature status of the charge coupled device CCD camera Figu
360. t an ROI from the ROI drop down list in the ROI Properties dialog box Figure 8 18 Figure 8 18 Opening the ROI Properties dialog box T 11120050624145507 005 oe esa Units Counts Display Overlay z Options Info 5 w Subject 1 Background ROI Luminescence Image Number 10000 TLT20050624145507 ROI 1 2 342e 03 none 2000 6000 ROI 50 J 3 349e 05 4000 V Lock Position ROT 850 J 1 63e 05 Xc pix 106 81818 2000 Yc pix 163 63636 Resize Angle deg 0 0000 Rotate Counts Copy ROI sa a Color Scale Width pix 11 00000 Copy All ROIs fe pix Max 10348 j 11 0900000 Paste ROI Height pix 11 00000 Duplicate ROI Line Size Set Bkg ROI to none Line Color Set Subject ROI to none Set Subject ROI to Subject 1 Hide ROI Tag Delete ROI Delete All ROIs Properties Unlock Position Unlock Size 136 Living Image Software User s Manual Figure 8 19 ROI Properties Background ROI tab The items in the ROI Properties box depend on the type of ROI selected in the image For more details see Table 8 3 page 137 E BCEE Options 7 Info gy a TLT20050624145507_005 Luminescence BKG 1 1 891e 03 10000 8000 6000 ROT 50 J 3 349e 05 4000 ROT 8 50 J 1 63e 05 Py 2000 Counts Color Scale Min 586 Max 10348 p E TLT20050624145507_005
361. t etc A list of the open image windows Click a window in the list to make it the active window indicated by a check mark Window gt Other Windows lt window name gt Lists other windows that are open For example If the Living Image Browser is open use these commands to make the browser the active window and display it on top of all other open windows Help User Guide Displays the Living Image User Manual Help License information Displays the license information Help Plug in Information Displays a list of tool plug ins and Tool Palette plug ins Help IVIS Reagents Opens the Caliper LS web page for In Vivo Imaging Reagents Help About Living Image Displays information about the Living Image software and Caliper technical Support contact information Click this button then click an item in the user interface to display information about the item 330 Living Image Software User s Manual Table J 2 Keyboard shortcuts Keys Shortcut Description Ctrl B Opens the Living Image Browser Ctrl C Copies the active image to the system clipboard Ctri D Arranges open windows in a cascade Ctri O Displays a dialog box that enables you to open data Ctrl P Open the Print dialog box Ctrl S Saves the active file or window Ctrl T Tiles the open windows Ctrl W Closes the active window Shift F1 Changes the mouse pointer to
362. t Colors Color Theme Background Color Restore Defaults Restore Defaults Source Vowels Reverse Suface amp Text Color a Surface color EJ Top a Text Color TE Bottom Ji Text Size j8 Color Palette Table B 6 3D view preferences Item Color Theme Description Predefined color schemes available for the 3D View window shown here Click the button to restore the defaults for the selected color theme E7 Turz00ses24145507 SEQ col rg C sequexeview 5 Ver specra Background Color Settings that modify the appearance of the background in the 3D View window Solid Color Choose this option to apply a non gradient background color to the 3D view in the image window Gradient Color Choose this option to apply a gradient background color to the 3D view in the image window Top the color at the top of the window Bottom the color at the bottom of the window Surface amp Text Color Settings that modify the display of the surface and text in the 3D View window Color Palette Source voxels Choose a color table for voxel display Reverse Choose this option to reverse the min max colors of the selected color table Restore Defaults Click to apply the default settings Y Caliper B 5 Optical Properties Appendix B Preferences 273 Figure B 9 Set the default optical properties preferences left for the Properties tab in the Plan
363. t are acquired during the same session and are intended to be grouped together For example images taken at different time points or an image sequence for DLIT or FLIT 3 D tomographic analysis Images that were acquired during different sessions can also be grouped together to form a sequence for more details see page 117 For example a time series could be constructed from images acquired on different days following an experimental treatment Some types of analyses are performed on an image sequence see Table 2 3 The sequence requirements number and type of images depend on the type of analysis Table 2 4 shows the types of analyses that are possible on the different IVIS Imaging Systems Table 2 3 Analyses that require an image sequence Analysis Planar spectral image Description Page Computes the total flux and average depth of a luminescent source 10 below the surface fluorescent sources Display multiple fluorescent or Uses the Image Overlay function to display multiple luminescentor 110 luminescent reporters fluorescent images on one photographic image Subtract tissue autofluorescence Uses the image math feature to subtract a background image from 151 using blue shifted background filters the primary image Spectral unmixing Removes tissue autofluorescence from a fluorescence image 165 DLIT Reconstructs the brightness and 3D location of luminescent 193
364. t the depth of the luminescent cells can help quantify the source brightness and provide information on the location of the cells The Living Image software uses spectroscopic information from a single view image to estimate the depth of the luminescent cells The method takes advantage of the fact that firefly luciferase bioluminescence is emitted from 500 to 700 nm a region of the spectrum where there are major contrasts in tissue optical properties Figure G 1 In this portion of the spectrum tissue absorption drops off dramatically between 500 580 nm green yellow wavelengths and 600 750 nm red wavelengths due mainly to the presence hemoglobin As a result the luminescent signal observed on the surface of the animal is dependent on both the wavelength and the thickness of the tissue through which it travels The depth and absolute photon flux of a single point source can be determined from two or more images acquired at different wavelengths using relatively simple analytical expressions derived from the diffusion model of the propagation of light through tissue 310 Living Image Software User s Manual Figure G 1 Optical Properties of Mouse Tissue and Firefly Luciferase Spectra The luminescent signal from firefly luciferase right is emitted from wavelengths of 500 700 nm which spans a region of the spectrum where there are major contrasts In the optical properties of mouse tissue left The firefly spectrum was measured at 37
365. t these wavelengths The Living Image software controls the illumination YY Caliper per Chapter F Fluorescent Imaging 293 intensity level off low or high The illumination intensity at the low setting is approximately 18 that of the high setting Figure F 4 Relative spectral radiance output for the quartz halogen lamp with dichroic reflector Quartz Halogen Lamps with Dichroic Reflectors R Cut Off EJV Lamp 3400 K 10 mm EKE Lamp 3200 K Relative Spectral Radiance 400 600 800 1000 1200 1400 1600 1800 Wavelength nanometers The lamp output is delivered to the excitation filter wheel assembly located at the back of the VIS Imaging System Figure F 5 Light from the input fiber optic bundle passes through a collimating lens followed by a 25 mm diameter excitation filter The IVIS Imaging System provides a 12 position excitation filter wheel allowing you to select from up to 11 fluorescent filters five filters on older systems A light block is provided in one filter slot for use during luminescent imaging to prevent external light from entering the imaging chamber The Living Image software manages the motor control of the excitation filter wheel Figure F 5 Excitation filter wheel cross section Fused Silica Callimating Fiber Optic Bundle Input Lenses Fiber Optic Bundle Excitation Motor Filter Following the excitation filter a second lens focuses light int
366. tative comparison of fluorescent signals When you select efficiency for the image data Figure D 2 the software normalizes the fluorescent emission image to a reference image and computes Efficiency Radiance of the subject Illumination intensity Prior to instrument delivery Caliper Life Sciences generates a reference image of the excitation light intensity no emission filter incident on a highly reflective white plate for each excitation filter at every FOV and lamp power The data are stored in the Living Image folder Image efficiency data does not have units The efficiency number for each pixel represents the fraction of fluorescent photons relative to each incident excitation photon and is typically in the range of 102 to 10 When ROI measurements are made the total efficiency within the ROI is the efficiency per pixel integrated over the ROI area so the resulting units of total efficiency is area or cm2 D 3 Flat Fielding Flat fielding refers to the uniformity of light collected across the field of view FOV A lens usually collects more light from the center of the FOV than at the edges The Living Image software provides a correction algorithm to compensate for the variation in the collection efficiency of the lens This enables uniform quantitation of ROI measurements across the entire FOV To apply the correction algorithm choose the Flat Field Correction option in the Corrections Filtering tools The algorithm multipli
367. ter 14 3D Reconstruction of Sources 209 14 5 Measuring Sources This section presents a convenient way to measure the source total flux or total florescence yield or if calibrated the abundance in cells or picomoles The volume center of mass and depth at the center of mass are also reported in the 3D Tools Source tab NOTE If the surface contains voxels pasted from other reconstruction results choose a source in the 3D Source tools Figure 14 17 For more details on pasting voxels see page 213 Determine Source Center of Mass HE 1 Click the toolbar button and then drawing a box around the source 2 Click Center of mass in the 3D tools Source tab Figure 14 17 to obtain measured source information Quantification The integrated intensity within the selected sources Volume The total volume of the selected sources Depth The perpendicular distance from the source center of mass to dorsal surface Center of Mass The weighted average x y and z coordinates of the selected voxels where the weights are the flux of each highlighted voxel Host Organ The organ in which the selected sources are located This information is available if organs are displayed with the reconstruction For more details on displaying organs see 3D Tools Registration page 225 NOTE The coronal sagittal and transaxial planes intersect at the center of mass of the selected source Figure 14 18 210 Livin
368. terest Make sure that you click the transformation tool so that it is highlighted before you use it Otherwise the dragging operation is applied to the optical data structured light surface 5 To return the 3D volumetric data to the default position and size click the Reset Registration button J 6 For details on saving the registration information see page 246 Registration information is saved with the results for the volumetric data and is specific for a particular optical data set 250 Living Image Software User s Manual 15 4 Classifying 3D Volumetric Data The 3D Multi Modality tools provide a histogram based method to classify the 3D volumetric data The histogram represents the distribution of voxel intensities in the 3D volumetric data and their color opacity values The goal of classification is to set color and opacity values for different intensity ranges so that the color opacity map shows the volume regions you are interested in opaque in the map and hides unimportant regions transparent in the map For example Figure 15 15 shows how the histogram tool designed a color opacity map that shows both the skin and bone The histogram tool enables you to easily re design the color opacity map to show just the skin or just the bone The 3D Multi Modality tools also enable you to classify the volumetric data by specifying color and opacity values for different intensity ranges so that you can easily view or hid
369. ters in the system before turning on the lamp Subsequent changes to the filter popup menus will have no effect until another Inspect operation is performed Field of View Sets the size of the stage area to be imaged by adjusting the position of the stage and lens The FOV is the width of the square area cm to be imaged A smaller FOV gives a higher sensitivity measurement so it is best to set the FOV no larger than necessary to accommodate the subject or area of interest The FOV also affects the depth of field range in which the subject is in focus A smaller FOV results in a narrower depth of field Select the FOV by choosing a setting from the drop down list For more details on the calibrated FOV positions A E see Table A 3 page 263 Service Moves the stage to a position for cleaning the imaging chamber below the stage Only available on the IVIS Imaging System 200 Series and IVIS Spectrum Mouse Imaging Shuttle Choose this option if the subject will be contained in the Mouse Imaging Shuttle during image acquisition Note The Mouse Imaging Shuttle or the Ventral Imaging Chamber can be used with the IVIS Kinetic but not both at the same time Load Moves the stage from the cleaning position back to the home position XFOV 24 Note This check box is only available on an IVIS System that includes the XFO 24 lens option When the XFO 24 lens is installed choose the XFOV 24 option For more details on ho
370. than the minimum color table setting are not displayed As a result the lowest intensity color in the table is transparent and this enables you to view the underlying photographic image in regions where the luminescent light emission is low While the pixels less than the minimum color table setting are not displayed they still exist in the image data Y Caliper Appendix D Image Data Display amp Measurement 281 D 2 Quantifying Image Data Counts The Living Image software can quantify and display scientific image data for several types of measurements Table D 1 Data display units Data Display Description Recommended For Counts An uncalibrated measurement of the Image acquisition to ensure that photons incident on the CCD camera the camera settings are properly adjusted Proper image parameter adjustment should avoid image saturation and ensure sufficient signal greater than a few hundred counts at maximum Radiance photons A calibrated measurement of the photon Luminescence measurements emission from the subject Radiance is in units of ohotons second cm2 steradian Radiant Efficiency _Epi fluorescence A fluorescence Fluorescence measurements emission radiance per incident excitation power fluorescence Transillumination fluorescence Fluorescence emission radiance per incident excitation power Efficiency epi Fluorescent emission normalized to the Epi fluorescence measurements fluo
371. the Analyze tab click Start 9 The Data Preview window appears and displays the image data that will be included in the reconstruction Usually no data adjustment is required However it is possible to exclude or include user selected pixel data from the analysis For more details see page 198 You can also include or exclude image data by adding or removing the check mark next to the images listed in the Analyze tab Figure 14 10 10 Click Reconstruct The reconstruction normally requires less than one minute depending on the reconstruction volume parameter settings and computer performance When the analysis is finished e The 3D View window displays the surface and the reconstructed sources e In the Tool Palette the Results tab displays the results data and the algorithm parameter values Figure 14 13 e The 3D Tools appear in the Tool Palette For more details on the 3D Tools see page 220 231 For details on managing results for example save load or delete see page 205 Figure 14 12 FLIT results 3D View window and Results tab For details on the 3D View toolbar see Table 14 3 page 197 3D View toolbar T CK20080407145405 SEQ i i i X Tool Palette J ra Sequence View LA 3D View Spectra ERETI 4 a Photon Density Maps Export Results Save Results Name FLIT_45405 X ke prol M em O Source Intensity bject Height 19 6mm Perspective
372. the control panel specify the imaging settings for the first luminescence or fluorescence image in the sequence and the photograph For details on the imaging parameters in the control panel see page 259 NOTE If you choose the photograph Reuse option in the control panel Figure 4 25 the IVIS System acquires only one photograph for the entire sequence If this option is not chosen the system acquires a photograph for each image in the sequence 5 Click the Add button G The acquisition parameters appear in the sequence table Figure 4 25 6 Repeat step 4 to step 5 for each image in the sequence 7 To set a time delay between each acquisition enter a time minutes in the Delay box in the sequence table 8 To save the sequence setup information xsq a In the sequence table click the Save button fed b In the dialog box that appears select a destination directory enter a file name and click Save Figure 4 25 Control panel and sequence table with image settings Each row in the sequence table specifies the acquisition parameters for one image in the sequence e Time inni F Excitation Filter Emission Filter a Di ra e Ce Se inni FStop Excitation Emission Structure FOV Height 0 20 i 1 Block Yes 1 50 Block No 1 50 Block No 1 50 Block No 1 50 Block No 1 50 Field of View cr E Mouse Imaging Shuttle Ba Jm Subject height 1 50 e m ro ie r E Pinte oem i or 00 em 1 i
373. the image based on subject height If you do not want to use the automatic focus feature you can manually set the focus 1 In the control panel choose Manual Focus in the Focus drop down list The Manual Focus window appears Figure A 2 Opening the Manual Focus window nen OO nd MS Acquisition Control Panel x Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter Field of View System Status T Mouse Imaging Shuttle Subject height 1 50 2 To mark the center of the camera in the window put a check mark next to Display CCD Center Select the size of the step increment that the stage moves Coarse Normal or Fine 4 Click Up or Down to move the stage and change the focus If necessary select another F stop setting from the drop down list and adjust the light level using the arrows 6 Click Update to apply the settings The resulting focal plane cm above the stage is automatically entered in the Subject height box 7 Click OK when the image is focused YY Caliper Appendix B Preferences General Preferences 2 ee ee ee a a 266 User Preferences 2 nk ea Rew HERES CRG ROH EW HG 268 ACOUGO s te KHER EMKEERREREEHR DEH ESSERE RES HH DS 269 TGS 2 Fe PRESSOR DES DOMES ee ee Se ee eee hee 270 Optical Properties 62 464 8a dae ee eRe RROD ee HDR e BE HO 273 You can manage user IDs and specify defaults for some parameters that are
374. the rectangle drag a green handle M so that it includes only the area of interest 7 Click Next The purple data mask appears The mask is an overlay on the subject image that defines the area of interest for the surface topography reconstruction The mask should match the underlying photograph of the subject as closely as possible without including any area outside the subject image Y Caliper Chapter 12 Reconstructing a 3D Surface 179 Figure 12 3 Data mask purple EJE O A Single View Surface Topography Analysis Threshold image e Threshold 22 8 If itis necessary adjust the threshold value so that the mask fits the subject image as closely as possible To change the threshold do one of the following e Press the left or right arrow keys on the keyboard e Move the Threshold slider left or right e Click the arrows or enter a new value in the box 9 Click Finish The surface and 3D tools appear in the Tool Palette For more details on the Tool Palette see page 220 Figure 12 4 3D view and 3D tools in the toolbar and Tool Palette Too Palette 8 E 11120050624122348 seo ROI Tools E Sequence view lA 3DView Spectra _ Planar Spectral Imaging 3 i l gt Surface Topography _ DUT 3D Reconstruction Cronal z 11 0 gt 3D Multi Modality Tools 7 3D Tools Pi Ne Surface Source Registr
375. the sequence table and starting the Imaging Wizard j IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter puto feeca ream pi wfc foren a puto Field of view c M System Status c B X Rays will be produced Idle when energized Acquire Subject height 1 50 lcm Sequence Setup Focus use subject height v Temperature M Locked Select the type of subject and probe from the drop down lists Exposure Time Binning F Stop Excitation Filter Emission Filter F Display Photographic Settings E sea Medium vfs ify Medium vie no Sabet Probes Sequence table Field of View Gs System Status Acquire Sequence XFOV 24 Zoom Ide C er Subjectheight 1 50 m imags isin Focus use subjectheight v Temperature M Locked Initialize T ber of Segments i Delay 0 0 a E Imaging Wizard K Imaging Mode Double click here to set up a bioluminescence image sequence Select this option for imaging bioluminescent or Bioluminescence chemiluminescent reporters such as firefly luciferase Double click here to set up a fluorescence SEEEN image sequence Fluorescence Imaging click beetle luciferase renilla or bacterial luciferase Select this option for imaging fluorescent proteins dyes or nanoparticles in the wavelen
376. tical Zoom Lens attachment avoid touching the optical glass Installing the Zoom Lens Attachment 1 Choose the Zoom option in the control panel Figure 4 28 You are prompted to insert the Zoom Lens attachment Figure 4 28 Control panel Zoom option selected IVIS Acquisition Control Panel l IE Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter T d Field of View a System Status al Fov EZ Zoom moo eats ee em Subject height as g cm Carefully insert the zoom lens in its position in the top center of the imaging chamber Close the door and click OK when done 46 Living Image Software User s Manual 2 After you install the Zoom Lens attachment in the imaging chamber click OK in the prompt The stage moves to the Z field of view position NOTE When the Zoom Lens attachment is installed single images or sequential images can only be acquired at the Z field of view setting During sequence setup if you attempt to change away from the Z setting the sequence table will be cleared If you attempt to change from another field of view setting to the Z setting the sequence table is cleared of all previous settings before the camera settings for the Z position are added Removing the Zoom Len Attachment The imaging system is set to the Z field of view until the Zoom Lens attachment is removed 1 Remove the check mark next to Zoom in the
377. ting in an image where denser material appears lighter and intensity measurements are proportional to the tissue density Figure 5 18 Figure 5 18 X ray images raw left and in absorption units right The Living Image software displays transformed X ray images by default To display raw X ray images remove the check mark next to X Ray Absorption in the Corrections Filtering tools Figure 5 19 Figure 5 19 Corrections Filtering tools Tool Palette Read Bias Subtraction Flat Field Correction Cosmic Correction If this option is not selected no mathematical transformation is applied to the data and raw X ray images are displayed Figure 5 18 Smoothing Mone Image Information ROI Tools d When the X ray data has been corrected for absorption you can evaluate relative bone density by comparing the signal intensities of measurement ROIs The ROI intensity increases with increasing tissue density When acquiring X ray images for bone density evaluation it is best if the subject does not fill the entire field of view since the X ray absorption measurements are referenced to the exposed scintillator plate 66 Living Image Software User s Manual To determine relative bone density 1 Load an X ray image For more details on opening image data see page 83 2 Confirm that the X ray absorption correction default is applied Figure 5 19 3 Inthe ROI tools sele
378. tions info yim Epi Fluorescence Radiance oisecicm2 sri Color Scale Min 4 51e8 Max 4 69e9 ias yeme Accumulate Bee Extract I 122 frames selected out of 231 frames f Table 6 4 Image window kinetic data Item Description gt Play Starts playing kinetic data Stop Stops playing kinetic data 7 Edit and Save Shows or hides the bottom sliders that enable you to select a range of options data and the Extract button that provides save options for the user selected image or data Accumulate If this option is chosen the software computes and displays the cumulative intensity signal Choose this option and playback the kinetic data to visualize accumulation as it happens Extract Click to select a save option for the current image or selected data Extract Current Image Displays the current image in a new image window The software prompts you to save the image when you close the image window Extract Accumulated The software computes the cumulative signal for each image sum of Image the signal in all images up to and including the current image then displays the cumulative signal of the current image in a new image window The software prompts you to save the image when you close the image window Extract Kinetic Data Choose this option if you want to save a particular range of images Opens the Browse For Folder dialog box that enables you to select where t
379. to an 8 bit representation to reduce memory overhead and for easier color mapping The 3D Multi Modality tools provide an 8 bit color opacity map for volume visualization which maps each voxel to an RGB color or a color and opacity value A histogram of voxel intensities appears in the Multi Modality tools and the software sets a default air noise boundary Figure 15 3 3D Volumetric Data Browser Click a row to preview data playback Click a column header to sort the browser Double click a row to load the data contents in ascending alpha numeric order Click the column header again to sort in descending alpha numeric order E Browse 3D Volumetric Data Name Folder Path iGEM Mouse3_3_Day 28 145522 C Share Caliper LS Caliper Data CoReg_Demo j IF mouse 3_3_20100528_140051_FOV60mm_Dynamic C Share Caliper LS Caliper Data Multi Modality Datasets TIFF 512 t v Load in a new window Load V Add to list Browse Toview a particular slice stop playback then move the slider or enter a slice number l Starts data playback a Stops data playback 1 517 To select a range of slices for playback move the left and right sliders or enter the first and last slice numbers Table 15 2 3D Volumetric Data Browser Item Description Add to List If this option is chosen the data selected in the Browse for Folder box is added to the 3D Volumetric Data Browser If this
380. to include in the image label E Units Counts v Display Overlay l Options v ro r a Image TLT20050624145507_005 Series Male Nn nu Fri Jun 24 2005 07 56 46 Experiment DOB 03 21 05 Em Filter 640 Bin M 8 FOV 12 6 f2 1s i wa dia Ima ge label Camera IVIS 200 Beta II SI620EEV Comment dorsal Luminescence 10000 8000 6000 4000 2000 Counts Color Scale Min 586 Max 10348 Y Ca i cies Chapter 7 Working With Optical Image Data 91 Table 7 2 Image window continued ltem Info Description Click to show or hide the image label The image label includes information you enter in the Edit Image Labels dialog box see page 23 and other information automatically recorded by the software Opens all of the images in a sequence Closes all open images of a sequence Opens the Edit Sequence dialog box that enables you to add or remove images from a sequence Opens a dialog box that enables you to export the active view as a graphic file TIIE Display single image Takes a snapshot that is displayed with the data in the Living Image Browser For more details on the browser see page 83 C Living Image Browser TLT20050624145507_SEQ Click Number EX Filter EM Filter Illumination Mode User ID Experiment Commenti CF750 dye in pillows 0 25 pmol uL 2 uL bead 12 Ber r rrr oe oo coe eee ee ee cee ee eee vever
381. ts Table To export the table 1 In the ROI Measurements table click Export 2 In the dialog box that appears select a folder and enter a name for the file txt then click Save To copy the table to the system clipboard Copy selected rows Select the rows of interest and click Copy Alternatively select the rows then right click the table and choose Copy on the shortcut menu All rows Click Select All and then click Copy Alternatively press Ctrl A then right click the table and choose Copy on the shortcut menu Figure 8 32 Copy all rows in the ROI Measurements table to the system clipboard TLT20050624145507 002 palo ies 04 1 402e 02 os Sa TLT20050624145507 003 Copy Ctrl C IPR eee Eee TLT20050624145507 003 ROI4 tye o5 la SelectAll Ctri A POPRETIE sooo TLT20050624145507 004 ROIS poi 1621e 06 4 053e 04 8 599e 03 2976e 04 5 899e 04 Eee ELE ea 0050624145507 004 ROI6 Overy _ 7 273e 05 8 456e 03 1 784e 03 6 007e 03 1199e 04 ESN 4 a Selections Me Types Attribu ROI Dimensions 150 Living Image Software User s Manual This page intentionally blank YY Caliper 9 Image Math Creating a New Image Using Image Math 4 4 151 Subtracting Tissue Autofluorescence 0 082 8 ee eee es 153 The Living Image software provides tools that enable you to mathematically combine two images to create a new image The primary use of image math is to
382. ty is usually more important than the spatial resolution Further since in vivo signals are often diffuse due to scattering in tissue little 1s gained by increasing spatial resolution For more background on the propagation of light through tissue see Diffusion Model of Light Propagation Through Tissue page 310 In such cases high levels of binning may be appropriate up to 10 or 16 depending on the CCD of the VIS Imaging System If signal levels are high enough that sensitivity is not an issue then it is better to image at a lower binning level two or four in order to maintain a higher degree of spatial resolution For application specific questions regarding the appropriate binning level please contact Caliper Corporation The VIS System Control panel provides several binning options The actual binning numbers associated with these settings depends on the CCD chip and type of image Table C 1 These choices should satisfy most user needs However if you want to manually 278 Living Image Software User s Manual control binning you can specify Manual Binning in the Living Image Tools Preference Camera Settings box Table C 1 Binning settings Binning IVIS System 100 200 Spectrum Lumina Lumina XR Lumina Kinetic Small high resolution Lumin Bin 4 Bin 2 Bin 2 Bin 2 Medium Lumin Bin 8 Bin 4 Bin 4 Bin 4 Large high sensitivity Lumin Bin 16 Bin 8 Bin 8 Bin 8 Small high resolution Photo Bin 2 Bin 1 Bin 1 Bi
383. ubject e A sequence of two or more images of the light emission from the surface of the subject acquired at different filter bandwidths Table 14 1 Use the Imaging Wizard to acquire the images The input data to the FLIT algorithm for 3D reconstruction of fluorescent light sources includes e A surface that defines the surface of the subject 192 Living Image Software User s Manual e A sequence of images acquired at different transillumination source positions using the same excitation and emission filter at each position Use the Imaging Wizard to acquire the images Table 14 1 IVIS System filters for luminescence amp fluorescence tomography IVIS Imaging System Filters Bandwidth 200 Series 6 emission filters 550 670 nm 20 nm Spectrum 10 excitation filters 415 760 nm 30 nm 18 emission filters 490 850 nm 20 nm Figure 14 1 shows an example 3D reconstruction workflow Figure 14 1 Basic workflow for 3D reconstruction of sources C Trasen sog kelem D teracese24145507 SEQ hla CI Segue View Soectn Sequence View lt lt SView Spectra Units Courts Lee Saved Cys tora x Info J e a b E a S H e Load an image sequence see page 193 Generate or load a surface for image sequence requirements Tool Palette _ z C TLT20050624145507_SEQ rc C eS _ ROI Tools gt Sequence View A 30 View Spectra gt Planar Spectral Imaging E t Ea s ale MDE
384. unit GPU must meet the minimum specifications shown in Table 15 1 If the appropriate license is not installed or the GPU does not meet these specifications the 3D Multi Modality tools will not appear in the Tool Palette If you do not have the 3D Multi Modality tools you can still view DICOM data see page 258 however the 3D Multi Modality tools are required to register optical and volumetric data Table 15 1 Minimum graphics card specifications Specification Description OpenGL Version Requirement OpenGL 2 0 and above OpenGL Extension Requirement GL EXT texture3D Graphics Card Memory Minimum 256MB Dedicated Shared Recommended 1GB Dedicated Consumer Graphics Cards Desktop Supported Mobile Windows Mac NVIDIA GeForce 8 Series and above 8 9 100 200 300 and 400 series ATI Radeon HD 4000 Series and above 4000 and 5000 series Recommended Desktop NVIDIA GeForce GT 240 and above Mobile NVIDIA GeForce GT 230M and above Workstation Graphics Cards Desktop Supported Mobile Windows Mac NVIDIA Quadro NVS Series and Above NVS amp FX series ATI FireGL V5600 and Above FireGL FirePro amp CrossFire series Recommended Desktop Quadro FX 1800 and above Mobile Quadro FX 880M and above If these specifications are not met the 3D Multi Modality tools do not appear in the Tool Palette 240 Living Image Software User s Manual Figure 15 1 Steps t
385. ure time click the arrow Alternatively manually set the exposure binning and F Stop For more details on the control panel settings see page 259 5 Make a selection from the Energy drop down list Table 5 1 Lumina XR energy options Energy Option Suitable For X Ray Energy Level Animal Living subject 35 Kv 100 A filtered X rays Specimen Non living specimen 28 Kv 100 A unfiltered X rays NOTE A Caliper field service engineer can customize the default X ray energy levels Table 5 1 to settings from 5 0 40 Kv and from 1 100 A with or without the low energy X ray absorbing Al filter 6 Set the Field of View Make a selection from the Field of View drop down list For more details on the field of view see page 261 NOTE To view the subject s inside the chamber before image acquisition take a photographic image uncheck the luminescent or fluorescent option choose the Photographic and Auto options and click Acquire 7 Set the Focus e Select use subject height from the Focus drop down list and use the arrows or the keyboard arrows to specify a subject height cm NOTE The subject height for X ray images is restricted to 2 8 cm or less The subject height is critical to ensure proper optical and X ray overlay The subject height is used to determine the X ray resizing coefficient Select a subject height which suits the region of interest or Y Caliper 8 9 Chapter 5 I X Ray Im
386. urface Results Aa kof oO v Display Volume Level Of Detail Y Volume Performance _ Quality Color Opacity Map Grays Click a button to save or load a color opacity map Intensity V Logarithmic Histogram E Maximum Intensity Projection MIP EF Gradient Illumination 3D Tools j C gt Spectral Unmixing 15 5 Volume Display Options Adjusting the Image Quality By default the color opacity map displays the volumetric data at original 1x resolution This means for example if the volume comprises 512 slices then all of the 512 slices are displayed You can increase or decrease the resolution of the data display from 0 5x to 3 0x resolution for examples see Table 15 3 If you increase the resolution the software interpolates the data and adds slices to the volume If the processing performance is impacted at the original resolution you may want to reduce the resolution to improve performance Reducing the resolution down samples the data and fewer slices are displayed To adjust the image resolution 1 Move the Level of Detail Slider to the left or right The color opacity map is updated 2 To return the resolution to 1x click the Reset button a YW Caliper Chapter 15 3D Multi Modality Tools 255 Figure 15
387. utofluorescence Autofluorescence may be generated by the system optics plastic materials such as microplates and by animal tissue Filter leakage which may also occur is another source of background light The optical components of the IVIS Imaging Systems are carefully chosen to minimize autofluorescence Pure fused silica is used for all transmissive optics and fiber optics to reduce autofluorescence However trace background emissions exist and set a lower limit for fluorescence detection To distinguish real signals from background emission it is important to recognize the different types of autofluorescence The following examples illustrate sources of autofluorescence including microplates other materials and animal tissue 300 Living Image Software User s Manual Microplate Autofluorescence When imaging cultured cells marked with a fluorophore be aware that there is autofluorescence from the microplate as well as native autofluorescence of the cell Figure F 12 shows autofluorescence originating from four different plastic microplates The images were taken using a GFP filter set excitation 445 490nm emission 515 575nm Figure F 12 Examples of microplate autofluorescence emission The black polystyrene plate emits the smallest signal while the white polystyrene plate emits the largest signal Imaging parameters GFP filter set Fluorescence level Low Binning 8 FOV 15 f 1 Exp 4sec White polystyrene
388. vantage of working with image data in radiance mode is that camera settings can be changed during an experiment without having to adjust the images or the measured ROI data Images or ROI data can be quantitatively compared across different IVIS Imaging Systems Caliper Life Sciences calibrates the CCD response and lens of each IVIS Imaging System for all the emission wavelengths The response of the CCD 1s relatively flat 10 over the range from 500 700 nm which includes the spectral variation found in bacterial or firefly luciferase Therefore calibration is accurate over this range The fluorescent signal detected from a sample depends on the amount of fluorophore present in the sample and the intensity of the incident excitation light The excitation light incident on the sample stage is not uniform over the field of view FOV The profiles for all stage locations peak near the center of the FOV The illumination intensity profile varies by up to 30 across the entire FOV Figure D 4 Figure D 4 Illumination profiles at different FOVs Measurements were taken at the center of the FOV on the IVIS Lumina Series Normalized Intensity FOV A 5cm FOV B 7 5cm FOV C 10cm FOV D 12 5cm FOV cm 284 Living Image Software User s Manual Displaying fluorescent image data in terms of efficiency eliminates the variable excitation light from the measurement and enables a more quanti
389. venient way to browse and preview optical data view information about the data and load the data To start the browser 1 Click the Browse button Alternatively select File gt Browse on the menu bar 2 In the dialog box that appears select the folder of interest and click OK The Living Image Browser appears Figure 7 1 84 Living Image Software User s Manual Figure 7 1 Opening the Living Image Browser File Edit View Tools Window Help s aaazaerw gt J Point Source Fitting b J TL720041215133517_SEQ gt J TLT20050624122348_SEQ b J TL720060406164950_SEQ b Jy TLT20060510114512_SEQ gt M Transillumination data b B TwoColor gt E XPM 2 MIS3D b Js XQA20050609145636_SEQ gt X Ray Datasets SF LS eS Location C Share Caliper LS Caliper Data Sample Data CK20050420175030_SEQ SequenceInfo txt Living Image Browser NOTE The next time you start the Living Image software and open the Browse For Folder box the software automatically returns to the last folder visited The Living Image Browser displays the selected data along with the user ID label information and camera configuration information ME image SE image sequence bch kinetic data or image exported as DICOM file y7 Calipe Der css Chapter 7 Working With Optical Image Data 85 Figure 7 2 Living Image Browser To expand a sequence click Click a column header to sort To view data properties th
390. w row appears in the spectrum list 2 In the new row select a probe name from the drop down list Figure 11 7 Selecting a library spectrum to display E TL720060406164950_SEQ Unmixed Images Ji Normalized Legend Red Shift a UMIE 2 a AF s0Erm Lad Hormahzed Arnphtuce e Tissue AF On Type ami Label Color l TissueAF E umx 2 E AF630Em AF 750Ex ALFoodEm ALFoodEx ALFreeEm ALFreeEx 700 750 Wavelength rom To add spectra from an ROI 1 Create an ROI on an image and apply it to the sequence 1 Click the button arrow and select From ROI A new row appears in the spectrum list 2 In the new row select an ROI name from the drop down list To remove a spectrum 1 In the spectrum list select the spectrum row that you want to remove 2 Click the button Y Ca i a Chapter 11 Spectral Unmixing 173 11 4 Spectral Unmixing Parameters The Results tab in the Spectral Unmixing Tool Palette shows the optimized fit parameters used by the software to derive the spectral unmixing results Figure 11 8 Figure 11 8 Tool palette Spectral unmixing tools Results tab Tool Palette Number of Iterations Number of Components Number of Wavelengths Number of Samples Lack of Fit WPCA Lack of Fit 7o EXP Denoise PCA Save Results Name SPUM_7 Delete 3 84347 Divergence counter g Medium Table 11 5 Spectral unmixing tools Results tab Item
391. w to install the XFO 24 lens see the XFOV 24 Lens Instructions OL ae ALERT If you remove the XFO 24 lens from the system be sure to remove the check mark from the XFOV 24 check box VIC Select this option when using the Ventral Imaging Chamber to acquire ventral kinetic images on the IVIS Kinetic Imaging System Note The Mouse Imaging Shuttle or the Ventral Imaging Chamber can be used with the IVIS Kinetic but not both at the same time 262 Living Image Software User s Manual Table A 1 IVIS acquisition control panel continued ltem Zoom Description Select this option to install and acquire images using the Zoom lens on the IVIS Lumina IVIS Lumina XR or IVIS Kinetic Imaging System After the Zoom lens is installed the stage automatically moves to the Z position adjusted by the specified subject height The Zoom lens is focused to this position Subject height cm Sets the position of the focal plane of the lens CCD system by adjusting the stage position The subject height is the distance above the stage that you are interested in imaging For example to image a mouse leg joint set the subject height to a few mm To image the uppermost dorsal side of a mouse set the subject height to the 1 5 2 0 cm The default subject height is 1 5 cm OL ae ALERT The IVIS System has a protection system to prevent instrument damage however always pay close attention to subject height particu
392. wn arrow to select a color table for the image data For more details on color tables see Pseudocolor Images page 279 Reverse Choose this option to reverse the selected color table Logarithmic Choose this option to apply a log scale to the relationship between numerical data and the color range in the color table A log scale increases the range of meaningful numerical data that can be displayed Palette label To include a brief line of text next to the color scale enter text in the palette label box then press the Enter key To remove the text from the image window delete the text in the palette label box and press Enter Scales per Column Sets the number of color scales to display in a column YW Caliper 7 10 Rendering Intensity Data in Color Chapter 7 Working With Optical Image Data 113 The colorize tool renders luminescence or fluorescence data in color enabling you to see both intensity and spectral information in a single view The tool provides a useful way to visualize multiple probes or scale probe signals that are not in the visible range To view colorized intensity data 1 Load an image sequence Figure 7 28 Microplate images Images were acquired using different combinations of excitation and emission filters The samples are quantum dot nanocrystals 700 or 800 nm File Edit View Tools Acquisition Window Help a amp ia amp R Units Counts v C Apply to all
393. y compare DLIT and or FLIT analysis results ZlS Voxel color scale with transparent as the minimum or maximum in a reverse color table 224 Measure voxels which have been copied from one surface and pasted to another using the 221 3D Source tools Enables you to view all voxels original and pasted using one color scale Working With Volumetric Data Dedicated 3D Volumetric data browser with preview and playback capabilities Enables 241 loading of volumetric data without first loading an optical data set Save registration information for a particular volumetric and optical data set 246 Save a color opacity map that can be applied to volumetric data 203 2 Living Image Software User s Manual 1 2 About This Manual This user manual explains how to acquire optical image data on an IVIS Imaging System and analyze images using the Living Image software The manual provides detailed instructions and screenshots that depict the system response NOTE Sometimes the screenshots in the manual may not exactly match those displayed on your screen For more details on your IVIS Imaging System please see the appropriate system manual Conventions Used In the Manual Convention Example Menu commands are bolded To open image data select File Open Dataset on the main bar Toolbar button names are bolded To open image data click the Open Dataset button Gar Numbered steps explain how t
394. y imaging 63 64 histogram 103 image adjusting appearance 97 cascade 88 correct or filter 99 100 correcting filtering tools 99 100 export 114 116 histogram 103 information 93 Y Ca ais line profile 104 magnify or pan 98 measurements 107 pixel data 102 print 114 116 tag 96 tile 88 image data colorize 113 114 export 47 open 87 Save manually 46 image layout window 115 116 image math 151 153 image overlay tool 110 112 image sequence application specific 10 create from individual images 117 118 edit 116 117 include X ray images 54 61 manual setup 41 45 image window 89 imaging modes 7 9 imaging system capabilities 10 Imaging Wizard 33 36 import organ atlas 230 surface 183 information about an image 93 infusion pump 323 infusion syringe injection system control panel 324 infusion pump 323 initialization See system initialization instrument fluorescent background background instrument fluorescent background 304 IVIS Imaging System fluorescence imaging components 291 294 K kinetic acquisition settings 71 acquisition window 70 kinetic data acquire 69 save 77 78 view and edit 75 76 kinetic ROI plot 134 kinetic ROI draw 133 Index 333 kinetic ROI draw 132 L lens aperture 275 line profile 104 Living Image browser 86 Living Image software starting 15 Longitudinal Study window 213 luciferase spectrum 311 luminescence reconstruct 3D sources 193 200 luminescent imaging single image 21
395. y the change to all images choose the Select All option 3 Click Data Adjustment 4 In the window that appears enter a new Threshold value The new Threshold appears in the Analyze tab 5 To reset the Threshold to the default value for the selected images click Restore Threshold Ee YW Caliper Chapter 14 I 3D Reconstruction of Sources 199 Figure 14 7 Adjusting the Threshold Tool Palette Ed Data Preview window rz FO GO Ie EES a ee le E TLT20050624145507_SEQ l Sequence View LA 3D View Spectra Data Preview Analyze Properties Results Sequence 72720050624145507_SEQ Tissue Mouse Tissue Source Firefly Select Filters Filter Threshold V Image Label V Median Filter Restore Threshold Data Adjustment E Select All Cancel Reconstruct F tataAdyustenent Set the Threshold here Note Min Counts translates the Threshold to the minimum counts required for reconstruction To select particular regions for reconstruction 1 Open the Data Preview window as shown in Figure 14 7 2 Click Data Adjustment 3 In the window that appears choose the Draw option and put the mouse pointer over the image so that the pencil tool if appears 4 To automatically select all pixels in a source right click with the region with the pencil tool

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