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LI 3.2 User Manual.book

1. Mesh tab Mesh 170 Render Mesh Opacity J 60 E Render Photon Density Map Apply Simulated v Images 1 v Threshold Intensity 7 10e 3 Color Table CO C Reverse i C Logarithmic Scale Slice Coronal J 11 0 amp Sagittal J 0 0 F Transaxial J 7 0 B Volume tab f volume 172 Render Volume Min 0 0015 Render voxels As Max 0 1616 Cubes J Threshold Intensity 0 0081 Color Table a Reverse C Logarithmic Scale Source Yoxel Measurement Fluorescence Yield 0 0775 Ai mm 2 Yolume 2 0305 mm Center of Mass 2 5977 14 8917 9 8416 Host Organ Unknown Export Yoxels Center OF Mass 162 Living Image Software User s Manual v r Y Ca PS as KECIK LE See Page Organs tab Animation tab Organs Render Organs Opacity J Iso Organ Atlas CT Female Dorsal v gt Organs Wadrenal WBbladder bone brain cecum P Weolon MB eyestxterior eyesInterior heart Bieu MI inm Animation Preset Animations Presets Spin CW on Current Axis Frame Factor 1 Animation Setup Time Scale o 99 le Key Frame 1 A Key Frame 2 F Key Frame 3 Key Frame 4 Key Frame 5 Key Frame 6 4 XO Play Record i Frames Per Second 10 lt gt lt gt r Total Duration secs 5 Load Save 175 182 163 12 3D Reconstruction of Sources
2. Slice Coronal 10 5 Sagittal S 1 8 Transaxial 8 1 i photons sec Perspective Source Intensity a View name 169 12 3D Reconstruction of Sources Front Back Left Figure 12 6 Alternative views of the surface 12 5 3D Tools Mesh Tab The Mesh tab includes tools for viewing the reconstructed surface photon density maps and planes through the 3D reconstruction Roar EPAR E eG gt F Render Mesh v Render Photon Density Map Apply Simulated ka Images H Threshold Intensity Color Table E O oO Reverse C Logarithmic Scale Sie Coronal Sagittal Transaxial 7 0 E Figure 12 7 3D tools mesh tab 170 Ti i Living Image Software User s Manual Y Caliper Table 12 5 Mesh tab Item Description Render Mesh Choose this option to display the surface in the 3D view window It may be helpful to turn off the surface to better view the photon source 3D reconstruction Opacity Controls the surface opacity A lower number makes the surface more transparent This may be helpful for viewing the photon source 3D reconstruction Photon Density Choose this option to display the photon density map If the DLIT Map reconstruction of the bioluminescent source has not been g
3. Available items column headers Items column headers that can be added to the table currently in the table 2 Do either of the following eSelect a configuration that you want to modify from the User Lists drop down OR eSelect Customized Unsaved from the User Lists drop down to create a new configuration 3 To add an item to the table click an item in the Available Item list and then click Add 4 To remove an item from the table select the item that you want to remove in the selected Items list and click Remove Configure Measurements User Lists Customized Unsavec M Name My ROI Table Available Items Total Counts Analysis Comment A A Avg Counts Analysis User ID soles Stdev Counts Angle Animal Model Max Counts Animal Number ROI Pixels Animal Strain Areatccd Pixels Area cm2 Xc pixels Avg Dark Charge Counts Y c pixels Avg Efficiency Width pixels Avg Fluorescent Bkg Counts Height pixels Avg Radiance p s cm2 sr Binning Cell Line Comment Comment Comment2 Selected Items 109 6 Working With ROI Tools Copying or Exporting the ROI Measurements Table 110 5 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 6 To save the table configuration enter a name in the Name box and click Save Note You cannot overwrite a fact
4. s hreshold i jo gt Planar Spectral Imaging gt Surface Topography DLIT 3D Reconstruction k ROI 2 4 627e 05 Counts Color Bar Min 1122 Max 19546 mac IIS an en Game j ROI Measurements 5 x ROI Measurements Image Number ROI Image Laye Total Counts Avg Counts Stdew Coun Min Counts Max Counts TLT20050624145507_006 ROI 1 Overlay 1 735e 06 5 074e 03 4 461e 03 1 178e 03 2 245e 04 ROI Measurements table Customized Selections Copy Measurements Types Image Attributes ROI Dimensions Counts _none_ vi nne Selected cose Figure 6 1 Example measurement ROIs and ROI measurements table 81 6 Working With ROI Tools Table 6 1 Types of ROIs Type of ROI Measurement ROI Average Background ROI Subject ROI Measures the signal Measures the average signal intensity Identifies a subject animal in an intensity in an area of an in a user specified area of the image image image that is considered background Note Using this type of ROI is Note Using this type of ROI is optional optional It provides a convenient NEN If the animal has significant way to automatically associate link Description autoluminescence or autofluorescence a measurement and average you can determine a background background ROI for background corrected signal in a measurement ROI corrected ROI measurements when by subtracting an
5. The items in the ROI Properties box depend on the type of ROI selected For more details see lable 6 3 page 99 98 Ti Living Image Software User s Manual Y Caliper Table 6 3 ROI properties Item Description ROI 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 ROI Label Click to edit the selected ROI label name Click Number A drop down list of open images Subj ROI The Subject ROI tab shows a drop down list of all subject ROIs in the click BKG ROI number selected above that can be linked to a user specified measurement ROI or average background ROI selected from the drop down list at the top Info tab of the dialog box The Bkg ROI tab shows a drop down list shows all average background ROls 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 coordinate of the ROI selected in the image Yc Y coordinate 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 t
6. 6 Click Generate Mesh Coefficients 7 Enter a name for the atlas and click Save Organ Atlas The organ atlas atlas is created The atlas name appears in the Organ Atlas drop down list in the 3D tools Organs tab 181 12 3D Reconstruction of Sources 12 8 3D Tools Animation Tab The Living Image software can produce an animation from a sequence of 3D views or key frames For example an animation can depict e Magnifying zooming in on the 3D view e Spinning the 3D view on an axis e The surface or organs fading out decreasing opacity or fading in increasing opacity The animation can be saved to a movie mov mp4 or avi In the Animation Tab You Can See Page View a preset animation generated from a predefined animation setup 184 Create a custom animation generated from your custom animation setup 185 Edit amp save an animation setup 186 File Edit View oe Aa amp Acquisitia Window Help amp rk Units Counts vO Apply to all Tool Palette Ea EE gt Surface Topography Sequence view 3D View 4 07 gt 2 E Lae m Mesh Volume Organs Animation Preset Animations Presets sjs pe humel anal fi 1V20080208142757_SEO Frame Factor Animation Setup Time Scale o g Key Frame 1 Key Frame 2 Key frame box shows the key frames Key Frame 3 Key Frame 4 in the current animation setup Key Frame 5 Click a key frame
7. 5 Working With Data 5 2 The Tool Palette amp Image Window The tool palette contains information about the data and organizes the image analysis tools The tools available in the tool palette depend on the type of data that is active Tool Palette Tool Palette a a mA g m a Photo Adjustment Brightness lt gt Gamma _ on lt gt lt gt Opacity Color Scale Mn u2 Max 19546 lt gt lt gt Color Scale Limits Auto Full Manual Individual Color Table poron Reverse C Logarithmic Scale gt Corrections Filtering gt Image Information gt ROI Tools gt Planar Spectral Imaging gt Surface Topography _ Point Source Fitting gt DLIT 3D Reconstruction Figure 5 1 Tool palette An image image sequence or kinetic data set is displayed in an image window Multiple image windows can be open at the same time The options available in the image window and tool palette depend on the type of image data fe W Wee toe Wed feb siut BS R miran Dawes LANAG Aer Mute Actuators Figure 5 2 Image window kinetic data 50 Living Image Software User s Manual Y Caliper 5 3 Working With an Image Sequence When you open an image sequence the image window displays thumbnails of the images in the collection A single color table is applied to the images For details on how to open an image sequence using the Li
8. Apply Individual Color Scale for Sequences Display ROI Label As Measurement Show Transillumination Locations Photons Total Flux v _ Shou dow Winda Counts Total Count E w Civ Show Advanced Options Cancel 2 In the dialog box that appears put a check mark next to Show Advanced Options and click OK The point source fitting tools appear in the tool palette Figure 11 1 143 11 Point Source Fitting 144 Analyze tab Tool Palette gt Swiace Topography J Z Point Source Fitina Analysis Params Properties Results Sequence CX200702227839972_ SEQ Tissue Muscle Source AA Select Single Image Source Image ExwL EmWL MinRadiance a 1 675 720 2 44e 07 DO 0 1 mm 720 8 70e 06 720 2 62e 06 720 2 62e 06 B FLIT 3D Reconstruction _ gt 3D Tools Analysis tab shows the active image sequence Click the sign to display the position of the bottom illumination source read from the click info x y or x y z depending on the image model Properties tab Tool Palette gt Image Adjust B Surface Topography Point Source Fitting Tissue Properties Muscle Internal medium index of refraction Wavelength nri gt FLIT 3D Reconstruction E Spectral Unmixing Select other starting values for the optical properties here Params tab Tool Palette E Surface Topography f Point Source Fitting
9. Target Max Count A user specified intensity Range Values 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 F Stop image acquisition Binning Exp Time sec 199 B Preferences B 4 Theme Preferences General User Theme Tissue Properties 30 Analysis 3D View Background Color Solid Color i te Gradient Color Top C I Click to apply the poton J factory set defaults Preferred Color Palette Luminescent Rainbow wt Reverse Fluorescent YellowHot wt C Reverse C Use saved colors while loading data Restore Defaults Preferred ROI Line Color Luminescent a Fluorescent a Restore Defaults Click so select a different color or define a custom color Figure B 5 Theme preferences Table B4 Theme preferences Item Description 3D View Background Color 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 Figure B 6 Top the color at the top of the window Bottom the color at the bottom of the window Preferred Color Palette Luminescent The color scale that is used to display luminescent images Click Reverse to reverse the color scale Fluorescent The color scale that is used
10. 2 Initialize the IVIS System and confirm or wait for the CCD temperature to lock For more details see page 11 IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter al Field of view System Status Idle Acquire aim Subject height 150 m Sequence Setup 3 Putacheck mark next to Luminescent 4 Confirm that Excitation Filter setting is Block and the Emission Filter setting is Open 5 Select the Auto exposure time click the arrow Alternately manually set the exposure binning and F Stop For more details on the control panel settings see page 191 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 160 15 3 Acquire an Image or Image Sequence 16 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 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 or e Select Manual focus from the Focus drop down list For more details on manual focusing see page 34 A Set the photographic image settings a Choose the Photographic option b Enter an exposure time or choose the Auto option c Co
11. The sequence setup appears in the Sequence Editor j Imaging Wizard Open Filter Bioluminescence Bioluminescence DLIT Diffuse 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 bioluminescent light source distribution inside the subject To generate a 3D reconstruction of bioluminescent sources the Living Image software requires a photographic image a structured light image and j Imaging Wizard i om Fluorescence Spectral Unmixing Type Epi Illumination Trans Illumination Spectral unmixing uses a mathematical operation to separate the tissue autofluorescence from the fluorescent signal of interest It can also be implemented to separate fluorescent signals from two or more probes bioluminescent images obtained at three or more 2D wavelengths using filters from 560 to 660 nm f This technique requires fluorescent images taken with multiple excitation and or emission filters A photographic Fliter scan image is also necessary i Unmixed Autofivor Unmixed AF680 Spectral unmixing Planar Spectral Measurement Reconstruction Unmixed AF 750 pg d A Source 1 Spectral unmixing h be Source 2 Dorsal View For more information on DLIT see Living Image User Guide Appendix F page 189 aU _ 2 For more information on Spectral Unmixing see
12. Lock Size Widtht pix d To select a custom color for the ROI line click a ve custom color swatch and click OK eset ph Line Size j Select color Basic colors Brightness slider TT e TT 1 i Cross hairs in the custom color field Custom colors BEEREEHEEEE H EENEN NN Define Custom Colors gt gt Sat 83 gt Green 124 Val 11 Sl Blue 95 Add to Custom Colors 102 ad Living Image Software User s Manual Y Caliper Moving or Editing an ROI Label To move an ROI label 4 TLT20050624145507_006 Units Counts v Display Overlay v mo 1 Place the pointer over the ROI label 2 When the pointer becomes a h drag the label Max 22447 3 Click to release the label at the new location EKS 1 1 051e 03 fm YY ROI 2 4 009e 05 Counts Color Bar Min 1122 Max 19546 To edit an ROI label 1 Double click the ROI Alternately right click the ROI Ctrl click for Macintosh users and select Properties on the shortcut menu 2 Inthe ROI Properties box that appears edit the name in the ROI Label box and click Done 4 11T20050624145507_006 Sel Units Counts v Display Overlay ROI Properties mS ROI ROIZ ROI Label My ROI BkgROI SubjROI Info Image Number TLT20050624145507_006 v ROI _none_ N Max 22447
13. Sort Choose this option to automatically sort the unmixed spectra in ascending order of their center wavelength Force Choose this option to force the first component to non zero throughout the image Denoise by PCA Determines how much of the data will be filtered by principal component analysis Stronger denoising means less principal components will be used in the data and more details will be lost Stronger denoising also may slow down the unmixing Unimod Tolerance The threshold for the unimodality constraint It is the percentage of overshoot allowed for the second spectral peak PCA Mode PCA can be performed on the original data the correlation matrix of the original data or the covariance matrix of the original data Click to display the explained variance Click to display the biplot graph 135 9 Spectral Unmixing 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 j 11720060406164950_SEO BHA Sequence view Spectra Distribution PZA Biplot tos Component 1 Oxis Componente Figure 9 4 PCA biplot 136 Ti Living Image Software User s Manual Y Caliper PCA Explained The PCA Explained Variance histogram shows the part of variance y axis that can be Variance explained by a number of principal components x axi
14. Specifies the minimum per cent of peak pixel intensity that a pixel must have to be included in an ROI identified by the software 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 5 To show the ROI Measurements table click the Measure button in the tool palette 6 Working With ROI Tools j ROI Measurements ROI Measurements Image Number Image Layer Total Counts Avg Counts Stdey Counts Customized Selections Measurements Types Image Attributes _ ROI Dimensions Counts Vv _none_ v The ROI Measurements tab 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 106 Manually Drawing a 1 Open an image or image sequence and in the ROI tools select Measurement ROI Measurement ROI from the Type drop down list 2 To specify the ROI shape a Click the Circle OJ Square OJ 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 RO s and intensity label s appear on the imag
15. _none_ _none_ l ROI Measurements EAR v RorfRor falFrames Image Laye Total Flux p Avg Radian Stdev Radia Min Radianc Max Radian ROI Pixels Kinetic ROIs are displayed in a separate tab Customized Selections Measurements Types n ROI Dimensions Select All Photons All Possible Values Pixels vi 106 Living Image Software User s Manual j Y Ca Be ins Table 6 4 ROI Measurements table Item Description Measurement Types None Counts Photons Efficiency Make a selection from the drop down list to specify the type of ROI measurements to include in the table Exclude 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 213 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 ophotons sec in each pixel summed or integrated over the ROI area cm x 4r Av
16. G Planar Spectral Imaging This page intentionally blank 246 Living Image Software User s Manual Y Caliper Appendix H 3D Reconstruction of Light Sources Determining Surface Topography naoa 0002 eee ee ee es 247 Algorithm Parameters amp Options 2 0 ee ee 252 Diffuse 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 bioluminescent light source distribution inside the subject Fluorescent Tomography FLIT analyzes images of surface light emission to generate a 3D reconstruction of fluorescent light source distribution inside the subject To generate a 3D reconstruction of bioluminescent sources the Living Image software requires a photographic image a structured light image and bioluminescent images obtained at two or more wavelength filters from 560 660 nm To generate a 3D reconstruction of 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 mesh from structured light images The mesh is defined by a set of connected polygons or surface elements e Maps the surface radiance photons s cm2 steradian to the photon dens
17. Volume of voxel size To convert the Fluorescence Yield measurement to the number of fluorescent compounds divide the Fluorescent Yield values by measured quantities of Fluorescence quantum efficiency of excitation wavelength conversion to emission wavelength Excitation photon absorption cross section for the fluorophore Ti i Living Image Software User s Manual Y Caliper Table 12 3 3D reconstruction results continued Final vsize The voxel size length of a side mm that produces the optimum solution to the DLIT or FLIT analysis Number of sources 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 x2 value indicates a better quality of fit Starting vsize The voxel size at the start of the analysis Kappa best DLIT result The kappa value that produces the optimum solution N surf 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 Threshold angle 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 For more details see Angle Limit page 253 Kappa li
18. You can view information about the active image using the Image Information tools Figure 5 10 Tool Palette gt Image Adjust gt Corrections Filtering lt Image Information laa Of 7 EE BB units Image Binning amp Width 12 6cm Height 12 6 cm Image Y 5 852 7 770 on Image Data 27406 counts Crop Distance A ifaoaedy B 6 00 0 00 0 000 008 Distance 0 00 gt ROI Tools Figure 5 10 Tool palette Image Information tools 67 5 Working With Data Table 5 7 Tool palette Image Information tools Tool Description See Page Click this button to display a histogram of pixel intensity 70 P Click this button to display a line profile 71 a a Click this button to display the distance measurement cursor in 72 the image window Click this button to draw and measure a rectangle on an image 73 Click this button to display hide a scale on the x and y axis of the image window E C Click this button to display hide a grid the image window 3 lt Choose the units cm or pixels for distance measurements in the image window Image Binning The binning applied to the image Note If soft binning is applied 69 to the image data and the binning level is changed from 8 to 16 the new binning is indicated as 8x2 Image X Y The x y pixel coordinates of the mouse pointer location in the image Image Data The intensity counts or ph
19. lt lt Reactivate Browser Images JJH200506301 42125 _002 JJH200506301 42125 _003 Single images in the Living Image Browser Retired Images Images that have been removed from the active sequence Close The updated image sequence is displayed Creating an Image Sequence from Individual Images 1 on browsing see page 45 NOTE Browse for individual images which may or may not be part of a sequence not image sequences 5 To add an image to the sequence select an image and click Copy To remove an image from the sequence select an image and click Retire The image is removed retired from the sequence To restore a retired image to the sequence select the retired image and click When you are finished editing the sequence click Close You can create a sequence from images acquired during different sessions In the Living Image Browser browse for the images of interest For more details 5 Working With Data 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 amp Living Image Browser click Number EX Filter EM Filter Illumination Mode UserID User Group Series Experirr MBHX20070321091157_001 Reflective XFM 2 No 4 AF680 Testing Scan Emission at Ex605 ME HX20070321091157_002 Mi HX20070321091157_003 Mis HX20070321091157_002 M
20. Analysis Params Properties Results Model Type Epiillumination Fluorescence ngle Limit deg zo Spatial Filter 01 Parameters starting values 0 140 Ef mm 20 628 ch coef Powerphoton s ie10 gf MuaEm 1 cm 054 ef MusEm 1 em 881 ef Restore Defaults Mask No mask Statistics Weighting LMF Fitting B FLIT 3D Reconstruction gt 3D Tools Starting parameter values Results tab Tool Palette gt Surface Topography 7 Point Source Fitting Analysis Params Properties Results Error Estimation Starting Chi 2 8 58e 09 Ending Chi 2 7 03e 08 Point Source Fitting Results Parameters Fitted value 0 143 cm 6 676 cm 1 710 cm 0 049 cm 2 22e 11 photons x location of the source 0 181 cm sa A A Ce a Photon Density Maps Export results Save Results Name LMFIT_13 Delete gt FLIT 3D Reconstruction gt 3D Tools Point source fitting results Figure 11 1 Tool palette point source fitting tools amp results If the image sequence does not include a structure light image the point source fiting tools do not appear in the tool palette Living Image Software User s Manual Y Caliper ifeSciences Table 11 1 Tool palette point source fitting Item Description Analysis Tab Image ExWL EmMWL MinRadiance X Y Params Tab Model Type Angle Limit deg Spatial Filter Parameter starting valu
21. Analyze Properties Results Spectral Results Unsaved Analyze Properties Results Analyze sequence ILIZ0050624145507_5EQ Tissue Properties Muscle Tissue Musol Source Sires Cc a Internal medium index of refraction 1 40 ROI Depth mm Total Flux phat s Select Filters Se Source Spectrum Firefly a ROIZ 2 341 0 269 2 66e7 5 186 Plot Tissue Properties m p em 1 pef 2 Plot Linear Fit Plot Intensity Save Results SpIm_2 v Imageset TLT20050624145507_006 Rieter ROI List Al Delete i Figure 8 1 Tool palette planar spectral imaging tools 123 8 Planar Spectral Image Analysis 124 Table 8 1 Planar spectral imaging tools amp results Item Description Analyze Tab Sequence Tissue Source Select Filters ROI List Analyze Name of sequence used for the analysis The tissue properties and source spectrum selected in the Properties tab 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 A drop down list of the ROIs in the active image Click to perform the spectral analysis Properties Tab Tissue Properties Internal m
22. EKG 1 1 051e 03 i 7 Wi Lock Position xc pix 105 00000 Yc pix 163 00000 ROI 1 BKG 1 1 687e 06 Angle deg 0 My ROI 4 009e 05 Lock Size width pix 13 00000 Counts Color Bar Min 1122 Max 19546 Line Size 2 Line Color I C 103 6 Working With ROI Tools Saving ROIs The software automatically saves ROIs with an image The ROI measurements are saved to the AnalyzedClickInfo txt file associated with the image ROIs are saved per user and can be 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 File Edit view Tools Acquisition Window Help SARRE R Units Counts vo Apply to all Tool Palette gt Image Adjust r c psn aaa ae Units Counts v Display Overlay v gt Corrections Filtering E Se I gt Image Information ROI Tools Min 48 C Apply to Sequence E i Hira A ype Average Bkg ROI Save ROIs ame ROI_2_KSA 3 KG 1 5 412e 02 Delete Load oon gt Planar Spectral Imaging gt Surface Topography _ DLIT 3D Reconstruction ROI 1 1 369e 06 10000 5000 Counts Color Bar Min 1122 Max 19546 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
23. Plot Apply to C DLIT FLIT C Planar Spectral Imaging Tool Palette Tissue Properties gt Image Adjust gt ROI Tools v Preview gt Planar Spectral Imaging v gt Surface Topography ay 0 baton Source Spectrum i Analyze Params Properties Results Tissue Properties Muscle Internal medium index of refraction 600 800 Plot Source Spectrum Wavelength rm 1 00 Normalized Amplitude i 0 0 D 600 300 Wavelength nra gt Spectral Unmixing Figure B 7 Set tissue properties preferences left for the Properties tab in the Planar Spectral Imaging DLIT or FLIT tools Table B5 Tissue properties preferences Item Description Tissue Properties Choose a default tissue type that is most representative of the area of interest Source Spectrum Choose the default luminescent source 201 B Preferences Table B5 Tissue properties preferences continued Item Description Index of Refraction The software automatically sets the internal medium index of refraction based on the selection in the Tissue Properties list Plot Tissue Properties Choose this option to display a graph of the absorption coefficient ua effective attenuation coefficient ues and reduced scattering coefficient u Or usp Source Spectrum Choose this option to display the source spectrum Apply to DLIT FLIT Choose this opti
24. j JJH20050630142125_006 DER Draw a subject ROI that Units Counts Display overy w _ info i includes the measurement ROI and the average background ROI For details on how to draw a subject ROI a see page 90 h F R k ROI 3 9 709e 04 Image PI oom Min 64 Max 44260 Subject 2 wa BKG 2 1 617e 04 ROI 2 4 742e 05 i 4 e ROI 1 2 358e 06 2 Counts A 7 4 y i Color Bar Min 2213 Max 36749 Method 2 j JJH20050630142125_006 BAR Right click the measurement furts counts _vnisplay lovertay vI info aa ROI and select Set BkG ROI to Bkg X on the shortcut i H 4 menu that appears Max 44260 4 BKG 1 1 042e 0 cus l BKG 2 1 617e 04 ROI 2 BKG 1 4 742e 05 REFS d b gt i Fso share m ROI 1 BKG 1 2 358e 06 E gt Copy ROI Copy All ROIs Duplicate ROI Max 36749 Set Bkg ROI to BKG 1 Set Bkg ROI to BKG 2 Set Bkg ROI to none Set Subject ROI to none Hide ROI Tag Delete ROI Delete All ROIs Properties Unlock Position Living Image Software User s Manual v i Y Ca Be ens Method 3 1 Right click a background ROI and select Properties on the shortcut menu 2 In the ROI Properties box that appears click the Bkg ROI tab and put a check mark next to Use as BKG for future ROIs in 3 Choose the image name or the Entire sequence option j JJH20050630142125_006 TBS Units Count
25. 0 2 00 0000008 189 Appendix A IVIS Acquisition Control Panel 2 088 ee eae 191 Appendix B Preferences 1 2 2 eee et ee 195 B rena Pe ENS amp aa ea hee eee SRE ee ee ee ee hae ae 196 B 2 User PreterenCes 2 522545 22 bed oe ke ORE Se EEO See ER Sw ee ee ee 198 Do ACUSION se rasere nss nsista nedad eee eee ee eee eee ee 199 De Teme tee enaeauaeee eee eeaeeen Ce eee eh ehaee ewe Fase eee 200 B gt Tissue PIOperies 24 ne ee eee ewe Reese Keb eee hha ee eeeenaacs 201 B 6 3D Analysis 1 aoaaa aa a 202 Appendix C Detection Sensitivity 2 202008 ee ee eee 205 C1 CCD Detection EMICIENCY oa 4 asin bho we Re ee EER eS ew ee ee E 205 C2 BDN annae ee beeeans tee eee ee eee eee wede ese eees aes 206 Co SMODE gn2 ane ke oe seed eee keh bew Ree Ge ERR See EHS eae ee 208 Appendix D Image Data Display amp Measurement 588 5 211 DI Imee Dala co uuseteeeatereesaseevaeee ns Geode eee ada dae ox 211 D 2 Quantifying Image Data 2 a 213 D3 Flat Fielding s s ccs 4865 be ERGO EER OEE SEES OES ee DRE ESE Ho 216 DA Cosmic Ray Corrections ibs eke a tbe eee wee ee eS Ewe ee ee ee 216 Appendix E Luminescent Background Sources amp Corrections 217 E 1 Electronic Background 2 ee 217 E 2 Background Light On the Sample 0 0 00 eee eee eee 218 E 3 Background Light From the Sample 0 0 0 0 0 00 eee ee eee 220 Appendix F Fluorescent Imaging 02 0082 ee eee ene
26. 12 6cm Height 12 6 cm Insge X Y 6 RY AEN om Crop box position amp dimensions Crop box 2 When the mouse pointer changes to a draw a rectangle on the area of interest 3 To change the size or position 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 r71 button 73 5 Working With Data Table 5 11 Crop box position amp dimensions Item Description pen 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 5 10 Rendering Intensity Data in Color 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 NOTE The colorize tool is only available if Show Advanced Options is selected in the general preferences see page 196 To view colorized intensity data 1 Open an image sequence Fie Ek View Tock Apa Wires Heip ei a amp amp Ne Units Counts C Apply te al l g TSCA Wey a menka E O vee Saved Colors In this example images were acquired using different combinations of excitation and emission filters The samples are quantum dot nan
27. 8 Y Ca l n Living Image Software E 4 User s Manual Version 3 2 2002 2009 Xenogen Corporation All rights reserved PN 125112 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 and Living Image are either registered trademarks or trademarks of Xenogen Corporation 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 Ti i Living Image Software User s Manual Y Caliper Contents 1 WOICOMG xe ssa ERR eR EHS Pee eS eee wee 1 1 1 What s New In the Living Image 3 2 Software 0 0 00 02 eae 1 LZ Aboul Tms Minual s ess se eee eee eee ee eee Phe eee ee ee Ge oe 1 1 3 Contacting Caliper Technical Support aooo 3 2 Getting St rted o a a Bet ee eevee bee etek eeetetes owe a 5 2 1 Overview of Imaging amp Image Analysis aoaaa ee 5 2 2 Starting the Living Image Software ooo a 9 2 3 Initializing the
28. ARW20050826124002_001 Color Scale Type White Levels Color Scale Per Column 3 ARW20050826124002_002 Fluorescent Images Rw 200508261 On ARW 200508261 C Edit Layer Properties Opacity Color Table 100 BlackBlue a Reverse Logarithmic Sca e 119 7 Image Math 7 To remove all fluorescent or luminescent images from the overlay click the button 8 To change the display of a fluorescent or luminescent image in the overlay a Select the image in the lower box b Choose the Edit Layer Properties option c Adjust the opacity select a different color table or edit the color table properties When finished clear the Edit layer Properties option Select white or black for the Set the number of color scales low end of the color scale displayed per column ij image Overlay for Sequence Sequence ARW20050826124002_ SEQ Photograph Images AR W200508261 24002_001 ARW20050826124002_002 Fluorescent Images ARW200508 2002 ARW 200508 0 C Edit Layer Properties Opacity e a everse Logarithmic Scale 9 To reorder the images in the list a Choose the Edit Layer Properties option b Select an image c Click the or arrows 10 To copy the overlay image to the system clipboard click the Copy button ERT 11 To export the overlay image to a graphic file click the Export button ig 120 X Living Image Software User s Manual Caliper feSc
29. Alfalfa known to be An alfalfa free diet reduced background emission slightly but phosphorescent was eliminated not significantly from 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 chemiluminescent 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 is 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 cm sr and 1000 photons s cm sr respectively Since 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 6 page 91 Note that the background light emission is not uniform over the entire animal In Figure E 2 image
30. Apply Individual Color Scale for Sequences Display ROI Label As Measurement Show Transillumination Locations Photons Total Flux v C Show Advanced Options Counts Total Count v 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 195 B Preferences B 1 General Preferences l Preferences General User Acquisition Theme Tissue Properties 3D Analysis Start Up Defaults Show Activity Window on Warnings Errors vi Dock Tool Palette Left Right Window Size Save Settings Color Selections Folder Locations Width 55 Restore Defaults 7o 55 G window Size amp Position ns x Height 65 _ Most Recently Used Dataset History IS 2 Apply Individual Color Scale For Sequences Display ROI Label As Measurement Show Transillumination Locations Photons Total Flux C Show Advanced Options Counts Total Count Figure B 1 General preferences Table B1 General preferences Item Description Start Up Defaults Dock Tool Palette Sets the position of the tool palette in the application window Choose left or right Note Io 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 Set
31. Corrections Filtering tools to avoid filtering out single cell images Ti Living Image Software User s Manual Y Caliper Appendix E Luminescent Background Sources amp Corrections Electronic Backgro nd 4446444 e4 he eo eS Se wR ew ER ESS 4 217 Background Light On the Sample 2 002 eee eee ee 218 Background Light From the Sample 00858 ee eee 220 The background sources of light from bioluminescent 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 229 E 1 Electronic Background Read Bias amp Drift 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
32. Flux K 12 10 mueff crn 1 Figure G 4 Planar spectral analysis results 1 00 Normalized Flux a u WA Plot of Intensity Vs Lambda FS Units Photons 5 Display Overlay w Bc Image Min 1 26e3 Max 9 76e5 6 0 5 0 40 x10 3 0 RO 1 3 091e 06 f ve 2 0 10 p sec em 2 st Color Bar Min 6 97e4 Max 6 44e5 DAR Intensity vs Lambda Plot for ROI 1 a 600 Wavelength nm 620 243 G Planar Spectral Imaging 244 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 relative 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 O Fie Edit view Image Window Help JAX og QAM Lao amp h Units Photons C Apply t
33. HX20070321091157_007 Label Set All v Z Add to List Load as Group Location C Program Files Xenogen Sample Data Dataset Skull HX20060809153031_SEQ Sequencelnfo txt Images loaded in the browser as part of a sequence highlighted pink in this example These images cannot be selected for grouping into another sequence 2 In the browser select the images that you want to group together elo select adjacent images in the browser press and hold the Shift key while you click the first and last file in the selection elo select non adjacent Images in the browser PC users Press and hold the Ctrl key while you click the images of interest in the browser 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 For details on how to save or export the image data see Chapter 3 page 34 54 ad Living Image Software User s Manual Y Caliper 5 4 Working With a Single Image Table 5 3 explains the items in the image window For details on how to browse data and open images see page 45 Choose the image display units Select a display mode from the drop down list el TLT20050624145507_006 BR Units Counts v Display Overlay Overlay Photograph Luminescent Bias Image Min 48 Max 22447 Counts Color Bar Min 1122 Max 19546
34. Lexan 232 ad Living Image Software User s Manual Y Caliper _ Despite the presence of various background sources the signal from most fluorophores exceeds background emissions Figure F 13 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 13 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 Exo 4sec Reference standard TR 613 Red is available through Precision Dynamics Co http www pdcorp com healthcare frs hAtm Animal Tissue Autofluorescence Animal tissue autofluorescence is generally much higher than any other background source discussed so far and is likely to be the most limiting factor in in vivo fluorescent imaging Figure F 14 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
35. M Surface Kappa Limits o z Starting Voxel Size i Backgound Uniform Surface Sampling C NALS Weighted Fit Restore Defaults A Surface Limits CI NNLS Simplex Optimization NALS Weighted Fit Restore Defaults Ts 3D Tools gt 3D Tools Figure H 6 3D reconstruction tools Parameters tab DLIT left and FLIT right Angle Limit The angle limit refers to the angle between the object surface normal and the optical axis The optical axis can be considered to be a line perpendicular to the stage The surface normal is a line perpendicular to a plane tangent to the surface point For example in a dorsal view of a mouse the highest point on its back would have a normal line perpendicular to the stage In this case the angle is zero The side of a mouse abdomen would have a normal line parallel to the stage so the angle here would be close to 90 The software uses luminescent image data for surface elements that are less than the angle limit The default angle limit setting is 70 for the IVIS Imaging System 200 253 H 3D Reconstruction of Light Sources 204 Series or the VIS Spectrum For VIS Spectrum or 200 Series data if there is significant signal on the side of the subject a larger angle of 70 85 can be used Kappa Limits DLIT Kappa x is a parameter that is searched during a reconstruction to determine the best fit to the image data Small values of kappa tend to favor d
36. Size minimum size is set at 50 then ROIs created on the image must be greater than 50 pixels in size Preview If 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 For more details see page 91 Replace ROls 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 6 3 Measuring ROls in an Image To obtain the intensity signal in a user specified area of an image draw a measurement ROI on the image There are three ways to draw measurement ROIs Drawing Method Manual Automatic Free draw See Description Page Places one or more ROIs circular square or grid shape on the image 84 The software automatically locates and draws a contour ROI s on the 88 image To do this the software locates the peak pixel intensities in the image and searches the neighborhood around a peak pixel A pixel is incl
37. arrow in the 3D tools 2 o zoom in on the image magnify right click Ctrl click for Macintosh users and drag the toward the bottom of the window To zoom out on the image right click and drag the Ek toward the top of the window Ba 2 2 Living Image Software User s Manual Y Caliper Selecting a Drawing or You can choose a different drawing and lighting style to change the appearance of the Lighting Style for the surface Surface To choose a drawing style make a selection from the Drawing style drop down list in the 3D tools SA e BS NP ontelcons vO atoa i s Sequence View 4 3D View Planar Spectral Imaging loian Topogtaphe gt DLIT 3D Reconstruction Z 3D Tools C Render Photon Density Map Apply Wavelenath v Threshold Intensity 407e 6 Color Table Pai ily Reverse Logarithmic Scale Slice Coronal 10 5 Wire frame amp Surface face a Point cloud surface Wire frame surface Surface face surface EN ih Figure 12 4 Surface drawing styles 167 12 3D Reconstruction of Sources To choose a shading style make a selection from the Shading style drop down list in the 3D tools ig A ae BNP inis couts vC aiytoa E E Segere Vien gt Planar Spectral Imaging O Surface Topography DUT 3D eS e a 3D Tools aa are 3
38. j TLT 200506 PAT ASOT OOF maga TL 2ouo4 L4SS0 tats Seren Mae N Pri dun ot 205 OF 56 15 Em Alter s600 Enc Mig AOw 12 6 A 13 Camera WIS 200 Beta 11 SI620Ery Edit Image Labels UserID ksSA LabelSet Check any 5 fields For display Ta Enarari GSR 05 01 05 Labal kidney Comment doraa xenogen Universal User Group Experiment Commenti Comment2 C Time Point C Animal Number C Animal Strain C Animal Model C Sex C view C Cell Line C Reporter C Treatment C Luc Injection Time C IACUC Number Apply y To Sequence Image label Open an image double click the thumbnail to determine whether the signal of interest is above the noise level and below CCD saturation Check the image min and max in the color bar A signal greater than 600 counts and less than 60 000 counts is recommended If the signal level is unacceptable adjust the exposure time or binning level For more details on the image window see Table 3 7 Edit the label information here Figure 3 2 Image window amp Edit Image Label box Y Caliper Living Image Software User s Manual ifeSciences Table 3 7 Image window Item Description Units Select the measurement units for the image display counts photons or efficiency from this drop down
39. see Appendix A page 191 The IVIS Imaging System is ready for image acquisition after the system is initialized and the operating demand temperature of the CCD camera is reached locked 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 2 5 About the IVIS Acquisition Control Panel amp Auto Exposure Feature 12 The control panel Figure 2 5 provides the image acquisition functions For details on the imaging parameters in the control panel see Appendix A page 191 The auto exposure setting is useful in situations where the signal strength is unknown or varies widely for example during a time course study When you choose auto exposure Figure 2 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 w Living Image Software User s Manual Y Caliper _ In most cases the default auto exposure settings provide a good bioluminescence or fluorescence image However you can modify the auto exposure preferences to meet your needs For more details see page 199 To acquire an image using auto exposu
40. 12 4 3D Tools Click to select a tool to work with the surface or 3D results perspective view only to select a surface drawing style to select a shading style for the surface File Edit view Tools Window Help IS X PEE Ez Sequence View A 3D View Mesh Volume Organs Animation Render Mesh Opacity tt 15 Render Photon Density Map Apply Simulated v Wavelength 560 v Threshold Intensity j 2 s2e5 Color T able Rainbow x Reverse C Logarithmic Scale Slice Coronal A E 02 Sagittal Up 3 0 Transaxial 23 i T photons sec Perspective Source Intensity Figure 12 3 3D tools and DLIT results in the 3D view window Table 12 4 3D Tools Item Description Image Tools A drop down list of tools for viewing and working with the surface or DLIT results Select ie 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 173 Select eg to zoom in or out on the image use a click and drag operation S p fae Select h 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
41. 25 If you plan to manually set up the sequence Table 12 2 shows the recommended image sequence Acquire the images using transillumination on the VIS Spectrum using the same excitation and emission filters from at least four source locations Table 12 2 Example image sequence for FLIT analysis Image Type Source Location First location Subsequent locations Photographic J Use previous photo Structured light J Fluorescent J A For instructions see page 151 After the surface is generated the 3D reconstruction of the light sources can proceed 1 In the tool palette click FLIT 3D Reconstruction Tool Palette Surface Topoorenty _____ Analyze Params Properties Results Sequence CX200707 177710905 SEQ pias Afuncia Source ASEAUEm Analyze tab Select Image Sources eee Source 01 corresponds to the first aE a i o image of the sequence source 02 ca 70 28606 corresponds to the second image 640 720 286e 06 and so on E40 T20 206e 06 E40 r20 2 06e 06 E40 T20 206e 0 E40 720 493e 0 640 1 16e 07 Reconstruct gt 3D Tools l gt 5 pectral Unmixing 2 Inthe Properties tab make a selection from the Tissue Properties drop down list Muscle is usually the best choice of tissue properties for general in vivo reconstructions Note The internal medium index of refraction is automatically entered when you select a tissue Living Image Software User s Manua
42. 258 infusion pump 257 initialization See system initialization instrument fluorescent background background instrument fluorescent background 235 IVIS Imaging System fluorescence imaging components 223 225 K kappa 159 kappa limits 159 254 kinetic acquisition settings 39 acquisition window 38 kinetic data acquire 37 save 43 44 view and edit 42 kinetic ROI plot 95 draw 95 94 v L Y Ca PS cas L lens aperture 205 line profile 71 Living Image browser 46 Living Image software starting 9 luciferase spectrum 241 luminescence reconstruct 3D sources 152 155 M magnify image 65 manual focusing 34 save data 34 manual conventions 2 maximum vs time graph tracking infusion 259 maximum vs time graph 41 measurement ROI automatically draw 88 89 measurement ROIs 82 84 89 free draw 90 measurements 72 menu commands 261 mesh drawing style 167 lighting style 167 miscellaneous material autofluorescence 231 233 multiple reporters per photograph 118 120 N N surface limits 159 254 NNLS optimization 159 255 weighted fit 159 255 normalization 225 O open image data 47 49 optical density 226 optical properties for planar spectral imaging 241 organ atlas 181 organs 176 180 overlay 212 overlaying images 118 120 overview 267 Index imaging amp image analysis 5 8 P pan image 65 PCA biplot 136 PCA explained variance 137 photon density 160 photon density map measured 160
43. Choose this option to apply a logarithmic scale to the color table Source Voxel Click the button then click a voxel in the 3D reconstruction to Measurement display measurements for the voxel Total Flux DLIT The total flux measured for the voxels selected using the voxel tool results Total N mm measured for the voxels selected using the Measure Voxels Fluorescence tool 23 Yield FLIT results Volume Volume of the selected voxels mm ad Living Image Software User s Manual Y Caliper Table 12 6 3D tools Volume tab continued Item Description 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 Organ location of the selected voxel s Center of Mass Click to compute the center of mass for the selected voxel s Export Voxels Enables you to export the voxel measurements csv To display voxel measurements 1 Click the Measure Voxels button Ea 2 On the surface click a voxel Alternately draw a box around a group of voxels The Voxel tab displays the selected voxel data File Edit Yiew Tools Acquisition Window Help ao YN H a amp r Units Counts Oo Apply to all Tool Palette Pj V20080208142757_SEO DER Ea J 5 Vi 3D View 3D Tools pum Te A AE Lsm Mesh volume Organs Animation M Render Volume Min 5 26e 6 Render voxels As Max 1 14e 3 C
44. Confirm that the 3D view shows the properties of interest for example organs voxels surface or photon density maps Edit View Tools Acquisition Window Help ee gt Tool Palette EJ 1 gt ROI Tools e A W R amp R Units Counts vO Apply to all fi 1V20080208142757_SEO gt FLIT 3D Reconstruction i 3D Vie gt Surface Topography Eisneltubian 2 aa G r ka 07 2G Organs Animation Preset Animations Presets ja paan A sill la g lniae pal Frame Factor Animation Setup Time Scale o ee k J Key Frame 1 Key Frame 2 Key Frame 3 Key Frame 4 Key Frame 5 w P P O Nmm 2 Source Intensitas A In the 3D Tools click the Animation tab If necessary clear the key frame box click the button and select Delete All To view a preset animation make a selection from the Presets drop down list See Table 12 10 for a description of the preset animations A list of the key frames appears To view the animation click Play 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 To save the animation to a movie 1 2 Click Record In the dialog box that appears choose a directory enter a file name mov mp4 avi and click Save B Li
45. Density Map option to display the surface without the photon density map Figure 12 1 Viewing DLIT 3D reconstruction results Results tab top and 3D tools bottom Living Image 3 1 File Edit View Tools Window Help SAH p RA WD tnits counts vC Apply to all Tool Palette osi Pi 1L120050624145507_SEO Analyze Params Properties Results 4 DLIT Results DLIT_solution Loaded Key Value Living Image 3 1 File Edt View Tools Window Help ow Qh amp A GB R Units Counts v C Apply to all gt ROI Tools gt Planar Spectral Imaging e o m Mesh volume Organs Animation V Render Mesh C Render Photon Density Map Apply Simulated Wavelengths 560 Threshold Intensity 1 97e 5 Color Table a Reverse Logarithmic Scale w i adi A TLT2005062414550 7_SEQ Perspective Image window 3D view g photons sec Source Intensit photonsisac Source Intensi Light sources voxels 155 12 3D Reconstruction of Sources 12 2 3D Reconstruction of Fluorescent Sources Set Up amp Acquire an Image Sequence on the IVIS Spectrum Generate the Surface Topography Set the FLIT Parameters amp Reconstruct the Sources 156 Use the imaging wizard to setup the image sequence required for FLIT analysis For more details on the imaging wizard see page
46. Figure C 1 Quantum efficiencies Bialkali photocathode A back illuminated CCD and front illuminated CCD m IVIS systems use back illuminated CCDs IVIS Imaging Systems are equipped with a high light collection f 1 lens The sensitivity of the VIS 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 205 C Detection Sensitivity Image Exposure Time Field of View FOV C 2 Binning 206 At f 1 the depth of field ranges from 0 2 cm at FOV 3 9 cm VIS Imaging System 200 Series only to 2 cm at FOV 25 cm You can use the manual focus option on the Control panel to easily assess the depth of field at any f stop and FOV setting For more details on manual focusing see page 34 Generally f 1 is recommended for low light luminescent images and f 2 or f 4 is recommended for brighter luminescent or fluorescent images 1 B _ d pa ee i j i j narrow focus Fi deep focus Figure C 2 Lens f stop positions Left le
47. Halogen Lamps with Dichroic Reflectors IR Cut Off EJV Lamp 3400 K 10 m FKE Lamp 3200 K Relative Spectral Radiance 400 600 800 1000 1200 1400 1600 1800 Wavelength nanometers Figure F 2 Relative spectral radiance output for the quartz halogen lamp with dichroic reflector The lamp output is delivered to the excitation filter wheel assembly located at the back of the IVIS Imaging System Figure F 3 Light from the input fiber optic bundle passes through a collimating lens followed by a 25 mm diameter excitation filter The TVIS 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 bioluminescent imaging to prevent external light from entering the imaging chamber The Living Image software manages the motor control of the excitation filter wheel Fused Silica Fiber Optic Bundle Collimating Input Fiber Optic Bundle Excitation Motor Filter Figure F 3 Excitation filter wheel cross section Following the excitation filter a second lens focuses light into 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
48. IVIS Imaging System 2 2 11 2 4 Checking the System Temperature 2 0 0 0 0 eee ee ee 11 2 5 About the IVIS Acquisition Control Panel amp Auto Exposure Feature 12 3 Acquire an Image or Image Sequence aoaaa a a a 588888 15 3 1 Acquire a Bioluminescence Image 2 a a a 15 3 2 Acquire a Fluorescence Image With Epi Illumination 18 3 3 Acquire a Fluorescence Image With Transillumination a a aaa aa 19 3 4 Acquire an Image Sequence nonono a 22 3 5 Manually Setting the Focus 1 34 3 6 Manually Saving Image Data 2 20 0 0 2 ee 34 3 7 Exporting Image Data aoaaa ee 35 4 Acquire Kinetic Dala i2e aten eis eee eG Ohm S Ow ee ew ww Ee 37 4 1 Kinetic Acquisition aoaaa a 37 4 2 Viewing amp Editing Data Kinetic Acquisition window s oaoa e 42 AS Savine Dad sssr eb wae eee ee ee Shee Se ee Oe eee eG See od 43 5 Working With Data cece s teaches e tbe abi ea ee we eee ee Ee 45 5 1 Browsing amp Opening Data oaao ee 45 5 2 The Tool Palette amp Image Window onoo a a 50 5 3 Working With an Image Sequence ononon 2 0 eee ee ee 51 5 4 Working With a Single Image 2 a 55 5 5 Viewing Image Information 2 0 ee 59 5 6 Image Layout Window 1 ee 62 5 7 Adjusting Image Appearance 63 5 8 Correcting or Filtering Image Data aooaa a a a a 66 5 9 Image Information Tools 4 2 s sac eee ee hae we ee naidata EEE SHEER
49. Properties Results Value K Val 4 Ee alue Total fluorescence yield N mm 2 1 722 09 A enhe 0 75 Total source flux phot p 82e 10 Number of sources 24 2 00 Reduced Chi2 7 98e 07 Number of sources 30 Starting size 6 00 Reduced Chiz 2 38e 03 Neur best 200 Starting vsize best 8 00 Total surf samples 6000 i 2 00 Threshold angle 70 00 S00 Uniform Surface Sampling TRUE Total surf samnles TAN lt E Photon Density Maps Export So at 1 Save Results Photon Density Maps Espot 3 all Save Results SS EE Name DUIT_noSimplDLmsh F E amiak i Ovennrite C 5pecual Unmixing e Figure 12 2 3D reconstruction results FLIT left and DLIT right For more details on the DLIT and FLIT algorithm parameters see Appendix H page 247 Sometimes adjusting the DLIT algorithm parameters improves the fit of the simulated photon density to the measured photon density data Table 12 3 3D reconstruction results Item Description Optimized fit parameters Total source flux phot s DLIT The sum of the bioluminescent source intensities result Total fluorescence yield N mm The total sum of the fluorescent yield The quantity FLIT result measured is Fluorescence quantum efficiency for the excitation wavelength to emission wavelength photons Excitation wavelength photon absorption cross section Fluorophore number density
50. ROI parameter thresholds or draw one ROI at a user specified location Automatically Drawing Measurement ROls To automatically draw all ROls detected by the software 1 Open an image or image sequence and in the ROI tools select Measurement ROI from the Type drop down list 2 Click an ROI shape button Circle OJ Square G or Contour and select Auto All from the drop down list The ROIs appear on the image or thumbnails eRe mM A R uscar D Aptos Le JJH200506 30142719_SEO a Sequence View Units Counts Ea g Use Saved Cokas Fuko RO Por aneters Threshokd h Lower Lovet teu Se wu Trenew Use Sho Offipet Replace ROl estore Defeats 3 Click the Measure button Y The ROI table appears For more details on the table see Managing the RO Measurements Table page 106 l ROI Measurements EAR ROI Measurements Image Laye Total Counts Avg Counts Stdey Coun Min Counts Max Counts N 1 075e 07 8 034e 04 Image Number ROI 6 568e 03 6 052e 02 2 598e 04 Customized Selections Copy Measurements Types Image Attributes _ ROI Dimensions Counts _none_ v _none_ cere 33H20050630142719 003 ROL2 Overlay 11 282e 06 6 573e 03 88 _ a Living Image Software User s Manual Y Caliper 4 Double click a thumbnail to view the ROI measurements in the image To automatically draw an ROI at a user specified location 1 Open an i
51. Sources 12 1 3D Reconstruction of Bioluminescent Sources General Considerations Generate a Surface Topography Set Up amp Acquire an Image Sequence 152 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 3D reconstructions are currently not possible on black or dark colored furred mice In this case it is recommended that you shave the animals or apply a depilatory Luminescent Exposure vs Luciferin Kinetic Profile It is important to consider the luciferin kinetic profile when you plan the image 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 For details on generating the surface see page 139 Use the imaging wizard to setup the image sequence required for DLIT analysis For more details on the imaging wizard see page 25 If you plan to manually set up the sequence Table 12 1
52. Stop For more details on the control panel settings see page 191 6 Set the FOV To adjust the field of view FOV make a selection from the Field of View drop down list For more details on FOV see page 160 e 3 Living Image Software User s Manual Y Caliper _ NOTE To view the subject s inside the chamber before image acquisition take a photographic image clear 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 or e Select Manual focus from the Focus drop down list For more details on manual focusing see page 34 8 If necessary click mage Setup jn 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 22 9 When you are ready to acquire the image click Acquire During image acquisition the Acquire button becomes a Stop button To cancel the acquisition click Stop The image window appears Figure 3 1 If this is the first image of the session you are prompted to choose an autosave location Living Image 3 2 Do you wish to enable auto saving of acquired data for this session wy This can be ch
53. 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 233 F Fluorescent Imaging 234 Regular Rodent Food GFP DsRed youn ICG Alfalfa free Rodent Food GFP DsRed Cy5 5 ICG Figure F 14 Images of animal tissue autofluorescence in control mice Nu nu females Animals were fed regular rodent food top or alfalfa free rodent 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 Figure F 15 shows a comparison of fluorescence and bioluminescence emission in vivo In this example 3x 10 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 bioluminescent and fluorescent expression The fluorescence signal level is 110 times brighter than the bioluminescence signal However the autofluorescent tissue emission is five orders of magnitude higher In this
54. The point source fitting results are saved with the image To view results 1 Select the results of interest 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 Name LMFIT_13 Tool Palette E E gt Surface Topography E i 7 Point Source Fitting pe Analysis Params Properties Results Eror Estimation k Starting Chiz 8 58e 09 Ending Chive 7 03e 08 Paint source Fitting Results Parameters Fitted value 0 143 cm 6 676 cm 1 710 cm 0 049 cm Total source flue 2 22641 1 photonss s location of the source 0 151 cm EF er Photon Density Maps Export results Save Results ad Delete Load sae gt FLIT 3D TEER i i gt 3D Tools a 1 Right click the results row of interest and select Copy Selected 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 select Copy All from the shortcut menu that appears All of the results table is copied to the system clipboard 149 11 Point Source Fitting This page intentionally blank 150 Ti Living Image Software User s Manual Y Caliper 1 2 3D Reconstruction of Sources 3D Reconstruction of Bioluminescent Source
55. VIS camera setup 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 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 75 5 Working With Data 5 11 Viewing Transillumination Data 76 The transillumination overview feature combines the images of a FLIT sequence a fluorescence sequence acquired in transillumination mode into a single image All of the individual fluor 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 photon units and can be analyzed using the tools in the tool palet
56. Y Caliper Appendix IVIS Syringe Injection System Controlling the Infusion Pump nanoa oe PH ee EEK DERE EO 257 Tracking Infusion in the Maximum vs Time Graph naana aaa 259 Closing the Infusion Pump Control Panel 0 0002 ee eee ees 259 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 3 2 installation CDROM The IVIS Syringe Injection system can be used during kinetic or still image acquisition however subjects must remain immobile 1 1 Controlling the Infusion Pump After the IVIS Kinetic imaging system is initialized and locked you can access the infusion pump controls 1 Select Acquisition Infusion Pump Setup on the menu bar The Infusion Pump control panel appears above the IVIS acquisition control panel ie ce ae i Bhgn Li Fluorescent Bachgaung P iuair tuted Ti 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 IVIS Syringe Injection System
57. and Non Negative Least Squares are 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 minimum radiance and only keeps the voxels that contribute significantly to these surface elements m B Living Image Software User s Manual Y Caliper Source amp Tissue Properties DLIT analysis of spectrally filtered images requires knowledge of the spectral dependence of bioluminescent light emission Table H 1 shows the factory set source spectra provided by the software NOTE The source spectra is not an input to the 3D reconstruction of fluorescent sources Tool Palette gt Planar Spectral Imaging ie E Surface Topography X A DLIT 3D Reconstruction Analyze Params Properties Results Select a bioluminescent source spectrum Select a tissue or organ from the Tissue Properties drop down list The associated 5 7 internal medium index of refraction is automatically Source Spectun ee Pat Internal medium index of refraction 1 40 Normalized Amplitude Choose the Source Spectrum trom the Plot drop down list to display the selected spectrum 1 00 600 800 Wavelength rir Catos S _ zd Figure H 4 DLIT 3D reconstruction tools Properties tab Table H 1 Source spectra Source Spectrum Description Bacteria Bacterial lu
58. average background there is significant ROI from a measurement ROI autoluminescence or autofluorescence Available ROI e Manual e Manual e Manual e e Automatic e Free draw e Automatic Drawing Methods e Free draw e Free draw Available Shapes Circle square grid or Circle or square Square contour NOTE For a quick guide to drawing a measurement ROI see page 84 6 2 ROI Tools To display the ROI tools 1 Open an image or image sequence and click ROI Tools in the tool palette File Edit View Tools Acquisition Window Help 3 ik Ex i A amp R Units Counts vo Apply to all 117 20050624145507_006 Seca Units Counts v Display Overlay v ROI tools ype Measurement ROI Save ROIs Name ROI_1_KS54 Delete Load Auto ROI Parameters hreshold Lower Limit 0 0 Minimum Size 20 Preview Use Bkg Offset Replace ROIs Restore Defaults Save Load Color Bar Min 1122 Max 19546 82 Living Image Software User s Manual a Y Ca Be ens Table 6 2 Tool palette ROI tools Item Description Click to select the number of circle ROIs to add to the active image Click to select the number of square ROIs to add to the active image Click to specify the grid pattern for a measurement ROI that you want to add to the active image This tool is useful for an image of a multi well culture plate or microtiter plate S H e Click and sele
59. 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 5 A steep slope is required to avoid overlap between the two filters Typically the slope is steeper at shorter wavelengths 400 500 nm 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 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 Ti Living Image Software User s Manual Y Caliper 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 The 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 System
60. can obtain a background corrected ROI measurement by subtracting an average background ROI from a measurement ROI The software computes Background corrected intensity signal Average signal in the measurement ROI Average signal in the average background ROI 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 Image Layout Window page 62 and Magnifying or Panning in the Image Window page 65 To measure background corrected signal 1 Draw one or more measurement ROIs on the subject For more details see page 90 2 Draw an average background ROI on the subject a Select Average Bkg ROI from the Type drop down list b Click the Square Z or Circle O button and select 1 The ROI is added to the image For more details on adjusting the ROI position or dimensions see page 100 and page 101 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 three methods to associate the average background ROI with one or more measurement ROls 91 6 Working With ROI Tools 92 Method 1
61. down list For more details on the different types of image displays see Figure 5 6 page 57 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 Color bar Shows the minimum and maximum pixel intensities in the image as well as the color bar Pixels less than the color bar minimum or greater than the color bar maximum are not displayed in the image Image label Information about the image that the software automatically records and user specified information entered in the Edit Image Label dialog box 3 2 Acquire a Fluorescence Image With Epi Illumination 18 Epi illumination uses an excitation light source located above the stage For more details on fluorescence imaging see page 169 1 Start the Living Image software double click the icon on the desktop 2 Initialize the IVIS System and confirm or wait for the CCD temperature to lock For more details see page 11 E IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning FiStop Excitation Filter Emission Filter i a hl inl lt M lt Field of View System Status Acquire 0 2 Subject height 150 Sem Sequence Setup 3 Putacheck mark next to Fluorescent 4 Confirm that the Fluorescent Lamp Level is set to the appropriate level High or Low 5 Select the Auto exposure time click the arrow Alternately manually set the exposure binning and F
62. ee 132 9 4 Spectral Unmixing Options a aoa a 134 10 Generating a Surface Topography 2 2585858008 139 10 1 Generate the Surface Topography 0 0 0 0 00 ee ee 139 10 2 Managing Surfaces s siesati eae e dd See eee Ska eae wea we 142 11 Point Source Fitting 2a2 e es ee Bee ke ee ee we ee ew 143 11 1 Displaying the Point Source Fitting Tools 2 2 2 2 2 00 2 ee eee 143 11 2 Point Source Fitting a oaoa e ee 146 11 3 Checking the Point Source Fitting Results noaoae aa 148 11 4 Exporting KResuts 5e2ec 544405 6464 Sew eee eee ee ee oe oes 148 11 5 Managing Point Source Fitting Results 2 2 ee ee 149 12 3D Reconstruction of Sources 2 0 2 0 0 ee eee ee ee 151 12 1 3D Reconstruction of Bioluminescent Sources 1 2 a a 152 12 2 3D Reconstruction of Fluorescent Sources ooo 156 Ti i Living Image Software User s Manual Y Caliper PoE CCPL RESUS aera a e aT 158 PASD TOO gaan ee eb GREE OES AREER EDERAL EERE OEE EER eS OD Ge 164 12 3 3D Tools Mesh Tab 222 635 8240246484688 24 44648554 CRE Bese eS 170 12 6 3D Tools Volume Tab s sa s amosa koema eo SH ee Pee ee PRR H He EE 172 12 7 3D Tools Organs Tab 2 ee 175 12 8 3D Tools Animation Tab 2 2 ee 182 12 9 Managing DLIT FLIT Results 0 0 0 0 2 0 2 00 0 187 13 Troubleshooting Guide 0 000 2 ee eee ee ee 189 13 1 DLIT FLIT Analysis Troubleshooting 0 0 0
63. enables you to select an instrument luminescent background This background measurement is subtracted from luminescent images Opens a dialog box that enables you to view the dark charge measurements for the system Opens a dialog box that enables you to remove the dark charge measurements from the system Opens a dialog box that enables you to acquire a dark charge measurement Opens a dialog box that enables you to select an instrument fluorescent background measurement for the active image data If the Sub Fluor Bkg option is chosen in the control panel the background measurement is subtracted from the image data Starts a measurement of the instrument fluorescent background Opens a dialog box that enables you to select a fluorescent background measurement Living Image Software User s Manual W caliper Table J 1 Menu bar commands and toolbar buttons continued Menu Bar Command Toolbar Description Button Acquisition Fluorescent Background Clear Available Fluorescent Background Acquisition Fluorescent Background View Available Fluorescent Background Window Close Window Close All Window Cascade Window Tile Window 1 xx Window 2 xx Window etc Window Other Windows gt Browser Window Help User Guide Help About Living Image 3 Table J 2 Keyboard shortcuts Keys Shortcut Description Opens a dialog box that enables you to
64. example fluorescent imaging requires at least 3 8x 105 cells to obtain a signal above tissue autofluorescence while bioluminescent imaging requires only 400 cells Fluorescent Bioluminescent 9 5x10 1 0x10 7 8x10 71x10 photons s cm sr Signal Background 7 8 Signal Background 7500 Min detectable cells 3 8 x 10 Min detectable cells 400 Figure F 15 Fluorescent left and bioluminescent right images of stably transfected dual tagged PC3M luc DsRed cells The images show the signal from a subcutaneous injection of 3x10 cells in an 11 week old male Nu nu mouse NOTE When you make ROI measurements on fluorescent images it is important to subtract the autofluorescence background For more details see Subtracting Tissue Autofluorescence page 115 al E Living Image Software User s Manual Y Caliper 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
65. file Copies the line profile graph to the system clipboard Opens the Print dialog box To measure distance with the measurement cursor 1 Open an image and in the Image Information tools click the Distance Measurement Cursor button ie A measurement cursor 4 ______sE appears on the image The tool palette shows the position and length of the cursor Fle Ed View Tools Acquistion Window Hels g 3 u Ra A amp W Units Courts 1 Apply toal v Weh 2em Heit 12 6cm bmage X Y 12 295 617I cm image Data 73 counts ony etarnce Ta leensen B omnes Jose Detence 1 S300 Measurement cursor T ier 7 Living Image Software User s Manual Y Caliper 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 Fa button Table 5 10 Measurement cursor position amp length Item Description pan Pixel x y coordinates of position 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 To measure distance using the crop box 1 Open an image and in the Image Information tools click the Image Crop button 1 e ES in amp A 3 R Unts Courts Apply to al Wah
66. 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 104 ad Living Image Software User s Manual Y Caliper Deleting ROIs You can delete ROIs from an image or permanently remove ROIs from the system To delete ROIs from an image In the ROI tools click the button _ Bo 11720050624145507_006 See Units counts Display overlay vmo _ a Tool Palette gt Image Adjust gt Corrections Filtering gt Image Information ROI Tools oO ey X C Apply to Sequence Image Min 48 tod Max 22447 ype Measurement ROI Save ROIs ame ROI_1 KS4 z ma Ci save All Autos si mia ave AIIBKGs All Measurements BKG 1 1 253e 03 Auto ROI Parameters All Subjects Lg e e hreshold o J 9 5 Lower Limit J o es al ROT 1 Minimum Size tf 20 ROI 2 Preview Use Bkg Offset C Replace RO Save j Load ROI 2 BKG 1 Restore Defaults Save Load Auto ROI Parameters Name KSA_AUTO_MEAS ROI SET_1 v gt Planar Spectral Imaging Color Bar Min 1122 gt Surface Topography Max 19546 _ gt Point Source Fitting gt DLIT 3D Reconstruction 2 Make a selection from the drop down list of dele
67. intensity of shorter wavelength data 203 B Preferences This page intentionally blank 204 Ti Living Image Software User s Manual Y Caliper Appendix C Detection Sensitivity CCD Detection Efficiency 0 ee ee 205 PM 6 nee ee Oe eh Pee eae Oe eee So Re 206 IIQGUNIN 6 gee hoo aR Ree REDRESS RSS HSS 208 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 visible 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 Back Ilhuminated E front Whaminated Image Intenafer Quantum Efficiency 200 400 600 BOD OOO Wavelength nm
68. is initialized and the CCD camera reaches operating demand temperature locked 4 1 Kinetic Acquisition 1 Start the Living Image software double click the icon on the desktop 2 Initialize the IVIS System and confirm or wait for the CCD temperature to lock For more details see page 11 3 When you are ready to begin imaging click Kinetics in the control panel The Kinetic Acquisition window appears IVIS Acquisition Control Panel fe fx Imaging Mode Exposure Time Binning FiStop EMGain Excitation Filter Emission Filter 1 00 Gece V vedun e Jom d i al Field of view D v System Status C xFoy 24 Idle 1S cm Subject height 1 50 cm Sequence Setup Focus use subject height v Temperature TG Locked Initialize Kinetic Acquisition Control Panel DEN Display Siieaerg 13 frames sec Acquisition Settings Luminescent v Overlay Dynamic Range 14 bit v Exposure Time msecs 35 F Binning 4 v F Stop 2 v EM Gain 50 v Excitation Filter Block Emission Filter Open v FL Lamp Level High Photograph Light Level 51 6 sie Frame C Accumulate Play h mm ss 0 0 00 00 0 ee E OG Color Scale EE Minimum Figure 4 1 Control panel top and Kinetic Acquisition window bottom 4 Select the type of data to acquire and set the acquisi
69. 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 phosphoresce 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 1s a major area of interest in in vivo bioluminescent 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 Ti Living Image Software User s Manual Y Caliper Xenogen Corporation has conducted tests to try to minimize the source of the background light emission in mice Test Description Observation Test 1 Subject animals were Background emission levels were not reduced A housed in the dark 12 hours prior phosphorescent component in mouse fur or skin is not the to imaging source of light emission Test 2 White furred animals No increase or decrease in background emission levels were shaved prior to imaging Test 3
70. j 11720050624145507_006 DAR Units Counts v Display Overlay EE Image TLT20050624145507_006 Series Male Nn nu Fri Jun 24 2005 07 57 18 Experiment DOB 03 21 05 Em Filter Open Label kidney To display or hide the Image Bin M8 FOV 12 6 f4 1s Comment dorsal i i Camera IVIS 200 Beta II SIB20EEV nfo rm atio n cl IC k Info 3 Image Min 48 Max 22447 15000 10000 5000 Counts Color Bar Min 1122 Max 19546 Figure 5 5 Image window overlay display mode 55 5 Working With Data Table 5 3 Image window Item Description Units Choose counts photons or efficiency from the drop down list for the image data For more details on counts photons or efficiency see Appendix D page 213 Display To choose an image display mode in the image window make a selection from the Display drop down list See Figure 5 6 for examples of the display modes Overlay A pseudocolor image of luminescent or fluorescent image data displayed over a grayscale photographic image Photograph A grayscale image that is captured when the IVIS Imaging System illumination lights are activated Luminescent A pseudocolor image of the luminescent data captured during an exposure when the IVIS Imaging System illumination lights are off Fluorescent A pseudocolor image of the fluorescent data captured during an exposure when the IVIS Imaging System illumination lights are off Background The CCD came
71. list Display Select the image type for example overlay that you want to display from this drop down list For more details on the different types of image displays see Figure 5 6 page 57 Info Click to display or hide the image label information a Opens a dialog box that enables you to export the image or thumbnails to a graphic Tile for example omp or DICOM format wj Creates a preview picture of the image or thumbnails that the Living Image browser displays when the data is selected amp Living Image Browser TLT20050624145507_5EQ Click Number User ID EM Filter Series Experiment Label Comment Analysis Comment 00H Spectral VIS 200 1 3 5 dorsal day 29 10 post luc 17 post luc 2 Location C Program Files enogen Sample Data I I5200 data TraserBeadsPC TLT20050624145507_SEQ Sequencelnfo txt Preview picture of the selected data Color bar Shows the minimum and maximum pixel intensities in the image as well as the color bar Pixels less than the color bar minimum are not displayed in the image Pixels greater than the color bar 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 33 3 Acquire an Image or Image Sequence 3 5 Manually Setting the Focus The IVIS Imaging System automatically focuses the image based on subject height see page 193 If you
72. lool Palette x Image Adjust E hi gt Corrections FRering 1 i E image information __ NT20050524145507 006 Fri Jun 24 2005 07 57 10 Em Ati Opin Bin M98 FOV 12 6 4 15 z dor Label set VIS 200 Beta IL SI6200 information amp acquisition parameters 59 5 Working With Data To view information about an image 1 Open the image or image sequence of interest For details on how to open data see page 45 2 Select View Image Information on the menu bar The Image Information window appears 3 To choose an image make a selection from the Sequences drop down list and the Images drop down list Drop down list of open sequences Drop down list of images in the selected Choose Individual Images from the sequence Or a list of single images if list to show the open single images Individual Images is selected in the in the Images drop down list Sequences drop down list Pj Image Information Sequences Individual Images V Images D420081107162612 v C Show All Sections Section Label Xenogen Universal luminescent TIF photographic image photograph TIF Peeeseeeeeseesessoeossesesesseesesesesssssessseosssssessssossssesesssesesesesesesesssesessssessssosopseseseseosnsoereseosossesesssseesssossssesessssossssesesssesesesdossssesssssessssssessoes Camera System Info feeeseseesseeeeotoeososseseseosseseseseseseseseosseseeseseessesesssesesssesesesesesssesesessessesesopesses
73. measured using the background filter to the autofluorescent signal measured using the excitation filter in a region on the animal where no fluorophore is present Compute k from ROI This option is useful for subtracting fluorescence background Draw the same ROI in both images on an area considered background In the Compute k from ROI drop down list select the same ROI in each image 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 Living Image Software User s Manual i 3 Y Ca PEI us Table 7 1 Image Math window continued Item Description Display Result for Measuring Opens the image generated by image math in an image window 7 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 Table 7 2 Table 7 2 Emission excitation and background filters used to acquire data that can be corrected for tissue autofluorescence Emission Filter Excitation Filter Primary Background Filter LLLE T CECCI EMEC Fluorophore Passband nm GFP 515 575 445 490 410 440 DsRed 575 650 500 550 460 490 Cy5 5 695 770 615 665 580 610 ICG 810 875 710 760 665 695 The obj
74. measurements dialog box appears F ROI Measurements ROI Measurements I Kinetic ROI Measurements Plot Kinetic ROI Measurements 7 Plot Multiple ROI Measurements 5 P 5 10 Total Counts Vs Time a 1 20090223153526 ROI 1 e a aai ll Mi gp Total Counts a 1V20090223153526 ROI 2 ROI Measurement Total Counts BIZ0090220041944 IROI 1 1 20090220113928 ROI 1 S 1 20090223153526 V ROIL 1 ROI2 k r s4 M A May Aan nA iv Sten APA Ss bw Plot ROI Measurements 10 1 Time secs Configure Export Close 4 Click the Plot Kinetic ROI Measurements tab 5 Make a selection from the Measurement Unit and ROI Measurement drop down lists 6 Select a data set and an ROI 7 Click Plot ROI Measurements 8 To add other ROI data to the graph repeat step 6 to step 7 96 ad Living Image Software User s Manual Y Caliper 6 6 Managing ROIs Viewing ROI Properties 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 To view ROI properties do one of the following eDouble click the ROI of interest 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 6 2 j 11120050624145507
75. not enough data N Surface This can occur because It is desired that all wavelengths DLIT and all trans illumination source positions FLIT are represented in the data equally In the DLIT case if the signal in a short wavelength is very low then the sampled data can be too few for sufficient refinement of the voxel gridding To Increase the data to be included determine the photons sec cm2 sec level for 30 counts on the low signal image this is the level of noise using Image Adjust in the Tool Palette Enter in the photons sec cm2 sec value into the Minimum Radiance column next to the wavelength image number in the Analyze Tab in the DLIT FLIT 3D Reconstruction menu of the Tool Palette e t is best to include the low signal data as it adds constraining information to the solution Note Using the Imaging Wizard should reduce the occurrence of low signal images in the data sequences No sources in solution e In DLIT or FLIT this can occur if the surface is not correct That is if a Surface is imported into the 3D View from another source other than from the Structured Light Analysis e In FLIT if you choose an emission Source Spectrum in the Properties Tab an Advanced User feature which is incorrect for the fluorophore of interest this can also result in no sources e In FLIT if there is very little signal detected at the surface and the Background box is checked in the Params tab then it is possible
76. 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 IVIS Imaging Systems implement a subtraction method based on the use of blue shifted background filters that emit light at a shorter wavelength see Table 7 2 page 115 The objective of the background filters is to excite the tissue autofluorescence without exciting the 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 7 page 115 The assumption here is 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 17 shows an example of this technique using a fluorescent marker In this example 1x 10 HeLa luc PKH26 cells were subcutaneously implanted into the left flank of a 6 8 week old female Nu nu mouse Figure F 18 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
77. one cm margin around the subject if possible 139 10 Generating a Surface Topography 5 Click Next to display the mask The mask is a purple overlay on the subject image that defines the area of interest tor 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 6 If you want to smooth the surface confirm the default surface generation options and surface smoothing parameters or enter new values For more details on the parameters see Table 10 1 7 If you want to save the results confirm the default name for the results or enter a new name 8 If necessary adjust the threshold value so that the mask fits the subject image as closely as possible without including any area outside of the subject 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 For more details on the tools see page 164 File Edit View Tools Acquisition Window Help ase W a amp h Units Counts v O Apply to all Tool Palette x gt ROI Tools LJ Sequence View 3D View gt Planar Spectral Imaging 2 seq Surface Topography Surface Reconstruction Structure Light TLT20050624145507_001 V SL B
78. operation 164 Ti Living Image Software User s Manual Y Caliper Table 12 4 3D Tools Item Description Drawing Styles A drop down list of drawing styles for the surface for examples see Figure 12 3 page 164 The Surface face drawing style a is the default Point cloud Pa A A Wire frame Surface face Wire frame and surface face Shading Styles A drop down list of shading styles for the surface for examples see page 166 The Reflect smooth surface face shading style ip is the default Smooth face Smooth surface face Reflect surface face Reflect smooth surface face 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 12 6 Ad Select this tool from the drop down list to display the perspective view a Click to show or hide measurement cursors in the coronal sagittal or transaxial views Click to display the manual transform tool Automatic atlas registration tool Click a voxel in the 3D reconstruction then click this button to display measurements for the voxel in the 3D tools source voxel measurements gt 5 T 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 subject in the 3D view window Click to show or hi
79. 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 A ILI FOROS TODAS 00 Links Photons Display Creede p sec em 2 st Color Bar Min 1 26e9 Max 2 47e10 select Tools Image Math for xx SEQ on the menu bar ad Living Image Software User s Manual Y Caliper 4 In the Image Math window that appears select the primary image in box A Select the background image in box B Note For more details on items in the Image Math window see Table 7 1 page 114 5 Select the math function A B k in the Result drop down list Almage Math Window Sequence TLT20061220101746_SEQ A TLT20051221160239 TLT 20051 221160320 B TLT20051221160239 TLT20051221160320 Color Scale Limits for and B Full Auto Result Color Scale Limits Full Auto Result A B k k 1 00 Compute k from ROl C Min 0 6 Click and select the ROI created in step 2 from the drop down list The background corrected signal is displayed 7 To view the mathematical result overlay mode in a separate image window click Display Result F
80. remove the fluorescent background measurements trom the system Opens a dialog box that displays the fluorescent background measurements for the system If a fluorescent background is selected the Sub Fluor Bkg option appears in the control panel Choose the Sub Fluor Bkg option to subtract the user specified background measurement from the image data Closes the active image window Closes all image windows Organizes the open image windows in a cascade arrangement see page 59 Organizes the open image windows in a tiled arrangement see page 58 A list of the open data Select the data of interest to it the active window and display it on top of all other open windows If the Living Image browser is open makes it the active window and displays it on top of all other open windows Displays the online help Displays the online help index Click this button then click an item in the user interface to display information about the Item Ctrl B Opens the Living Image browser Ctri C Copies the active image to the system clipboard Ctrl D Arranges open windows in a cascade Ctrl 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 the What s This tool Click this button then click an item in the user interface to displ
81. right slider position selected data range left slider position Figure 5 11 Image window kinetic data 77 5 Working With Data File Edit View Tools Window Help o gt A amp R Units Photons wo Apply to all j DA20081107162612 Eak Units Photons Display Overlay v info a a mA galaa Photo Adjustment Color Scale Limits Auto Full O Manual Individual Color Table YellowHot v C Reverse C Logarithmic Scale gt Corrections Filtering gt Image Information gt ROI Tools AD 3 0 x10 2 0 10 pfsecicm 2 sr Max 4 63e9 gt JA C Accumulate J lo19 H Extract 136 frames selected out of 231 frames Figure 5 12 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 Table 5 13 Image window kinetic data Item Description gt Play Starts playing kinetic data Stop Stops playing kinetic data F 41 Edit and Save options Shows or hides the bottom sliders that enable you to select a range of 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 accumulations as it happens Extract Click to select a save option for the current image or s
82. 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 bar for an image Pixels less than the minimum value are not displayed Max The maximum pixel intensity associated with the color bar for an image Pixels greater than the maximum value are displayed in the maximum color Limits 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 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 is active 64 Living Image Software User s Manual v Y Ca Be cas Magnifying or Panning in the Image Window Table 5 5 Image Adjust tools continued Item Description Color Table Click the drop down arrow to select a color table for the image data For more details on color tables see Pseudocolor Images page 211 Rainbow Reverse Choose this option to reverse the selected color table Logarithmic Scale C
83. sequence If this option is not chosen the system acquires a photographic image for each image in the sequence A Click Lad The acquisition parameters are added to the table 3 Acquire an Image or Image Sequence Click to include the parameter settings Each row in the editor specifies the acquisition for the photographic image in the editor parameters for one image in the sequence a Imaging Wizard _ Display Photographic Settings Mode Exposure Binning FStop Excitation Emission Structure FOY Height Delay 1 pG 1 Medium 1 Block Open Mo E 150 0 00 Number of Segments Delay min Add The image s in the sequence editor comprise one segment You can acquire multiple segments with a user specified time delay between acquisitions This is useful for acquiring data for kinetic analysis 5 Repeat step 3 to step 4 for each image in the sequence 6 To specify atime delay between each acquisition enter a time in the Delay min box in the Sequence Editor 7 If you want to save the sequence setup information xsq a In the sequence editor click the Save button fa b In the dialog box that appears select a directory for the file enter a file name and click Save 28 fl Living Image Software User s Manual Y Caliper Editing an Image Sequence You can edit a parameter value as well as add or remove images from the sequence A shortcut menu of edit commands is available when you right c
84. 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 is After you acquire a fluorescent image inspect the signal to determine 1f a fluorescent background should be subtracted Figure F 16 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 background subtraction on and off using this check box The fluorescence background also contains the read bias and dark charge Dark charge subtraction is disabled if the Sub Fluor Bkg check box is checked Image Image Min 462 6 Min 41624 Max 47662 Max 46239 counts counts 40000 40000 30000 30000 20000 20000 10000 10000 Color Bar Min 900 Max 40000 Color Bar Min 900 Max 40000 Figure F 16 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 F 7 Adaptive Background Subtraction Adaptive back
85. that are installed Table A 1 IVIS acquisition control panel Item Luminescent Fluorescent Exposure time Binning Description Choose this option to acquire a luminescent image Choose this option to acquire a fluorescent image The length of time that the shutter is open during acquisition of a photographic or luminescent 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 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 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 binnin
86. that only the background fluorescence is simulated Surfaces are spiky e The most common source of spiky surfaces are folds in the animal skin or animal fur which corrupt the desired smooth lines projected on the animal from the laser galvanometer Adjusting the Path Averaging Size in the Surface Topography menu in the Tool Palette can help reconstruct an improved surface Smoothing the surface by using the Smooth feature in the tool palette Surface Topography menu can also help improve the surface 189 13 Troubleshooting Guide This page intentionally blank 190 v ae i Living Image Software User s Manual Y Caliper Appendix A IVIS Acquisition Control Panel The control panel provides the image acquisition functions Figure A 1 To acquire an image using auto exposure click the arrow and select Auto IVIS Acquisition Control Panel ER Lum INESCENCE Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter imaging settings Fluorescence imaging settings d Photographic Field of view System Status Imaging settings e aden Structured light imaging settings saratsa is Slm Focus Temperature B orea Figure A 1 IVIS acquisition control panel auto exposure selected Idle i Acquire NOTE The options available in the IVIS acquisition control panel depend on the selected imaging mode the imaging system and the filter wheel or lens option
87. 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 that 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 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 measurem
88. to access a shortcut menu of viewing options Table 4 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 View 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 Draw Scale Displays a scale along the x and y axis of the frame Insert Tag Displays a tag with x y pixel information at a user selected location of the image To insert a tag right click a location in the Image and choose Insert Tag on the shortcut menu Remove Tag Removes a user selected tag from the image Remove All Tags Removes all tags from the frame Display Color Bar Choose this option to display the color bar Display Color Min Max Choose this option to display the color bar minimum and maximum Display Image Min Max Choose this option to display the minimum and maximum signal The IVIS Kinetic instrument ena
89. to display fluorescent images Click Reverse to reverse the color scale Use saved colors while If this option is chosen data are displayed using a user specified loading data 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 Preferred ROI Line Color Luminescent Color of the ROI outline on a luminescent image Fluorescent Color of the ROI outline on a fluorescent image 200 Living Image Software User s Manual y Caliper File Edit View Tools Acquisition Window Help a A W A amp R Units Counts v C Apply to all Tool Palette I A TLT20050624145507_SEQ TER gt ROI Tools 4 riren z er as Sequence View 3D View gt Planar Spectral Imaging T gt Surface Topography a gt Point Source Fitting DLIT 3D Reconstruction Analyze Params Properties Results Sequence FL I20050624745507_SEQ Tissue Kidney Source refy Select Wave Filters Filter Minimum Radiance e se0 3 02e 05 6 06e 05 2 13e 06 3 02e 06 2 35e 06 gt Spectral Unmixing gt 3D Tools i photons sec Perspective Source Intensiti Figure B 6 Image window 3D view with gradient background B 5 Tissue Properties fi Preferences General User Tissue Properties Pancreas Source Spectrum Firefly Index of Refraction 1 40
90. voxels are also automatically displayed when the 3D reconstruction Is completed For more details on measuring the voxels see page 172 2 To display the measured and simulated photon density profiles a Select a wavelength b Drag the crosshair to the location of interest The horizontal and vertical photon density profiles are updated fi Photon Density Maps DE Wavelength EA v v Angle of View HATA E aT C Logarithmic Scale E7 f f i ie in Bacal photons a K Wie 1 photons mm 3 Horizontal Profile a Simulated Vertical Profile Simulated jot m Measured agi m Ileasured S 0 Photon density photonsimm 3 35 Photon density photonsimm 3 an In a good reconstruction the 20 measured blue and m simulated red photon H density curves are close 10 0 10 40 20 0 20 Position mm Position mm Position 4 0 Position FF Close 161 12 3D Reconstruction of Sources 3D Tools The 3D tools appear in the tool palette when a surface topography surface or 3D source is reconstructed or when you open saved results me am BY 1 11 ke LE See Page 3D tool buttons 164 Mesh volume Organs Animation Preset Animations Presets Spin CW on x Axis ri Frame Factor 1 Animation Setup Time Scale o J lt gt 4 XO Play Record Frames Per Second 10 Total Duration secs 5 Load i Save
91. x y coordinates and dimensions in cm in the table Copy Selected Copies the selected row s in the table to the system clipboard 107 6 Working With ROI Tools Configuring the ROI Measurements Table 108 Table 6 4 ROI Measurements table continued Item Description All Copies all rows in the table to the system clipboard Refresh Updates the table Configure Displays the Configure Measurements box that enables you to specify and organize the data categories column headers for the table Export geet Save Measurements box so that the data can be saved to a txt or csv Tile Note Grid ROI measurements exported to a csv Tile can be opened in a spreadsheet application like Microsoft Excel Close Closes the ROI Measurements table You can customize the data and information column headers in the ROI Measurements table Several predefined categories are available in the Measurement Types Click Attributes and ROI Dimensions drop down lists j ROI Measurements DER ROI Measurements Image Number Image Layer Subject Subject Lab Subject ID Bkg ROI Total Count Avg Counts TuT20050624145507_003 ROLL Overlay Subject 1 Subject i MMe 12826406 12 2898 04 TLT20050624145507 004 i 2 434e 06 3 202e 04 TLT20050624145507 006 ROIZ Overlay MO es BKG 1L 44 7506405 2 4616 03 TLT20050624145507_006 i Overlay _none_ 522e 05 11 272e 04 lt gt Customized Selection
92. 09 and change the paper frequently Cleaning the imaging chamber frequently is also helpful 1 IMPORTANT ALERT Use only Xenogen approved cleaning agents Many cleaning compounds phosphoresce Contact Xenogen 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 VIS Imaging System standards and interfere with measurements Black or clear plastic microplates do not phosphoresce making them better choices The Xenogen High Reflectance Hemisphere provides a more definitive way to determine the presence of an undesirable light source Figure E 1 Itis 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 219 E Luminescent Background Sources amp Corrections Figure E 1 Xenogen High Reflectance Hemisphere and a plastic marker pen Left Photographic image Right Photograph with luminescent overlay The hemisphe
93. 1 positions and dimensions of the selected ROI BkgROI SubjROI Info 2 Enter a new width or height value in the ROI septa agen TLT20050624145507_006 c Properties box e aint 3 Tolock the current ROI size choose the Lock Size option Note The ROI size cannot be changed until the C Lock Position Lock Size option is cleared Xe pix 120 47356 e e pix 109 36069 A v A v Angle deg 0 0000 C Lock Size Width pix 17 49013 E Height pix 17 04815 Line Size 2 E Line Color ee 101 6 Working With ROI Tools Editing the ROI Line 1 Double click the ROI that you want to edit The ROI Properties box appears ROI Properties TIK ROI ROI 2 v ROI Label ROI 2 2 To edit the ROI line thickness enter a new value in the Line Size box Alternately click the arrows BkgROI SubjROI Info Image Number TLT20050624145507_006 v ROI BKG 1 Ni 3 To change the ROI line color a Click the Browse button The Select Color box appears Lock Position Xe pix 105 00000 c pix 163 00000 Angle deg 0 0000 b To select a basic color for the ROI line click a basic color swatch and click OK c To define a custom color drag the crosshair in the custom color field adjust the brightness slider and click Add to Custom Colors
94. 11 4 Exporting Results To export all results 1 In the Results tab click Export results 2 To export user selected results 1 Right click the item of interest in the results list and select Export Results on the shortcut menu 2 148 D m p W Took Wide ao The Photon Density Maps window appears p Compare the simulated and measured photon In the dialog box that appears select the destination folder for the results and click OK The results include a txt cSv xsc Source information and a xmh surface mesh file In the dialog box that appears choose a folder for the results enter a file name txt and click Save name LmFiT_13 x Tool Palette R E Surface Topography Point Source Fitting Analysis Params Properties Results Error Estimation Starting Chi 2 8 58e 09 Ending Chi 2 7 03e 08 Point Source Fitting Results Parameters Fitted value Mua Em 0 143 cm Mus Em 6 676 cm Mueff 1 710 cm Diff 0 049 cm Total source flux 2 22e 11 photons x location of the source 0 181 crm ey ot U D EN u m a ae Photon Density Maps Export results Save Results Delete Load gt FLIT 3D Reconstruction 3D Tools Living Image Software User s Manual v r Y Ca Be ns 11 5 Managing Point Source Fitting Results To save results 1 Select the results of interest LMFIT_xx from the Name drop down list 2 Click Save
95. 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 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 Table 7 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 bar scale does not affect the image math result Color Ranges for Result Image Full See above Auto See above Min 0 Choose this option to set the minimum data value to zero Results Drop down list of mathematical 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 k Image Math window A user specified scaling factor applied in the results function k Fluorescent Background The software computes k the ratio of the autofluorescent Subtraction window signal
96. 208 2 Set the volume and flow rate Infusion Pump Control Infusion Pump Control Panel Wolunnie Flow Rate Syringe Type Diameter C Suto Start After Start Mow 10 uLjmin B D tcc 639 mr 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 P Information SHARING s Diameter not known for syringe type Custom Please enter a valid diameter For the syringe selected 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 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 m NOTE The information in the infusion pump control panel is saved in the click info file During acquisition if you start infusion then manually s
97. 223 F 1 Description and Theory of Operation 2 2 a 223 BZ Pater SpeCla o oo ee as eee we eK ee eae eee 225 F 3 Working with Fluorescent Samples 2 oa aaa a 221 F 4 Image Data Display aoaaa a 228 Contents F5 Fluorescent Backeround o 4 sse42c 5 e a dey 2 Bade Hee eee EE Es 229 F 6 Subtracting Instrument Fluorescent Background 2204 235 F 7 Adaptive Background Subtraction 2 1 a 235 F 8 Subtracting Tissue Autofluorescence Using Background Filters 236 Appendix G Planar Spectral Imaging 2 008822 eee 239 G 1 Planar Spectral Imaging Theory 2 0 0 0 eee eee ee ee 239 G 2 Optical Properties 2 iu ss ae Seba eae piked eee ee a eee ee ee 241 G3 Lu ciferase Spectrum ss s s sree bw Seba ee bee a ees eu bogus we bs 241 G 4 An Example of Planar Spectral Imaging 02004 241 G 5 Optimizing the Precision of Planar Spectral Analysis 004 245 Appendix H 3D Reconstruction of Light Sources 247 H 1 Determining Surface Topography o a aooaa a 247 H 2 Algorithm Parameters amp Options 2 2 ee 252 Appendix I IVIS Syringe Injection System 02 0008 ee a 257 I 1 Controlling the Infusion Pump 0 0 0 0 0 00 251 I 2 Tracking Infusion in the Maximum vs Time Graph a aaao 2 00000 259 I 3 Closing the Infusion Pump Control Panel n aoa aaa ee ee 259 Appendix J Menu Commands Tool Bar
98. 3D Reconstruction gt Spectral Unmixing Min 2140 Nax 36445 Min 2949 Max 51182 95 Max 10343 96 The image window displays thumbnails of sequence images using a single color table Double click a thumbnail to open the image ine Caris e Degi Gey a rfg a a Drage m TTS 48807 oo Sarl Made Mra Ph dun 4 2s OS Deien Gon Gores Gen After open Labet kiray Bc PAA M ii Dorani darz dama Pets 20h Bata i E c Lang For more details on working with a sequence in the image window see page 51 me 7 Living Image Software User s Manual Y Caliper _ Opening Data from the Menu or Toolbar 1 In the Open box that appears Click the Open button Gf on the toolbar Alternately select File Open on the menu bar Living Image 3 2 double click the file of interest File Edit View Tools Acquisition Window Help Alternately select the dataand fsan s nsa R click Open File types Choose a file to open Click txt an image Living Look in E Dataset emgem j aresoxrmz Image Tile format Fe ee Sequence txt an image kaikas Eon sequence Living Image file B E Other fo rma t Desktop 9 TRANS 2Comp dcm kinetic data My Documents My Computer M oe File name es Files of type Living Image Files Click txt Sequence txt det 7 Cancel A NOTE To open a recently viewed files select File Recent Files on the menu bar
99. 4 Figure F 8 Fluorescent image data displayed in terms of efficiency When 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 to 10 On every 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 229 F Fluorescent Imaging 230 level of autofluorescence 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
100. 9 1 Tool palette spectral unmixing tools Item Description me Opens a dialog box that enables you to select a spectrum to add to the spectrum plot La Opens a dialog box that enables you to edit a spectrum in the spectrum plot Note You can also double click a spectrum row in the tool palette Spectrum tab to open the dialog box b Deletes the spectrum from the spectrum plot Type The type of spectrum 131 9 Spectral Unmixing Table 9 1 Tool palette spectral unmixing tools continued Item Description SOL A spectrum generated by the spectral unmixing algorithm ROI A spectrum calculated for a user selected ROI LIB A user selected library spectrum The library includes spectra obtained of different sources obtained using excitation and emission filters Name The spectrum identifier used by the unmixing algorithm The name cannot be modified Label The spectrum name in the spectrum plot key The label can be edited Color The color of the spectrum in the spectrum plot For the SOL type spectrum it is also the color in the composite image Spectrum Preview Shows the spectrum selected above click a row above the preview pane Use this tool to pick up a pixel in an opened image and plot the spectrum at this pixel in the spectrum preview 9 3 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 res
101. Bkg the primary excitation filter DsRed and the emission filter DsRed Figure F 17 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 w S E Living Image Software User s Manual Y Caliper improvement reduces the minimum number of cells necessary for detection from 1 5x 105 to 6 7x 103 Primary Background Corrected excitation filter excitation filter image a0 200 m it m iii 3 g 6x10 3 tna 3 100 100 amp 5 See 49x10 17x10 Er PE Signal Background 6 3 Seale Factor 047 signal Background 100 Min detectable cells 1 6x105 Min detectable cells 1 0x10 Figure F 17 Example of the autofluorescent subtraction technique using a background excitation filter a primary excitation filter DsRed b blue shifted background excitation filter DsRed Bkg and c corrected data The corrected image was obtain
102. D View SO F Render Photon Density Map Apply a v Threshold Intensity 4 07e6 ia Color Table iv Reverse _ Logarithmic Scale Slice Corona J 105 Sagt _ 18 Transaxia 81 hel Surface face eS Smooth surface face ity Reflect surface face amp Reflect smooth surface face Figure 12 5 Surface shading styles 168 gt e Living Image Software User s Manual Y Caliper Changing the View You can view a 3D image from different perspectives Figure 12 6 shows examples of Perspective the other available views To change the view e Select to change the view e Alternatively click the surface then press the V key to cycle through the different views of the surface Figure 12 6 eSelect to display the perspective view Note Only the perspective view the default view can be rotated or moved in the 3D view window i a Qu A amp R Unitsi Counts_ vO Apply to all 2 _ gt Surface Topography areas i H Help gt DLIT 3D Reconstruction 3D Tools 4 Q lz 2G Mesh Volumes Animation V Render Mesh Opacity l al Render Photon Density Map Apply Simulated v Wavelength v Threshold Intensity 407e 6 S Color Table Paco i iv Reverse Logarithmic Scale Planar Spectral Imaging
103. Filtering tools The algorithm multiplies 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 System 100 Series The IVIS Imaging System 200 Series has a larger lens with a smaller flat field correction 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 216 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 3 0 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
104. ID Deletes the user selected from the Existing User ID drop down list Preferences Defaults Label Name Drop down list of factory installed label name sets Edit User label Opens a dialog box that enables you to edit a label set Choices Default Units Specifies the units photons or counts for image display 198 Ti Living Image Software User s Manual Y Caliper _ B 3 Acquisition Preferences General User Acquisition Theme Tissue Properties ap Analysis Auto Exposure Camera Settings Luminescent f Fluorescent Auto Exposure Preferences First Preference Second Preference Third Preference Target Max Count Luminescent eoo SI sip E Fluorescent 4000 Range Values Exp Time sec Binning Min oso in Min Max i Max Restore Defaults Figure B 4 Acquisition preferences Auto Exposure Table B3 Auto exposure settings Item Description Luminescent Fluorescent Auto Exposure Preferences First Preference During auto exposure the software acquires a bioluminescence or fluorescence image so that the brightest pixel is approximately equal to the user specified target max count Second Preference Third Preference If the target max 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
105. Imaging 242 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 ia CK20031215150449_008 elec Units Photons Display Overlay Info iy Image Min 1 26e3 Max 9 76e5 p sec em 2 sr Color Bar Min 5 69e4 Max 4 48e5 Figure G 2 Metastatic sites in nude mice Mice were imaged 13 days after a tail vein injection of 5x105 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 To perform the planar spectral analysis draw a measurement ROI that captures the entire signal of each site of interest without including a neighboring metastasis Figure G 3 After the ROI is defined start the planar spectral analysis for more details see page 121 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 Figur
106. Item Description Hide Browse View Closes the browser table Close Preview Closes the image preview box Label Set A drop down list of the available label sets which specify image information column headers that is displayed in the Living Image browser Add to List If you choose this option the data that you select in the Browse for Folder box is added to the Living Image browser If this option is not chosen the data that you select in the Browse for Folder box replaces the contents of the Living Image browser Browse Opens the Browse For Folder box Load as Group 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 images in the browser Note he Load as Group option is only available when two or more images 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 46 Living Image Software User s Manual W Caliper Opening Data Opening Data from the Livin
107. Living Image user Guide Chapter 9 Imaging wizard Fluorescence Imaging wizard Bioluminescence NOTE The imaging options available in the imaging wizard depend on the IVIS imaging system and the installed filter set Manually Setting Up a Sequence You can manually set up an image sequence in the control panel and save the information to a Xenogen Sequence Setup file xsq 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 26 e Living Image Software User s Manual Y Caliper _ IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning gt Open n Y w E Field of View System Status see Subject height 1 50 em Focus use subject height v 2 If itis necessary to clear the sequence editor that appears click and select All 3 Inthe control panel specify the settings for the first bioluminescence or fluorescence image in the sequence and the photographic image For details on the imaging parameters in the control panel see page 191 Reuse option If you choose the photograph Reuse option in the control panel the IVIS System acquires only one photographic image for the entire
108. 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 loading onto other images For more details see Saving ROIS page 104 Auto ROI Parameters Parameters that specify how the auto ROI tool draws an ROI Note These are advanced options that are only available if Show Advanced Options is selected in the general preferences Threshold If the Auto All or Auto 1 method is selected the 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 Note 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 ROIs 6 Working With ROI Tools Table 6 2 Tool palette ROI tools continued Item Description Lower Limit Specifies a multiple 1 to 10 of the color bar minimum that sets the lower threshold for identifying an ROI For example if the lower limit 2 and the color bar 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 Sets the minimum size of an ROI measured in pixels For example if the
109. S Kinetic The manual provides detailed instructions and screenshots that depict the system response sometimes the screenshots in the manual may not exactly match those displayed on your screen For more details on the VIS Kinetic please see the appropriate VIS Kinetic System Manual 1 Welcome 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 Lar Numbered steps explain how to carry 1 To start the Living Image software click the 7 out a procedure icon on the desktop A dash precedes the description he main window appears of the system response to a procedure Document names are italicized Living Image Software User s Guide Note information A note presents pertinent details on a topic or Note Notes also appear in this format es EX CAUTION CAUTION A caution note warns you that your actions may have nonreversible consequences or may cause loss of data OL a ALERT Important information advises you of actions that are essential to the correct performance of the instrument or software Important Information 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
110. S ES 67 5 10 Rendering Intensity Data in Color 0 0 0 0 0 0 0 00 ee ee ee 74 5 11 Viewing Transillumination Data aoaaa aa ee 76 Contents 5 12 Viewing amp Editing Kinetic Data 2 0 0 0 71 6 Working With ROI ToolS aoaaa a 0 0 ee eee et 81 Gl About ROIs 22444444642 565 6465864 dh4464 H4 5684668446688 Ba BES 81 6 2 ROL TOOI 2 eon bene eden ene theese hese eb eagecaeeeeesaeeacees 82 6 3 Measuring ROIs in an Image aoaaa aa 0 ee ee 84 6 4 Measuring Background Corrected Signal 2 aaa a 91 6 5 Measuring ROIs in Kinetic Data 2 94 0O Manace ROIS e eee a aa ae a e a a a eee 97 6 7 Managing the ROI Measurements Table o a a aaa a 106 MAGS Mal pea ghee paanan a Eee ee 111 7 1 Using Image Math to Create a New Image 0 020000 0 eae 112 7 2 Subtracting Tissue Autofluorescence oaoa a 115 7 3 Overlaying Multiple Images noaa a 118 8 Planar Spectral Image Analysis a nononono 2 008 2 ee eee nae 121 8 1 Planar Spectral Image Analysis aoaaa ee 121 8 2 Planar Spectral Imaging Tools 2 0 ee 123 8 3 Viewing amp Exporting Graphical Results 2 0 0 0 0000 ee eee 125 8 4 Managing Planar Spectral Imaging Results 0 0 0 000 2 ee eee 126 9 Spectral Unmixing 1 eee ee e 127 9 Special UnMIXINE 2 24 ace eb seen Peete we Geeeeeebneendaedauaes 128 9 2 Spectral Unmixing Results Window 2 ee 131 9 3 Spectral Unmixing Parameters aooaa
111. Sel ROI Measurements Image Number ROI Image Laye Total Counts Avg Counts Stdey Coun Min Counts Max Counts 3H20050630142719_001 ROI 1 Overlay 1 2 799e 04 2 501e 04 1 447e 02 8 034e 04 Delete Loae 4uto ROI Parameters hreshold Lower Limit i JJH20050630142719 003 ROIZ Overlay i 573e 03 16 568e 03 6 052e 02 2 598e 04 Customized Selections Copy Minimum Size 20 yy Preview _ Use Bkg Offset _ Replace ROIs Restore Defaults Save Load Measurements Types Image Attributes ___ ROT Dimensions Counts v _none_ _none_ v SavejLoad Auto ROI Parameters Name KSA_AUTO_MEAS_ROI_SET_1 w Dalta bac Select the type of ROI or image data to include in the table gt Planar Spectral Imaging gt Surface Topography gt Point Source Fitting gt DLIT 3D Reconstruction Pj ROI Measurements EAR cick Hx20070420121444_001 v v Measurement Total Counts 1 2 4 5 7 Grid ROIs are displayed in 1 2 066e 04 2 405e 04 2 319e 04 5 566e 04 3 199e 04 2 619e 04 7 763e 0 a separate tab 2 3 998e 04 6 967e 04 2 169e 04 3 571e 05 1 648e 05 2 494e 04 7 465e 06 3 1 667e 05 3 516e 05 1 760e 04 6 626e 04 3 102e 04 2 154e 04 5 629e 0 4 2 824e 05 5 753e 05 1 853e 04 3 314e 05 1 516e 05 2 074e 04 2 918e 0 lt Customized Selections Measurements Types Image Attributes ___ ROT Dimensions Counts
112. Software User s Manual Y Caliper 2 Select Tools lmage Math for xx_SEQ on the menu bar 3 Inthe Image Math window that appears select an image of interest from box A and box B The Image Math window shows a thumbnail of image A image B and the new image fj Image Math Window Sequence TLT20060510114512_ SEQ A TLT20060510114512_001 TLT20060510114512_ 002 TLT20060510114512_003 TLT20060510114512_004 TLT20060510114512_005 TLT20060510114512_006 B B TLT20060510114512_001 Note For more TLT20060510114512_002 Efficiency details on items in TLT20060510114512_003 z l the Image Math TLT20060510114512_004 window see Table TLT20060510114512_005 TLT20060510114512_006 7 1 page 114 Color Scale Limits for 4 and B Full Auto Result Color Scale Limits Full auto Min 0 Result 4 B k v k 1 00 0 Compute k from ROI with Photo from A v Display Result For Measuring Efficiency 4 Select a mathematical 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 Fy LT Mr00G0310114517 005 Deeply Grirl r oa l inka ThE SEO tb TLS a o New image generated by the Result function 113 7 Image Math 114 To save the new image 1 Click the Save button fgl Alternatively select File Save on the menu bar
113. URA NE Toolbar ej IVIS Acquisition Control Panel Imaging Mode oo Time Binning i op sate Gain Excitatioi Field of View System Status System needs initialization Click on Initialize button to proceed Subject height iso 3 50 EAN T n Sequence Setup Setup Focus juse subject height Temperature Unlocked Acquire The acquisition control panel when the workstation controls the IVIS Imaging System for more details see Appendix A page 191 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 only the analysis features of the Living Image software 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 button then click the item Press F1 or select Help User Guide on the menu bar to display the Living Image 3 2 Software User s Manual pdf 10 n Living Image Software User s Manual Y Caliper 2 3 Initializing the IVIS Imaging System The imaging 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
114. _006 Units Counts Display Overlay o M info ROI Properties o ROI ROI 1 ROI selected in the image To view properties a S4120 Mii for another ROI Image Number select another TLT20050624145507_006 ROI from the aheti W drop down list ROI Label ROI 1 bubiect 1 Max 22447 i BKG 1 1 822e 03 Subject 1 gt i q F ROI 1 BKG 1 1 763e 06 ROI 2 BKG 1 4 845e 05 ba Lock Position Xcl pix Yei pix Angle deg Lock Size Copy ROI Copy All ROIs Max 19546 Height pix Duplicate ROI Hide ROI Tag Line Size ure coor I C Delete ROI Delete AIROIs Lock Position To view properties for another ROI e Click another ROI in the image Alternately select an ROI from the ROI drop down list in the ROI Properties dialog box Figure 6 2 97 6 Working With ROI Tools ROI Properties efx ROI BKG 1 v ROI selected in the image ROI Label BKG 1 BkgROI SubjROI Info Use as BKG for future ROIs in TLT20050624145507_006 c Entire sequence Selected image C Lock Position xc pix 120 47356 Yet pix 109 36069 Angle deg 0 0000 C Lock Size Widtht pix 17 49013 Height pix 17 04815 Line Size w
115. a abet emer Ehei inte ends Tee apa 130q_ Mace oct ie MS i i i i TE pel a bes Lo oT pt Edit Image Labels box Figure 4 5 The Edit Image Labels box Image window and tool palette appear when you save an image or kinetic stream NOTE You can edit and analyze kinetic data in the Image window i Living Image Software User s Manual Y Caliper 5 Working With Data Browsing amp Opening Data a a oaoa e e e BY Seo GS eS OS ee RE OO 45 The Tool Palette amp Image Window anaoa a 50 Working With an Image Sequence nonono a ee 51 Working With a Single Image nonono a 55 Viewing Image Information 64 686 6 B44 eNO Hee OSES ES oD SSE HESS 59 Image Layout Window 5456805 61 bee secede bee cede bee caw Se Se ss 62 Adjusting Image Appearance 2 a 63 Correcting or Filtering Image Data 0 0 0c a 66 Image Information Tools 44 b44 bs 66 eo oe Oo eee eS eee ROE as 67 Rendering Intensity Data in Color 2 00 cee ee 74 Viewing Transillumination Data 1 1 a 76 Viewing amp Editing Kinetic Data n ooa es 77 5 1 Browsing amp Opening Data The Living Image Browser provides a convenient way to browse and preview data view information about the data and open a single image image sequence or kinetic data You can also navigate to data and open it without the browser see page 49 Browsing Data _ 1 Click the Browse button Altern
116. a brief description about an item in the user interface click the toolbar button then click the item e Press F1 or select Help User Guide on the menu bar to display the Living Image 3 2 Software User s Manual pdf ad Living Image Software User s Manual Y Caliper 1 3 Contacting Caliper Technical Support If you need technical support please contact Caliper at Telephone 1 877 LabChip 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 1 Welcome This page intentionally blank Ti i Living Image Software User s Manual Y Caliper 2 Getting Started Overview of Imaging amp Image Analysis a naonao a ee 5 For more details on sequence setup using the imaging wizard see page 24 8 Initializing the IVIS Imaging System 0 000 ce ee ee 11 Checking the System Temperature 0 000 eee eee et 11 This chapter provides a brief overview of images and image analysis It also explains how to start the Living Image software and initialize the VIS imaging system 2 1 Overview of Imaging amp Image Analysis For bioluminescence imaging the imaging system acquires a photographic image and a bioluminescence image The Living Image software automatically coregisters the images to generate an overlay image Figure 2 1 For fluorescence imaging
117. age data folder The selected folder is displayed in the Living Image browser File gt Browse Opens the Biotrue CDMS Browser Note The browser is only available if the system includes the Biotrue CDMS option File gt Browse Biotrue File gt Save Saves overwrites the active image data File 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 Import Organ Atlas Opens a dialog box that enables you to import an organ atlas atlas File Import DICOM Opens a dialog box that enables you to import dcm image data that can be viewed in the Living Image software File gt Import 3D Mesh Opens a dialog box that enables you to import a mesh xmh Note This command is only available if an appropriate sequence is active DLIT or planar Spectral imaging sequence File Import 3D Volume Opens a dialog box that enables you to import a source volume voxels xsc Note This command is only available if an appropriate sequence is active DLIT or planar spectral imaging sequence File Export DICOM Opens the Browse for Folder dialog box that enables you to export the active image data to DICOM format dcm File Export 3D Mesh Opens a dialog box that enables you to save the 3D mesh of the active data in Open Inventor format iv File Export 3D Volume Opens a dialog box that enables y
118. ages Accumulate Select this option to view the cumulative intensity signal in real time When this option is chosen the software computes and visualizes the cumulative signal in each frame Color Scale 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 file size of the kinetic stream dcm being acquired The file size display is only available in the Kinetic Acquisition panel ad Living Image Software User s Manual Y Caliper Table 4 1 Kinetic acquisition settings continued Item Description 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 Maximum vs Time The maximum vs time graph ap
119. ameters and options Tool Palette gt Planar Spectral Imaging Eal C Surface Topography EA BLIT 3D Reconstruction w Params Properties Results Surface Topography lt FLIT 3D Reconstruction Analyze Params Properties Results Face CK20050G24751158_ SEQ Active sequence Select Wave Filters Sequence CRM FT 10905 SEQ Tissue Afuscle source AFGELE Select Image Sources Source Srei 3D Tools Minimum Radiance See er Source EswL EmWwhL MinRadiang lt E40 ral 286e 06 E40 ral 2 86e 06 640 ral 2 06e 06 E40 r20 2 oBe 06 640 fell 206e 06 640 fell 2 86e 06 640 fe 2 06e 06 640 fe 4 93e 06 a sk Fe 1 16e 07 gt 3D Tools H El E El E 1 He gt i ib Gs Gs ce cs ibh H Figure H 5 3D reconstruction tools Analyze tab DLIT left and FLIT right 252 Wavelengths For FLIT reconstruction of fluorescent sources you must specify the transillumination source positions It is recommended that you acquire images at a minimum of four source positions All images are acquired using the same excitation and emission filters For DLIT reconstruction of luminescent sources you must specify the acquisition wavelengths for the image sequence It is generally recommended that you acquire image data using two to four wavelengths rather tha
120. amp Shortcuts 261 Ti Living Image Software User s Manual Y Caliper 1 Welcome What s New In the Living Image 3 2 Software 1 0 2 a ee 1 About This Manual a ea dk ER ROR EE Ce GES BERGA ES 1 Contacting Caliper Technical Support 2 00 eee ee ee 3 The Living Image 3 2 software controls image acquisition on the IVIS Kinetic The application also provides tools for optimizing image display and analyzing images or kinetic data 1 1 What s New In the Living Image 3 2 Software The Living Image 3 2 software enables kinetic data acquisition on the VIS Kinetic and provides tools for visualizing and analyzing kinetic data New Feature or Updated Tool See Page Acquire and analyze luminescent or fluorescent signals in real time 37 14 bit or 16 bit dynamic range 39 EM gain option multiplies the signal for fast imaging applications 40 Maximum signal vs time graph plots the maximum signal in each frame of the 41 kinetic data and provides a convenient way to see signal trends View the cumulative signal to track signal changes in real time 40 Kinetic ROIs are applied to each frame in a kinetic data set and are displayed during 94 kinetic data playback Edit kinetic data 42 Save kinetic data in DICOM format dcm or save kinetic images to a movie for 43 example mpg4 1 2 About This Manual This user manual explains how to acquire and analyze images or kinetic data on the IVI
121. and optimize the image display using the color scale Min and Max sliders in the Image Adjust tools 4 Select Tools Image Overlay for xx_SEQ on the menu bar The image overlay window appears and shows the first photographic image In the sequence 118 Living Ilmage Software User s Manual Y Caliper feSciences 5 Select a photographic image in the upper box iA Image Overlay for Sequence Sequence AR W20050826124002_SEQ Color Scale Type White Levels Color Scale Per Column 1 al Photograph Images AR W 20050826124002_001 ARW200508261 24002_002 Fluorescent Images ARW200508261 24002_002 C Edit Layer Properties Opacity Color Table 100 Reverse Logarithmic Scale 6 To select all of the fluorescent or luminescent images in the lower box click the button Alternately to select particular images do either of the following e To select non adjacent images in the list press and hold the Ctrl key while you click the images OR e To select adjacent images in the list press and hold the Shift key while you click the first and last image in the selection In the overlay that is generated the signal in each image is assigned a different color table The photographic image is at the bottom of the stack and the last fluorescent or luminescent image selected from the list is at the top of the stack ig Image Overlay for Sequence Sequence ARW20050826124002_SEQ Photograph Images
122. anged anytime From the Acquisition menu 10 To specify a folder for autosaved data click Yes in the prompt and choose a folder in the dialog box that appears 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 11 In the Edit Image Labels box that appears enter information for the image label and click OK Figure 3 1 If you do not want to enter label information click Cancel 3 3 Acquire a Fluorescence Image With Transillumination Transillumination uses an excitation light source located below the stage For more details on fluorescence imaging see page 169 NOTE Transillumination is only available on the IVIS Spectrum imaging system 3 Acquire an Image or Image Sequence 20 1 Start the Living Image software double click the icon on the desktop 2 Initialize the IVIS System and confirm or wait for the CCD temperature to lock For more details see page 11 IIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter oo Field of view System Status Idle Acquire Subject height 1 50 gt em Sequence Setup 3 Putacheck mark next to Fluorescent and Transillumination 4 inthe control panel select an appropriate excitation filter and emission filter from the drop down lists 5 Click Setup P IVIS Acquisit
123. as they pass over the object The parallel lines are projected onto the surface of the subject at an angle 8 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 SL projector B a T a f L iinn 1 Y i X lt lt j Figure H 2 Structured light projector and subject D and l form two perpendicular sides of a triangle giving tan D Together Ax and h comprise a smaller version of this triangle The height A can be determined from h Ax tan 8 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 Ti i Living Image Software User s Manual Y Caliper Converting Light Emission to a Photon Density Map Defining the Linear Relationship Between a Source and Photon Density The input data to the FLIT algorithm for 3D reconstruction of fluorescent light sources includes e A surface mesh 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 The input data to the DLIT algorithm for a 3D reconstruction of bioluminescen
124. 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 bioluminescent light emission The luciferase bioluminescence 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 Xenogen Corporation 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 VIS Imaging System 200 Series using filters at six wavelengths from 560 to 660 nm in 20 nm intervals 241 G Planar Spectral
125. atively select File Browse from the menu bar 2 In the Browse for Folder box that appears select the data of interest and click OK Browse For Folder Living Image Dataset Folder Dataset aresoxFm2 PRA bree 4x20070321092352_SEQ Hx20070321092919_5EQ O Co localized O EPI 3Comp E O invitro E O Other C Plate skull v ee E7 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 5 Working With Data 3 The selected data are displayed in the Living Image browser along with the user ID label information and camera configuration information ME an image SEQ an image sequence BEM a kinetic data 4 Click the sign to show the images of a sequence To view data properties right click the item and select Properties on the shortcut menu amp Living Image Browser DAR DA20080806111439 Click Number EX Filter EM Filter UserID Series Experiment Label Comment Analysis Comment Dat A BBMB120080724144830 Block Open I 07 10 post luc J 33H20050630142125_005 10 post luc bn M JJH20050630142125_006 Open JJH Expt 145 Spectral IVIS 200 1 3 5 dorsal 10 post luc lt Location C Documents and Settings Katherinescls Desktop K2 Data LI Flles D4200808061 11439 D420080806111439 dcm Table 5 1 Living Image browser
126. aults or enter new values For more details on the parameters see Appendix H page 247 DLIT algorithm user modifiable parameters g Tool Palette o n ea SS z gt Planar Spectral Imaging i gt Surface Topography j DLIT 3D Reconstruction Cs EE ETTE Analyze arame Properties Results Angle Limit deg lower Kappa Limits CHE Angle limit default is 70 for IVIS Imaging System 200 m ie Series or IVIS Spectrum data N Surface Limits soo soo Voxel Size Limits E 5 l Vowel Size Increment 1 B Uniform Surface Sampling C NNLS Simplex Optimization C NNLS Weighted Fit Restore Defaults gt 3D Tools Params tab 8 In the Analyze tab click Reconstruct The reconstruction requires about 1 5 minutes depending on the parameter settings and the processor speed Figure 12 1 shows example 3D reconstruction results 154 Living Image Software User s Manual The 3D View in the Image window displays the surface the 3D reconstruction of the bioluminescent light sources inside the subject voxels and the photon density map photons mm just below the surface Results tab displays the results data and the DLIT parameter values To best view the light sources voxels 1 In the 3D tools move the slider or enter an opacity value to adjust the surface opacity 2 Clear the Render Photon
127. autosave feature you can manually save data 1 Turn off the autosave feature select Acquisition on the menu bar and remove the check mark next to Auto Save 2 After you acquire an image or image sequence click the Save button fa Alternatively select File Save on the menu bar 34 o y Caliper Living Image Software User s Manual ie 3 Inthe dialog box that appears select a directory of interest and click OK NOTE The software automatically includes the user ID and a date and time stamp with the data 3 7 Exporting Image Data You can export the image data in different file formats for example bmp dcm 1 Click the Export Graphics button mg Fle Edt Ve Tools Amiin Window Hp et Re BB e a SO yom 2 Inthe dialog box that appears select a directory choose a file type and enter a file name 3 Click Save NOTE When you export to DICOM dcm format a directory that contains the dcm files and a Sequencelnfo txt is created at the specified location 3 Acquire an Image or Image Sequence This page intentionally blank 36 ir i Living Image Software User s Manual y Caliper feSciences 4 Acquire Kinetic Data RiISUCAMCOUISHION v 45 Gh oes OS ee OOS EES OH ERS S SHAS REEL Oe Os 37 Viewing amp Editing Data Kinetic Acquisition window n s aooo 42 I le EEE E E E E E E E E E E E E E ES 43 The IVIS Kinetic is ready to acquire kinetic data after the system
128. ave the active DLIT or FLIT results Overwrite If you reanalyze saved results click to save the new results and overwrite the previous results 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 DLIT or FLIT algorithm first converts the luminescent image of surface radiance to photon density just below the animal surface because this 1s what can be observed Then the DLIT or FLIT algorithm solves for point source locations inside the tissue which would produce the observed photon density near the surface To check the quality of the DLIT or FLIT construction it is useful to compare the measured and simulated photon density plots The photon density is closely related to the measured radiance Living Image Software User s Manual W Caliper _ To view the photon density maps 1 In the Results tab click Photon Density Maps The Photon Density Maps window displays the photon density maps for all wavelengths Select one or all wavelengths for display Tool ToolPalette si Photon vani Maps Move the wheel to the left or right _ to rotate the Surface on the vertical axis Key Value Total source flux phot s 5 82e 10 Final vsize 2 00 Number of sources 2 38e 03 8 00 2 00 800 TRAN Save Results Name DLIT E v 3D Tools Error Note The
129. avelengths Emission wavelengths of the sequence m 5 53e6 f 4 85e6 Vax 1 10e8 92 3 Max 9 66e7 E wy a tu a n 1 61e6 n 1 40e6 Vax 3 16e7 f6 Wax 2 69e7 Components lz Mask Photo Unmix Spectra 1 05e6 DFi Tool Palette C Image Adjust gt ROI Tools r Spectral Unmixing Analyze Options Spectrum Results 3 Inthe Components drop down list select the number of spectral components to unmix in the images the number of fluorophores 1 since tissue autofluorescence accounts for 1 component Select Wavelengths For example if the image data includes one fluorophore then there are two components to unmix the fluorophore signal and the tissue autofluorescence Components 2 Mask Unmix Spectra 128 me o Living Image Software User s Manual Y Caliper 4 Make a selection from the Mask drop down list PETERT Photograph Image AR pAn anena hapa lala e Photo Opens the Photo Mask Setup window The purple mask specifies the area for analysis a Use the Threshold slider or arrows to adjust the mask so that it matches the underlying subject photograph as closely as possible without Including any area outside the subject image b Click Set e All The entire image is analyzed This option is not recommended It introduces many off target pixels that might mislead and Threshold significantly slow down the analysi
130. ay information about the item 263 J Menu Commands Tool Bar amp Shortcuts This page intentionally blank 264 Living Image Software User s Manual Index Numerics 3D fluorescent sources 156 157 3D image rotate or move 166 3D luminescent sources 152 155 3D perspective 169 3D reconstruction fluorescent sources 156 157 parameters amp options 252 255 3D reconstruction results 158 160 3D tools 162 187 Animation tab 182 183 Mesh tab 170 171 Organs tab 175 176 Volume tab 172 173 A accumulate signal 38 40 acquire image sequence 31 32 kinetic data 37 single bioluminescence image 15 18 single fluorescence image epi illumination 18 19 single fluorescence image trans illumination 19 22 adaptive fluorescent background subtraction 235 adjusting image appearance 63 angle limit 253 animation 182 187 custom 185 186 edit an animation setup 186 187 preset 184 185 auto exposure feature 12 autofluorescence 91 229 miscellaneous material 231 233 See tissue autofluorescence subtract using background filters 236 237 well plate 230 231 autoluminescence 91 automatically draw ROIs 88 89 average background ROI 82 91 B background v j m Y Ca Be n adaptive fluorescent background subtraction 235 fluorescent 229 235 light on sample 218 220 tissue autofluorescence 236 237 background light from sample 220 222 on sample 218 220 background corrected sig
131. binning 1 At binning 4 16 pixels are summed prior to read out Binning significantly affects the sensitivity of the IVIS 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 sensitivity is usually more important than the spatial resolution Further since in vivo signals are often diffuse due to scattering in tissue little is gained by increasing spatial resolution For more background on the propagation of light through tissue see Diffusion Model of Light Propagation Through Tissue page 240 In such cases high levels of binning may be appropriate up to 10 or 16 depending on t
132. bles 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 4 3 shows how binning conditions affect the total number of frames that can be collected in overlay or luminescent fluorescent only mode Table 4 3 Frames collected per 1 GB DICOM file Binning Level Frame Size Overlay Luminescent or DICOM File Size WM fete l Fluorescent Only Total Frames Collected Bin 2 MB 250 500 Bin2 512 KB 975 1950 1 GB Bin 4 128 KB 3900 7800 Bin 8 32 KB 15600 31250 Bin 16 8 KB 62500 125000 43 4 Acquire Kinetic Data 44 To save data 1 In the Kinetic Acquisition window click the Save button and select a save option Save Option Description Save Current Image 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 Inthe 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 Fis p We io Winco Hele ae fe amp Ag MF ocom D aoe 5 WM PO EI 95 toe p
133. c radiation Isotropic radiation from a cell is called photon flux photons sec When cells occur in tissue photon emission from the tissue surface is called surface radiance photons sec cm2 sr 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 4r steradians Lens systems typically collect light from only a small fraction of the total 47 steradians When image data is displayed in photons 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 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 advantage of working with image data in photons mode is that camera settings can be changed during an experiment without having to adjust the images or the measur
134. cale of an organ s you can click Reset to restore the default size and position of the selected organs Manually Adjusting 1 Follow step1 to step 4 above the Scale or Location of Organs 2 Click the Transform tool button Bi The transform tool appears 177 12 3D Reconstruction of Sources Fe Eas wie Tae Atgeier Winco Fep SANA AG inea O roa Transform tool In this example Skin is selected from the organ list 3 To adjust the x y or z position of the organ drag the transform tool 4 To return the transform tool to the default location click Reset 178 e Living Image Software User s Manual aA Caliper _ 5 Press the Tab key to put the transform tool in scale mode A red cube ff appears at each corner of the transtorm tool 6 Toincrease or decrease scale the size of the organ drag a red cube ata corner of the transform tool Note 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 o ae S Use T ab key to switch between anstormation tools Use Y of Z keys to restrict scaling to only one axis Scaling On Z 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 Note lo return the organ drawing to the defa
135. camera controller a component of some VIS 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 the hardware manual for your IVIS Imaging System To initialize the imaging system 1 Start the Living Image software double click the icon on the desktop 2 Inthe control panel that appears click Initialize You will hear the motors move A IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop EM Gain Excitation Filter Emission Filter Field of View System Status Acquire System need initialization Click on Initialize button to proceed Subject height 1 50 Sem Sequence Setup Focus luse subject height Temperature C Unlocked NOTE The control panel is only available on the workstation that controls the imaging system The items available in the IVIS System control panel depend on the particular IVIS Imaging System and the imaging mode selected luminescent or fluorescent Image Setup or Sequence Setup mode 2 4 Checking the System Temperature The temperature box in the IVIS acquisition control panel indicates the temperature status of the charge coupled device CCD camera Figure 2 4 After
136. cence contribution to the photon density at the surface is forward modelled Simulated photon density data due to background at the surface is subtracted from the measured photon density so that the subsequent photon density used in the fit consists only of signal that is associated with the fluorophore The background option specifies whether or not the background fluorescence should be fit This is not necessary for the XFM 2 long wavelength data It is important when there is non specific dye in circulation or a lot of autofluorescence Uniform Surface Sampling If this option is chosen the surface data for each wavelength will be sampled spatially uniformly on the signal area If this option is not chosen the maximum N surface elements will be sampled for the data This means that the N brightest surface elements will be used as data in the reconstruction Typically non uniform sampling is recommended if there is a single bright source while uniform sampling is preferred if there are several scattered sources NNLS Optimization Simplex Optimization DLIT If NNLS Optimization Simplex option is chosen the software uses a linear programming algorithm to seed the solution followed by the NNLS optimization NNLS Weighted Fit Choose this option to weight the data in the NNLS optimization 255 H 3D Reconstruction of Light Sources This page intentionally blank 256 n Living Image Software User s Manual
137. ch 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 is 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 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 defines 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 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
138. ciferase CB Green Click beetle green luciferase CB Red Click beetle red luciferase Firefly Firefly luciferase XPM 2 LED LED in the XPM 2 mouse phantom hRenilla Sea pansy Renilla reniformis luciferase 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 of the luciferase spectrum please contact Caliper Life Sciences technical support 251 H 3D Reconstruction of Light Sources You can view tissue optical property values Mp in the Tissue Properties drop down list The tissue properties are plotted as a function of wavelength Select the tissue or organ most representative of the source location Muscle is a good choice for general reconstructions in vivo Default tissue optical properties and source spectrum are specified in the Preferences box For more details see Appendix B page 201 H 2 Algorithm Parameters amp Options Analyze Tab Tissue and source are specified in the Select the acquisition wavelengths for the DLIT analysis If DLIT analysis results are open the 3D tools are available gt Planar Spectral Imaging lt This section explains the user modifiable DLIT algorithm par
139. 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 ROls and viewing ROI properties 00 CO To measure the ROls click the Measure button in the tool palette The Kinetic ROI Measurements tab shows ROI information for the current image To view ROI measurements for all images click the arrow next to Current Frame and select All Frames then click the Refresh button Kinetic ROI measurements are displayed in a separate tab Fle Edt Wee Tooke Widow Hp sau wt A amp NP bras tore w C pets al DATION IO7TIG2617 aaa ree Units Photons A Displey warkay info ROL Measurements Kinetic HOC Meuugetendc Kinetic RC Piet Date Ekk CAME UOT aS BOL Rot La Eurih Frame Fraai Boda ee eee te ee ee S apuse J4 Sagie t 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 2 Open one or more kinetic data sets Draw kinetic ROIs on the data sets in which you want to measure and compare ROIs In the ROI tools click the ROI Measurements button LW 95 6 Working With ROI Tools The ROI
140. ct Auto All to automatically draw ROIs 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 88 Click to display the ROI Measurements table or compute intensity signal in an ROI B Click to display a drop down list of options to delete an ROI s in the active image For more details see page 105 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 this option to apply the selected ROI to all images in a sequence 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 associated a measurement and average bkg ROI included in the same subject ROI Using this type of ROI is optional Save ROIs Name Delete Load Save 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
141. cular Reflection Specular Reflection Figure F 10 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 Black polystyrene microplates are recommended for in vitro fluorescent measurements Figure F 9 and Figure F 10 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 Control 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 Xenogen part no 60104 on the imaging stage to prevent illumination reflections and to help keep the stage clean NOTE The black paper recommended for bioluminescent imaging Swathmore Artagain Black 9 x12 Xenogen part no 445 109 has a measurable autofluorescent signal particularly with the Cy5 5 filter set Figure F 11 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 leakag
142. de a bounding box around the subject Click to show or hide a grid under the subject E e e 165 12 3D Reconstruction of Sources Rotate Move or Zoom on a 3D Image 166 a Fie Edit View Tools Window Help r o a E a R 8 nel Units fe conts_ SD Apply to all SSECesECEcecececcercrsccrsrcccesrcrsrsberersrccsrsrcrcrsesrce Ed e EJ Sequence View t 3D View E gt Planar Spectral Imaging 5 i E 5 RS FEN gt DLIT 3D Reconstruction 33 1 an 3D Tools JAQ eH Y 2S gt R E Q z olume Organs Animation m f Mesh 20 7 17 1 1 0 J m Sacittal 1 8 C Render Photon Density Map Apply S Simulated 30 0 En x10 Wavelength 0 E 0 0 Threshold 55 4 39 1 Intensity 4 O7e6 S 05 Color T able a HEN Reverse Logarithmic Scale 30 0 Slice A Corona B na F J 2 Sagittal J 1 8 on oe photons sec Transaxial J 8 1 20 7 17 1 i Eerennedse Source Intensity Select a tool from the drop down list Axis shows image orientation To rotate the image 1 Choose the or 4 tool 2 Place the pointer in the 3D View window 3 Click and drag the pointer in the x y or z axis direction The x y z axis shows the orientation of the image To move the image 1 Select the fh arrow in the 3D tools and drag the image Alternatively press the shift key while you drag the image To zoom in or out on the image 1 Select the gt
143. 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 IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter TE Field of view System Status Switching OFF Motion safety Subject height 1 50 cm Sequence Setup Focus luse subject height v Temperature I use subject height manual focus scan mid range haa 2 To mark the center of the camera in the window put a check mark next to Display CCD Center 3 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 5 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 3 6 Manually Saving Image Data When you acquire the first image s of a session the autosave feature prompts you to choose the folder where you want to save image data During the session all images will be saved to this folder You can change the autosave folder at any time select Acquisition Auto Save To on the menu bar If you do not want to use the
144. e Living Image 3 2 Do you wish to enable auto saving of acquired data for this session This can be changed anytime From the Acquisition menu 11 To specify a folder for autosaved data click Yes in the prompt and choose a folder in the dialog box that appears 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 12 In the Edit Image Labels box that appears enter information for the image label and click OK Figure 3 1 If you do not want to enter label information click Cancel 3 4 Acquire an Image Sequence A sequence is a collection of images that are grouped together in a single folder A sequence may include images that were acquired during the same session and were intended to be grouped together for example images taken at different exposure times 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 example a time series could be constructed from images acquired on different days following an experimental treatment For more details see page 26 Some analyses are performed on an image sequence see Table 3 3 The sequence requirements number and type of images depend on the analysis The imaging wizard provides a convenient way to set up several types of image se
145. e 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 231 F Fluorescent Imaging image Min 1056 5 Max 13537 counts Figure F 11 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 exhibit non negligible autofluorescence The chart in Figure F 12 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 NOTE lt is recommended that you take control measurements to characterize all materials used in the IVIS Imaging System 200 180 160 140 120 100 80 60 40 Autofluorescence Relative to Black Lexan 20 Figure F 12 Comparison of autofluorescence of various laboratory materials to that of black
146. e If you are working with a sequence open an image to show the ROI intensity 3 Adjust the ROI position a Place the mouse pointer over the ROI When the pointer becomes a 5 click the ROI b Drag the ROI 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 J so that it becomes aX Drag the handle to resize the ROI 86 Living Image Software User s Manual Y Caliper feSciences File Edit View Tools Acquisition Window Help a H a amp gr Units Counts v C Apply to all Tool Palette fi TLT20050624145507_006 Units Counts Display Overlay v Max 22447 Auto All Auto 1 Free Draw Preview Use Bkg Offset Replace ROIs Restore Defaults Save Load Counts Color Bar Min 1122 Max 19546 NOTE You can also change the ROI position or size using the adjustment controls in the ROI Properties box see Moving an ROI page 100 and Editing ROI Dimensions page 101 5 Click the Measure button jj The ROI measurements and table appear For more details on the table see Managing the ROI Measurements Table page 106 Note For information on how to save ROIs see page 104 6 Working With ROI Tools The Living Image software can automatically identify all of the ROIs in an image or image sequence that meet the auto
147. e 137 results 131 132 starting the software 9 starting the system See system initialization steradian 214 subject ROI 82 90 surface topography 139 140 syringe injection system 257 259 Living Image Software User s Manual system X FOV 194 x y coordinates 174 initialization 11 optics autofluorescence 235 temperature 11 T tag an image 58 technical support 3 temperature 11 threshold angle 159 tile images 58 tissue autofluorescence 236 237 eliminate by spectral unmixing 127 130 subtracting with background filters 115 117 tissue properties 160 tool palette 50 3D tools 162 187 correcting filtering 66 67 image information 67 68 point source fitting 143 146 ROI tools 82 toolbar 261 total source flux 158 transillumination overview 76 troubleshooting guide 189 U uniform surface sampling 159 255 units See display units user preferences 195 203 V voxel 173 249 size increment 254 size increments 159 size limits 159 254 vsize final 159 starting 159 W well plate autofluorescence 230 231 v ir L Y Ca PS cas 269 Index 2 0
148. e 143 POM SOUS FIGs iire Babe OSD ROR DER Oe Ree whe R CaS SES ES 146 Checking the Point Source Fitting Results 2 0 0000 eee eee eee 148 EXON ECU Ss hae eR SS EMSS SDR RG ROS He OS OH SE ES ERR RES 148 Managing Point Source Fitting Results 0 0 000 eee eee ee ees 149 The point source fitting algorithm is a tool for advanced users that can be used to estimate the optical properties of tissue the location and power of a point source or the fluorescent yield of fluorophores The software analyzes the images in a sequence acquired in one of the following imaging modes e Bioluminescence e Transillumination fluorescence bottom illuminated fluorescence e Epi illumination fluorescence top illuminated fluorescence e Transmission NOTE The point source fitting algorithm requires an image sequence that includes one or more images and a structured light image 11 1 Displaying the Point Source Fitting Tools The default tool palette does not include the point source fitting tools To display the point source fitting tools in the tool palette 1 Select Edit Preferences on the menu bar fl Preferences General User Acquisition Theme Tissue Properties 3D Analysis Start Up Defaults Save Settings Dock Tool Palette Left Right Color Selections Folder Locations Window Size Window Size amp Position Width 55 Restore Defaults Most Recently Used Dataset History 5 E Height 65
149. e G 4 e Fits the normalized data to the analytical expression in Equation 1 page 240 where S absolute total photon flux emitted by the bioluminescence source and d source depth Plot of Linear Fit Results Figure G 4 v Y Ca PS cas Living Image Software User s Manual jw CK20031215150449_ 008 Image Min 1 26e3 Max 9 76e5 10 p sec em 2 st Color Bar Min 6 97e4 Max 6 44e5 Figure G 3 Metastatic site 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 femoral bone of the animal Fie Edit view Tools Window Help A K og QA A amp R units Photons 7 Apply to all Corrections 7 Fiterings Chare oo O Z Planar Spectal magna Analyze Properties Results 1 j Spectral Results 7 TETS AT ROI Depth mm Total Flux phot s JROI1 1 250 0 105 4 36e6 3 09e5 lt je Plot Linear Fit Dai Save Results After the analysis is completed Soin click a button to display graphical results __ amp Plot Intensity Name E pl m2 i ba j gt Surface Topography DLIT Load Delete A Plot of Linear Fit Results DAOR E B a Linear Fit Plot for ROI 1 Log Norm
150. e Topography gt FLIT 3D Reconstruction a Adjust the 3D view to show the properties of interest 3D Tools __ RM Or Po 53 B b Click the f button Lae E Mesh volume Organs Animation 2 Toreorder a key frame in the sequence select the key ree P frame and click the or arrow Frame Factor 1 Animation Setup Time Scale o J 3 Update a key frame Key Frame 1 a Select the key frame of interest Key Frame 2 Key Frame 3 Key Frame 4 b Adjust the 3D view Key Frame 5 Key Frame 6 c Click the ial button Key Frame 7 Play Frames Per Second 10 Total Duration secs 5 186 Y Caliper Living Image Software User s Manual feSciences 4 Delete a key frame a Select the key frame that you want to remove b Click the button and select Delete Current To save the animation setup 1 Click Save 2 Inthe dialog box that appears select a directory and enter a file name xml 12 9 Managing DLIT FLIT Results Tool Palette gt ROI Tools gt Planar Spectral Imaging gt Surface Topography BLIT 30 Reconstruction Analyze Params Properties Results DLIT amp nalysis Results Unsaved Key Value Total source Flux photss 1 17e 11 Final voxel size ron 1 75 Number of voxels 30 Reduced Chiz 2 50e 02 Starting vsize best 7 00 4 00 105 Total surf samoles 525 Iil gt Photon Density Maps Save Results i Default
151. e coor M C Bkg ROI tab average background ROI selected in the image ROI Properties TIR ROI ROI 1 ROI Label ROI 1 Bkg ROI Subj ROI Info Image Number Drop down list of subject ROIs TLT20050624145507_006 w i in the image Subject 1 Enter label information here for the subject ROI eens selected above xci pix 112 Yci pix 1 7 Angle deg 0 0000 Lock Size Height pix Subj ROI tab Figure 6 2 ROI properties BkgROI SubjROI Info ROI Properties mf ROI ROI2 ROI Label ROI 2 Label name of the ROI selected above Double click to edit Image Number TLT20050624145507_006 Ror BKG 1 H Drop down list of average background ROIs in the image Lock Position xci pix Yci pix Angle deg Lock Size width pix 1 Heightt pix 15 Line Size we coor M C Bkg ROI tab measurement ROI selected in the image ROI Properties efx ROI ROI1 ROI Label ROT 1 Bkg ROI Subj ROI Info Shape Contour Information about the ROI selected from the drop down list above Lock Position xci pix Yc pix 1 7 Angle deg Lock Size Height pix 2 Line Size Line Color m Info tab
152. e details on initializing the system see page 11 193 A IVIS Acquisition Control Panel Table A 2 Additional IVIS System Controls for the IVIS Imaging System 200 Series or IVIS Spectrum Item Description Alignment grid Focus Transillumination Setup IVIS Spectrum only 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 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 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 r
153. e f button The second key frame is added to the key frame box Repeat Step 6 until all key frames of interest are captured For details on how to edit 185 12 3D Reconstruction of Sources the key frame sequence see page 186 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 8 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 frames Otherwise the frames may not be properly animated 9 To view the animation click Play To stop the animation click Stop To save the animation to a movie 1 Click Record 2 Inthe dialog box that appears choose a directory and enter a file name mov mp4 avi and click Save To save the animation setup 1 Click Save 2 Inthe dialog box that appears select a directory and enter a file name xml Editing an Animation Open an animation setup Setup 1 To select a predefined setup make a selection from the Preset drop down list or To select a saved user defined setup a Click Load b In the dialog box that appears select the directory and animation setup xml of interest To edit the key frame sequence Tool Palette gt ROI Tools 1 Adda key frame gt Surfac
154. e following wW N 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 To select a particular range of kinetic data move the start and end frames selection handles Alternately 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 77 Kinetic Acquisition Control Panel ES Display Photograph F 14 Framesisec Acquisition Settings Luminescent Overlay i Dynamic Range mage ae Min 111 Haak bt Frame number 121 Max 15366 Excitation Filter Block Emission Filter Current frame number Enter a new number or use the top slider to view another frame Open Aas FL Lamp Level Color Bar High Min 111 Max 13458 i oo 0 00 17 Start frame in a user specified data range _ Ja SJ Soler Scale Auto Minimum EERI Position of the displayed current frame Maximum played bee i Position of the start frame File Size 110 9 MB a ji at Position of the end frame Tithe secs Time secs 17 05 ef Save J Done Figure 4 4 Acquisition window 42 Th Living Image Software Users Manual Y Caliper Viewing Options 4 3 Saving Data After acquisition has been stopped right click the image
155. e imaging wizard can automatically setup an image sequence for some applications Using the Imaging that analyze an image sequence Table 3 3 The wizard guides you through a series of Wizard steps prompting you for the information that the software requires to set up the image sequence Table 3 4 Imaging wizard sequence options Bioluminescence Acquires Description Image Sequence Open Filter J Planar Spectral J Spectral J Unmixing DLIT A Fluorescence Acquires an image at maximum sensitivity Analyze the sequence to compute the average depth and total photon flux of a bioluminescent point source in a region of interest ROI Analyze the sequence to determine spectral signature of different reporters in the same image and calculate the contribution of each reporter on each pixel in the image Apply the DLIT algorithm to the sequence to reconstruct the 3D surface topography of the subject and the position geometry and strength of the luminescent sources Description Tz 127 151 2D A J Filter Scan J Spectral J Unmixing FLIT J Use the data to make fluorescence measurements Helps you determine the optimum excitation and emission filter for a probe Analyze the sequence to extract the signal of one or more fluorophores from the tissue autofluorescence Apply the FLIT algorithm to the sequence to reconstruct the 3D surface topography of the subject and the position geometry and stre
156. e type of tissue Autofluorescence can occur throughout the animal but is strongest at the surface where the excitation light is strongest 221 F Fluorescent Imaging Specifying Signal Levels and f stop Settings p CCD Array gt Imaging Lens Tissue Autofluorescence Photon Scattering Mean free path 0 5 mm Bioluminescent or Fluorescent Source Figure F 6 Illustration of the n vivo fluorescence process 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 Fluorescent signals are usually brighter than bioluminescent signals so imaging times are shorter typically from one to 30 seconds The bright signal enables a lower binning level that produces better spatial resolution Further the f stop can often be set to higher values f 2 or f 4 is 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 205 F 4 Image Data Display Fluorescent Efficiency 228 Fluorescent image data can be displayed in units of counts or photons absolute calibrated or in terms of efficiency calibrated n
157. ecommended 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 FOV Setting IVIS Imaging System Lumina 100 Series 200 Series Kinetic FOV cm A 4 10 3 9 4 B 7 15 6 5 7 C 10 20 13 10 D 12 25 19 5 12 E 26 T i Living Image Software User s Manual Y Caliper Appendix B Preferences General Preferences oaoa a 196 User Preferences aoaaa a a 198 ACUTO eea a E a a a eB Qe a A A 199 VOCE NEEE EENE EEEE EEE EE EE E 200 THOUS FIQGOGIIGS coo 4 o5 o 4 amp 4 64 ee EH 6 OH 6 SOHO 9 Se Sw eS 201 SD ANalySIS Lhe tena hae ee hese Ob eee OEE EE ERE EO 202 You can manage user IDs and specify defaults for some parameters that are associated with the user ID selected at the start of a new session 1 After you log on to view the user modifiable preferences select Edit Preferences on the menu bar The Preferences box appears Pi Preferences General User Acquisition Theme Tissue Properties 3D Analysis Start Up Defaults Show Activity Window on Warnings Errors Y Dock Tool Palette Left Right Window Size Save Settings Color Selections Folder Locations Width Restore Defaults e ss window Size amp Position Height Most Recently Used Dataset History s g
158. ective 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 236 The software computes Background corrected signal A B x k where A primary image acquired using the excitation 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 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 3 page 22 115 116 7 Image Math To subtract tissue autofluorescence 1 Load the image sequence that Includes the primary and background fluorescent Images j 11720060510114512_SEO SEE Sequence View Units Photons v C Use Saved Colors TELT TLT20051221160239 TLT20051221160320 Min 1 2609 gt Min 2 24e8 Mac 247el0 Mac 4 4329 2 Open either the primary
159. ed Comment bea P Analysis Commen Pr 5 Save the image to save the updated image ao a information Apply To Sequence 61 5 Working With Data 5 6 Image Layout Window 62 The Image Layout window provides a convenient way to annotate and save an image to a graphic file 1 To open the Image Layout window EMGaBaeyanaiiney select View Image Layout hae kteaBoO O R Window on the menu bar 2 To paste the active image Into the Image Layout window click the button 3 To resize the image drag a handle W at a corner of the image 4 To reposition the image in the window drag the image Annotation Table 5 4 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 file Pastes the active image 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 Elliose drawing tool Pointer tool 7 O00 mp 0 Arrow and line drawing tool ae Sy Living Image Software User s Manual v Y Ca Be as Table 5 4 Image layout window cont
160. ed ROI data Images or ROI data can be quantitatively compared across different VIS Imaging Systems Xenogen Corporation calibrates the camera settings of each IVIS Imaging System at 600 nm The response of the CCD is 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 _ Living Image Software User s Manual Y Caliper Efficiency 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 At FOV 10 there is a slightly dished illumination profile due to the close proximity of the stage to the illumination reflectors while the profiles for the other stage locations are peaked near their center The illumination intensity profile varies by up to 330 across the entire FOV Figure D 4 100 BO A 60 v E o 40 Bs ames FOV 10 ome FOV 15 20 FOV 20 FOV 25 0 15 10 5 0 5 10 15 Width cm Figure D 4 Illumination profiles at different FOVs Measurements were taken at the center of the FOV on the IVIS Imaging System 100 Series Displaying fluorescent image data in terms of efficiency eliminates the variable excitation light from the measurement and enables a more quantitative comparison
161. ed 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 1x 106 HeLa luc PKH26 cells in the left flank 1 2 Em Filter Ex Filter Bkg Filter 1 0 Excitation TT Emission 0 8 Autofluorescence at l l 0 6 0 4 Normalized Intensity 0 2 400 450 500 550 600 650 700 750 Wavelength nm Figure F 18 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 Bkg the primary excitation filter DsRed and the emission filter used with this dye 23 F Fluorescent Imaging This page intentionally blank 238 Ti i Living Image Software User s Manual Ww Caliper Appendix G Planar Spectral Imaging Planar Spectral Imaging Theory 2 00 2 eee eee ee ee 239 Optical Properties 8 4428 4244 608 64 8 ee we Sw eR OR RE RS 241 Luciferase Spectrum 6144546 4 eo HR ERR De we ew Ge Bw 8 241 An Example of Planar Spectral Imaging 0 22 0086 241 Optimizing the Precision of Planar Spectral Analysis 245 The unique spectral signatures of the luciferase emission spectrum and the optical p
162. edium index of refraction Source Spectrum Plot Tissue Properties Source Spectrum Drop down list of the absorption and scattering properties of various tissues Tissue index of retraction that is automatically specified when you select a tissue property Drop down list of bioluminescent sources Click to display graphs cm vs nm of the absorption coefficient u effective attenuation coefficient u and reduced scattering coefficient u Click to display the spectrum of the selected bioluminescent source intensity versus wavelength normalized to one Results Tab Spectral Results ROI Depth mm Total Flux phot s Plot Linear Fit Plot Intensity Export Save Results Name Delete Load Save Name of the analyzed ROI Estimated depth of the point source Estimated total photon flux from the point source Displays a graph of normalized intensity versus the effective attenuation coefficient Lag 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 Displays a graph of normalized intensity versus wavelength Intensity is normalized by the selected source spectrum and filter transmission properties Opens a dialog box that enables you to save the results to a text file txt A drop down list of saved results Includes the default name for ne
163. eeper sources while large values favor more shallow sources The limits on kappa are minimum of 0 1 and a maximum of 10 The default range for kappa is 0 5 4 Kappa is doubled at each iteration so for a selected range of 0 5 4 the kappa values for each iteration would be 0 5 1 2 and 4 Choosing a large range for kappa produces the most reliable solution but requires more analysis time N Surface FLIT The number of surface intensity points to use in the reconstruction at a given source position N Surface Limits DLIT This is the maximum number of surface intensity points to use in the reconstruction at a given wavelength The range is 200 to 800 and the default is 200 The time required for reconstruction is shortest for smaller values of N for example 200 However a large N value may give a more accurate result because more data are included in the fit Starting Voxel Size FLIT Voxels are the small cubes of space inside a subject each of which contains a quantity of fluorescent yield The FLIT reconstruction begins with large voxels specified by the starting voxel size the length of a voxel cube side in mm At each iteration the algorithm reduces the size of the voxels by a factor of 2 until the optimum solution is determined Starting Voxel Size Limits DLIT Voxels are the small cubes of space inside a subject each of which contains a light source much like a pixel in a 2D image The DLIT reconstruction begin
164. elected 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 Image sum of 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 78 Ti Living Image Software User s Manual Y Caliper Viewing Kinetic Data Editing amp Exporting Kinetic Data Table 5 13 Image window kinetic data continued Item Description _ 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 to save the selected data Open the kinetic data To start playing the kinetic data click the gt button If you want to start the playback at a particular image first move the top slider to the starting image then click the gt button To stop playing data click the button 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 You can select a range of images for export to DICOM format includes photographs intensity signal and read bias or
165. els S is the value of the strength or flux in photons sec 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 249 H 3D Reconstruction of Light Sources Finding the Best Approximate Solution to the Linear System 250 The reconstruction method is based on the principle that there is an approximately linear relationship between the source strength in each voxel S and the photon density 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 YG 5 1 i 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 Once the Green s functions G are known the goal is to solve Equation for the source strength S in each voxel The DLIT algorithm attempts to minimize x Equation 2 while requiring that the source strength in each voxel is positive Equation 3 2 wl 2 X Dg Pj Wys 2 j i S 20 3 A combination of methods called Simplex
166. enerated this option is not available Apply Select the simulated computed by the DLIT algorithm or measured photon density for the photon density map Images A drop down list of the images used to reconstruct the surface Make a selection from the list to view the DLIT FLIT results associated with a particular Image Threshold Choose this option to apply a photon density threshold photons mm 3 to the photon density map Intensity Use the Intensity slider the arrows or enter a value in the box to set the minimum intensity threshold Color Table Specifies the color table for the source intensity scale Reverse Choose this option to reverse the color table For example the BlackRed color table represents the source intensity photons sec from low to high using a color scale from black to red If Reverse is chosen the source intensity ohotons sec from low to high is represented using the color scale from red to black Logarithmic Choose this option to apply a logarithmic scale to the color table Scale Slice Move a slider to change the position of the coronal sagittal or transaxial plane through the surface The intersection of the plane and subject slice is shown in the coronal sagittal and transaxial views in the 3D view window 171 12 3D Reconstruction of Sources 12 6 3D Tools Volume Tab 172 The voxels are automatically displayed when the 3D reconstruction is complete In t
167. ent Background Add or Replace Fluorescent Background 262 Copies the active image window to the system clipboard Opens the Edit Image Labels dialog box that enables you to edit the label set information for the active data Opens the Preferences box Choose this option to display the toolbar Choose this option to display the status bar at the bottom of the main window Displays the Activity window at the bottom of the main application window The Activity window shows a log of the system activity Choose this option to display the tool palette Choose this option to display the activity bar at the bottom of the main window The activity bar lists a history of the recent software activities Displays the Image Information box that shows the label set and image acquisition information for the active data Displays the ROI Properties dialog box Displays the ROI Measurements table Opens the Image Layout window that enables you to paste an image of the active data in the window Displays the control panel Opens the Image Math window for the active data Opens the Image Overlay window for the active data Opens the Colorized View tab for the active sequence Note This menu item is only available if Show Advanced Options is selected in the Preferences Combines the images of a FLIT sequence into a single image intensities are summed that can be analyzed using tools in the tool palette Opens a dialog box that
168. ent 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 instead of a dark charge background 217 E Luminescent Background Sources amp Corrections Dark Charge the read bias image is stored with the image data rather than the usual background information 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 t B2 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 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 cam
169. era 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 followed 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 218 An underlying assumption for in vivo imaging is that all of the light detected during a luminescent image exposure 1s 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 Xenogen 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 s
170. erage Radiance the sum of the radiance from each pixel inside the ROI 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 214 Available for fluorescent images only Includes Total Efficiency Average Efficiency Stdev Efficiency Min Efficiency and Max Efficiency in the table For more details on efficiency see page 213 Image Attributes None All Possible Values All Populated Values Xenogen 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 Xenogen 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
171. es x y OFZ F yield Power MuaEm MusEm MuaEx MusEx Restore Defaults Mask Statistics Weighting LM Fitting Properties Tab Tissue Properties Internal medium index of refraction Results Tab MuaEm MusEx Mueft Diff Image number in the active sequence Excitation wavelength Emission wavelength Minimum surface radiance used for model fitting x and y coordinates of the bottom illumination source The image acquisition mode The angle limit refers to the angle between the object surface normal and the optical axis For more details see page 253 Filters out the noisy data at the mouse edges A setting of 0 1 means that the analysis includes 90 of the data from the center of mass to the edges Note Selecting a tissue Properties tab automatically updates MuaEm MusEm MuaEx and MusEx in the Params tab Source coordinates Fluorescence yield strength of illumination or bioluminescence source Absorption coefficient at the emission wavelength Reduced scattering coefficient at the emission wavelength Absorption coefficient at the excitation wavelength Reduced scattering coefficient at the excitation wavelength Resets the model type algorithm starting parameters and algorithm options to the default values A drop down list of ROIs in the selected image Select an ROI to compute only the source in the ROI Choose this option to apply a statistical weighting technique to helo reduce the er
172. es corrections to a frame specified in the Corrections Filtering tool palette then draws the ROIs 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 84 1 Open the kinetic data and click ROI Tools in the tool palette 2 Inthe 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 SAAT A g intros vi artoa ej DA2000110TI otal nme ROTA KSA Dieja Ao ROU Parameters Tesha 4 Use the ring to move the create tool to the area of the ROI 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 ROls that can be distinguished based on the signal strength e Living Image Software User s Manual Y Caliper feSciences Plotting Kinetic ROI Measurements Ol O N Click Create on the tool The ROI and label appear on the image If it is necessary to adjust the ROI boundaries or change any of the auto ROI parameters use the slider or 4 arrows e 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
173. esesssesesesesesssesessssessssoessoseesesesssssesesesessseedoesesessessessssessssoen Kinetic Info User Label Name Set Choose the Show All Sections option to display all categories of image information value Friday November 7 2008 Key Acquisition Date 4 To view information of interest 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 60 ad Living Image Software User s Manual Y Caliper Editing the Image You can edit the image label information after acquisition Label 1 Open an Image A TLT20050624122348_001 Units Counts 7 Display Overlay k 2 site Soit lmage Labels on the Pamba pr DOB 1 2 Label Two traser beads Scruff Bin MJ8 FOV 12 6 f2 1s Comment Dorsal Camera VIS 200 Beta Il SIGB20EEV Analysis Comment test Image Min 48 Max 28314 Counts Color Bar Min 142 Max 18709 Edit Image Labels 3 In the Edit Image Labels box that appears Us TLT w LsbelSet KENEEN edit the information of interest You can Check any 5 fields also select a new label set to apply to the image or sequence Series Male Nn nu Experiment DOB 02 28 05 4 When you are finished click OK Shel Two taser beads Scull The image information is updat
174. f iterations allowed Indicates how much of the data was filtered by principal component analysis The normalization method used in the analysis The non negativity method used in the analysis The weighting method applied to the data Indicates if column wise weighting was used Indicates if row wise weighting was used Click to display the spectrum plot tab Click to display the concentration plot tab 133 9 Spectral Unmixing 9 4 Spectral Unmixing Options In the spectral unmixing tools the Options tab shows the user modifiable parameters in the spectral unmixing algorithm Figure 9 3 It is recommended that you first perform spectral unmixing using the default settings Then if necessary change the option settings and reanalyze the data Tool Palette gt Image Adjust gt ROI Tools gt Planar Spectral Imaging gt Surface Topography _ Point Source Fitting gt DLIT 3D Reconstruction Spectral Unmixing Analyze Options Spectrum Results 7 X Lock Unimod HP LP CO N NA NJA O Nia NA N A Denoise by PCA Medium Default y Unimod Tolerance 3 5 PCA Mode Correlation Explained variance Biplot Figure 9 3 Tool palette spectral unmixing options Table 9 3 Spectral unmixing options Option Description Constraints The constraints for unmixing the components Init The method for generating the initial guess of the spectrum for the selected component Auto means this
175. fic 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 Measuring ROIs in an Image page 84 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 on the associated grayscale photographic image Figure D 1 Pixels in the luminescent or fluorescent image that are less 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 emissi
176. 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 34 Temperature The temperature box color indicates the temperature and status of the system System not initialized E System initialized but the CCD temperature is out of range kE 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 11 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 Setup table Sequence Setup Click to display the Sequence Editor so that you can access the imaging wizard specify and manage sequence acquisition parameters or open sequence acquisition parameters xsq For more details on setting up an image sequence see page 22 Image Setup Click to close the Sequence Editor Initialize Click to initialize the IVIS Imaging System For mor
177. g Image Browser You can open data from the Living Image browser the toolbar or the menu bar Multiple data sets can be open at the same time NOTE To open recently viewed files select File Recent Files on the menu bar 1 To open data do one of the following e Double click the data row image SEQ sequence DCM kinetic data e Right click the data name and select Load on the shortcut menu e Select the data row and click Load e Double click the thumbnail The image s and tool palette are displayed Open data Is highlighted in green in the browser amp Living Image Browser DAR TEFEOOGOSIOLIAGIZ OLO ficie Number EX Filter EM Filter Illumination Mode UserID Series Experiment Label Location C Program Files Xenogen Sample Data Dataset EPI 3Comp TLT200605101 14512 oh TEETE 14512 et Leen txt 2 To show the images in a sequence click the sign next to a sequence SE 47 48 5 Working With Data 3 To open all images in a sequence click the Display All button in the image window To close all images click the Hide button 75 7 File Edit Yiew Tools Acquisition Window eka Raw Help Units Counts B oO Apply to all STITT i Tool Palette gt Image Adjust gt ROI Tools Pl TLT20050624145507_SEQ Sequence View io gt Planar Spectral Imaging Units Counts J C Use Saved Colors gt Surface Topography gt Point Source Fitting gt DLIT
178. g is often acceptable for in vivo images where light emission is diffuse For more details on binning see Appendix C page 206 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 191 A IVIS Acquisition Control Panel Table A 1 IVIS acquisition control panel continued Item Description F stop Excitation Filter Emission Filter Photograph Structure Overlay Lights Fluor Lamp Level Field of View Service Load Sets the size of the camera lens aperture The 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 f stop see Appendix C page 205 A drop down list of fluorescence excitation filters For fluorescent imaging choose the appropriate filter for your application For bioluminescent imaging Block is selected by default If you select Open no filter is present For systems equipped with spectral imaging capability choose the appropriate emissi
179. ge Table 2 2 image sequence Table 2 3 or kinetic data Figure 2 3 Example imaging and analysis workflow 2 Getting Started Table 2 2 Image or kinetic sequence analysis tools Tool Palette Use The Tools to See Page Image Adjust e Tune the photograph brightness gamma similar to contrast or opacity 62 e Set the image display color scale min and max e Select a color table for image display Corrections Filtering Subtract dark background from the image data 66 e Apply flat field correction to the image data e Specify pixel binning e Smooth the pixel signal Image Information e Display x y coordinates and intensity data at a user selected location on the image 67 e Display a histogram of pixel intensities in an image e Plot the intensity y axis at each pixel x axis along a user specified line in the image e Measure distance in an image ROI e Measure counts or photons in a user specified region of interest ROI and compute 81 measurement statistics for example average min max standard deviation Measure efficiency in the ROI and compute measurement statistics for fluorescent images only Table 2 3 Analyzing image sequences Analysis Description Wizard See Page Available Planar Spectrallmage Determines the average depth and total photon flux of a bioluminescent Yes 121 Analysis point source in a user specified region of interest Analyzes a sequence of bioluminescent images acquired using different emiss
180. ground 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 16 235 F Fluorescent Imaging 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 such 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 236 High levels of tissue autofluorescence can limit the sensitivity of detection
181. he 3D tools the Volume tab displays the voxel intensity and other data and provides tools for voxel measurement and display Mesh Volume Organs Animation Render volume Min 0 0015 Render voxels 4s Threshold Intensity coos Color Table Reverse Logarithmic Scale Source Voxel Measurement Fluorescence Yield c ari S A mm2 volume ones mmi Center of Mass 25417 14 8917 9 8416 Host Organ Unknown Export Voxels Center OF Mass Figure 12 8 3D tools Volume tab Table 12 6 3D tools Volume tab Item Description Render Volume Choose this option to display the voxels Min The minimum voxel intensity ohotons sec for DLIT results N mm for FLIT results Max The maximum voxel intensity ohotons sec Render voxels as A drop down list of shapes for voxel display Threshold Choose this option to apply a minimum threshold intensity to the voxel display Intensity Use the Intensity slider the Bi arrows or enter a value in the box to set the minimum threshold intensity Color Table Specifies the color table for the voxel intensity scale Reverse Choose this option to reverse the color table For example the BlackRed color table represents the source intensity ohotons sec from low to high using a color scale from black to red If Reverse is chosen the source intensity ohotons sec trom low to high is represented using the color scale from red to black Logarithmic Scale
182. he CCD of the IVIS 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 Xenogen Corporation 207 C Detection Sensitivity C 3 Smoothing 208 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 control binning you can specify Manual Binning in the Living Image Tools Preference Camera Settings box Table C 1 Binning settings EEV ROPER SiTe Andor Medium Lumin Bin 8 Bin 5 Bin 4 Bin 4 Small high resolution Bin 4 Bin 2 Bin 2 Bin 2 Lumin Large high sensitivity Bin 16 Bin 10 Bin 8 Bin 8 Lumin Medium Photo Bin 4 Bin 2 Bin 2 Bin 2 Small high resolution Bin 2 Bin 1 Bin 1 Bin 1 Photo 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 impro
183. he ROI selected in the image for more details on setting the units see ROI Dimensions page 107 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 Ei 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 ROI Using the Free Draw Method page 90 e ROI size dimensions or position e Subject ROI ID information 6 Working With ROI Tools Moving an ROI 100 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 NOTE You cannot move ROls created using the auto ROI feature To drag an ROI j 117 20050624145507_006 DER Units Counts v Display Overlay mj mo 1 Put the mouse pointer over the ROI so that it becomes a J arrow 2 Drag the ROI Max 22447 3 Release the mouse button when the ROI is properly positioned Max 19546 To move an ROI using the ROI Properties box j ROI Properties X 1 Double click the ROI in the image ROI BKG 1 v The ROI Properties box appears and displays the cate Sl position and dimensions of the selected ROI Beg RO OA _ l l U
184. he 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 3 To edit the minimum or maximum bin intensity enter a new value in the Min Bin or Max Bin box or click the arrows hl 4 To edit the number of bins enter a new value in the Bins box or click the uw arrows 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 5 8 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 ei Opens a dialog box that enables you to export the histogram csv Copies the histogram to the system clipboard ch Opens the print dialog box 70 Ti Living Image Software User s Manual Y Caliper Viewing a 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
185. he signal intensities range from O to 65536 counts per pixel Note The14 bit dynamic range enables faster imaging Exposure Time msecs The exposure time for the luminescent image Shorter exposure times 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 4 Acquire Kinetic Data Table 4 1 Kinetic acquisition settings continued Item Description 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 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 206 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 The 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 k
186. hing specified Save Results Name The name of the surface Delete Click to delete the surface selected from the Name drop down list Load Click to load the surface selected from the Name drop down list Save Click to save the surface to the name entered in the Name drop down list 141 10 Generating a Surface Topography 10 2 Managing Surfaces Saving a Surface Loading a Surface Deleting a Surface 142 The generated surface can be saved for shared use by the Point Source Fitting DLIT or FLIT tools Tool Palette ROI Tools Planar Spectral Imaging Surface Topography Surface Reconstruction Structure Light TLT20050624145507_O01 SL Binning Surface Smoothing Smoothing Level tow Restore Loss Recovery Height smooth Save Results Name SURFA amp CLE_1 wt gt BLIT 3D Reconstruction gt 3D Tools Surface name gt Spectral Unmixing Figure 10 2 Tool palette Surface topography tools 1 Confirm the default name or enter a new name 2 Click Save 1 Select the surface that you want to use for point source fitting DLIT analysis or FLIT analysis from the Name drop down list 2 Click Load 1 Select the surface that you want to delete from the Name drop down list 2 Click Delete ad Living Image Software User s Manual Y Caliper _ 1 1 Point Source Fitting Displaying the Point Source Fitting Tools 0000 eee eee e
187. hoose 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 To incrementally zoom in or out on an image eClick the Cry or 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 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 eClick the R button Alternatively right click the image and select Reset Zoom on the shortcut menu To pan the image window 1 Click the 4 button 2 When the pointer becomes a h click and hold the pointer while you move the mouse Note Panning helps you view different areas of a magnified image If the image has not been magnified you cannot pan the image Fie Ear View bos A amp gqibon Wiridow Heip wJ ia A amp R LmtsiCounts w C Apply to al tuU LS if t00 C Ato Coker Tab e Logarithmic Scale Corrections Filtering v Reverse gt DUIT 3D Reconstruction 65 5 Working With Data 5 8 Correcting or Filtering Image Data Use the Corrections Filtering
188. ick 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 7 Set the FOV To adjust the field of view FOV make a selection from the Field of View drop down list For more details on FOV see page 160 To view the subject s inside the chamber before image acquisition take a photographic image clear the luminescent or fluorescent option choose the Photographic and Auto options and click Acquire 8 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 or e Select Manual focus from the Focus drop down list For more details on manual focusing see page 34 9 If necessary click mage Setup jn 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 more details on setting up a Sequence see page 22 10 When you are ready to acquire the image click Acquire During image acquisition the Acquire button becomes a Stop button To cancel the acquisition click Stop The image window appears Figure 3 1 If this is the first image of the session you are prompted to choose an autosave location 3 Acquire an Image or Image Sequenc
189. iences 8 Planar Spectral Image Analysis Planar Spectral Imaging Tools 0 0 ee ee es 123 Planar Spectral Image Analysis 0 2 0 eee es 121 Viewing amp Exporting Graphical Results 20 0000 eee eee ees 125 Managing Planar Spectral Imaging Results 2 00002 ee ee eee 126 The Living Image software enables you to apply planar spectral image analysis to a sequence to determine the average depth and total photon flux of a bioluminescent point source in a user specified region of interest For more information on planar spectral image analysis see Appendix G page 239 Use the imaging wizard to setup the image sequence required for planar spectral image analysis For more details on the imaging wizard see page 24 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 8 1 Planar Spectral Image Analysis 1 Open the image sequence that you want to analyze File Edit Yiew Tools Acquisition Window Help A mi A amp R E Unitsi Counts vO Apply to all T A T1T20050624145507_SEQ waaa idi Sequence view 3D View erai 1 7 Planar Spectral Imaging Units Counts C Use Saved Colors Analyze Properties Results Sequence 1720090624149507 _ SEQ Tissue Muscle Source Stefy Select Filters Imageset lt lt No active ROI selected gt gt ROI List _
190. ies tab make a selection from the Tissue Properties drop down list b Choose the tissue type most representative of the area of interest Muscle is a good choice for a generic tissue type ee rooe a Tissue Properties Muscle The software automatically sets the internal medium index of refraction based on the selection in the Tissue Properties list Ieee medium index pf refracuon 140 Source Spectrum Firefly Plot Tissue Properties v 6 Make a selection from the Source Spectrum drop down list n era 1 m peff Usp 7 Click Analyze in the Analyze tab HA LD 600 800 Wavelength nm _ Surface Topography gt Point Source Fitting gt DLIT 3D Reconstruction 122 Living Ilmage Software User s Manual Tool Palette The Results tab displays the computed average depth mm and total flux ohoton sec of the bioluminescent point source in the specitied ROI s Analyze Properties Results Spectral Results Unsaved ROI Depth mm Total Flux phot s ROIZ 2 3414 0 269 2 66e7 5 1866 lt M gt Plot Linear Fit Plot Intensity Save Results Name SpIm_2 Delete 8 2 Planar Spectral Imaging Tools The planar spectral imaging tools are displayed in three tabs Tool Palette Tool Palette Tool Palette gt Image Adjust gt Corrections Filtering gt Image Information gt ROI Tools Properties Results
191. 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 bioluminescent 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 239 G Planar Spectral Imaging Optical Properties of Mouse Tissue Ex vivo muscle tissue integrating sphere measurement 1 0 9 0 8 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 45 550 650 fo gt Hla i mm Wavelength nm Intensity a u Firefly Luciferase Spectra PC3M Luc cells i a Tissue Transmission Window j vitro n vivo 0 8 0 6 0 4 0 2 400 500 600 700 800 Wavelength nm Figure G 1 Optical Properties of Mouse Tissue and Firefly Luciferase Spectra The bioluminescent 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 C us
192. inates are updated To display planes in the 3D image 1 Click a voxel 2 Click the button The Coronal Sagittal and Transaxial view shows the plane slice through the voxel 174 Living Image Software User s Manual F ile Edit View Tools Acquisition Window T Y Ca PET a Help SAHI BS R intsony apto Tool Palette Render voxels As Threshold Intensty J 1 00e 4 Color Table BlackRed v C Reverse C Logarithmic Scale Source Yoxel Measurement Fluorescence Yield 2 00 Af mm 72 Volume 2 00 mn F Center of Mass 2 00 2 00 8 00 Host Organ Unknown Export Yoxels Center Of Mass Fi 1V20080208142757_SEO Sequence View 3D View o L heim arc HS Nmm 2 Source Intensiti Coronal sagittal and transaxial views show the slice through the surface taken by the associated plane 3 To change the location of a plane move the coronal sagittal or transaxial slider left or right Alternatively click the slider then press the lt or keyboard arrow keys The Coronal Sagittal or Transaxial windowpane is automatically updated 12 7 3D Tools Organs Tab The Living Image software provides digital mouse atlases that enable you to display a 3D skeleton and organs on the 3D reconstruction Table 12 7 Select the atlas and organs that you want to display in the Organs tab The software automatically aligns the organs on the surface However you ca
193. inetic 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 205 EM Gain Multiplies the signal in real time This option is useful for boosting low signals 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 bioluminescent imaging Block is selected by default If you select Open no filter is present Lamp Level Sets the illumination intensity level of the excitation lamp used in fluorescent imaging Off Low High and Inspect Low This 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 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 Level Controls the brightness of the lights at the top of the imaging chamber that are used to acquire photographic im
194. ing Image Software User s Manual Y Caliper _ 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 L 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 bioluminescent 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 subtracting tissue autoluminescence see Appendix E page 220 It is also recommended that you inspect all images in the sequence to confirm that the bioluminescent signal is greater than the tissue autoluminescence If the bioluminescent signal does not exceed the tissue autoluminescence at a particular wavelength do not include that wavelength in the analysis 245
195. ing PC3M cells 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 is collected over the animal surface the total integrated intensity I A is reduced to a relatively simple expression I SKO exp Lepp d 1 where Sis the absolute total photon flux emitted by the bioluminescent source and d is the source depth The term uepis 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 is 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 uep 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 expo
196. inning ej TLT20050624145507_SEQ Surface Smoothing Smoothing Level Loss Recovery Save Results Name SURFACE_1 v Delete Load Save DLIT 3D Reconstruction Analyze Params Properties Results Sequence 720050624145507 SEO Tissue Marcie Source bum Sead 5 Select Wave Filters Minimum Radiance 3 02e 05 6 06e 05 2 13e 06 3 02e 06 2 35e 06 140 Ti Living Image Software User s Manual Y Caliper Table 10 1 Surface topography options amp parameters Item Description Surface Reconstruction Structured Light A drop down list of structured light images in the sequence Select one for use in reconstructing the surface SL Binning The binning level applied to the pixels that are used to construct the surface topography from the structured light image If Auto is chosen the software automatically determines the appropriate binning level The higher the binning level the lower the mesh resolution For more details on binning see page 206 Reconstruct Click to generate the surface Surface Smoothing Smoothing Level The amount of smoothing to apply to a reconstructed surface Restore Removes smoothing that was applied to a surface Loss Recovery Smoothing can cause loss in the surface volume or height Make a selection from the drop down list to reduce losses Height is recommended for IVIS 200 or IVIS Spectrum surfaces Smooth Initiates the smoot
197. inued Item Description 4 Bring omen Y Sendbackward Send to back D Select an the item in the Image Layout window To move the item to the front or back in the window choose an option from the drop down list x Deletes the selected image Layout Style Layout ee d A drop down list of formatting options for the Image Layout window For example the 2x2 layout style provides 4 separate layout areas In the window A different image can be pasted into each layout area Annotate pila To apply notes to an image enter text in the annotation box and press Enter Drag the text to the location of interest in the image 5 7 Adjusting Image Appearance Opens a dialog box that enables you to select a font or edit the font style and size 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 Use the image adjust tools to adjust the appearance of an image Figure 5 7 NOTE Not all tools are available for all image display modes Fie ER Yev Tok Ahn Wide Hao gt Ou h A g k scans O etaa gt mariae Topography a gt Tee Scarce Ring DLIT 30 Reconstruction i Image data Min amp Max Color bar Color bar Min amp Max Figure 5 7 Tool palette Image Adjust tools 63 5 Working With Data Table 5 5 Image Adjust tools Ite
198. ion Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter 1 00 feee Seam ole Jea Soen bag C Move Motors To Selected Spot Grid Type 15x23 v Update Photograph C Transillumination Lamp Source C Mask Grid Points To Subject Grid Display Points me Clear Selections Field of View C v System Status Service Subject height 1 50 S icm Temperature I Locked Focus use subject height v 6 Inthe Transillumination Setup box that appears choose the locations for transillumination and image acquisition and click OK For more details on the Transillumination Setup box see Table 3 2 Ti Living Image Software User s Manual Y Caliper Table 3 2 Transillumination Setup box Item Description Move Motors To Transillumination motors will move to the grid location selected in the Selected Spot Transillumination Setup dialog box Transillumination Choose this option to turn on the bottom illumination lamp Lamp Source Mask Grid points Choose this option to select only the grid locations within the subject To Subject Grid locations outside the subject are masked Grid Type Select a grid type from the drop down list 15x23 11x23 5x10 or 8x12 well plate Xenogen Sparse Mask 6x8x1cm Grid Display Select Points or Cross Hair to display the grid Update Photograph Cl
199. ion filters Image Math A method for mathematically combining two images add multiply or No 118 subtract Use image math to e Remove autofluorescence from a fluorescent image e Display multiple bioluminescent or fluorescent images on the same photographic image so that you can view multiple reporters in one image Spectral Unmixing Removes tissue autofluorescence from a fluorescence image Analyzes a Yes 121 sequence of fluorescence images acquired using the fluorophore excitation filter and several different emission filters Spectral unmixing can be applied to images acquired using epi illumination excitation light above the stage or transillumination excitation light below the stage Point Source Fitting Estimates the optical properties of tissue the location and power of a point Yes 143 source or the fluorescent yield of fluorophores 3D Fluorescence A 3 dimensional reconstruction of the image tomographic analysis that Yes 152 Imaging Tomography estimates the location and intensity of a fluorescent light emitting source FLIT Analyzes a sequence of fluorescent images acquired on the IVIS Spectrum at different transillumination points excitation light below the stage 3D Diffuse A 3 dimensional reconstruction of the subject tomographic analysis that Yes 156 Luminescence estimates the depth and intensity of a bioluminescent light emitting Tomography DLIT source For more details on sequence setup using the imaging
200. iption Copy row s Copies the selected row s to the system clipboard Select All Selects all rows in the sequence editor Delete row s Deletes the selected row s from the sequence editor Replace Row s Replaces the row s selected in the sequence editor 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 editor Paste Row s Adds copied rows to end of the sequence Alternate method to add a row 1 In the sequence editor 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 Cj insert To insert the new image below the selected row click lt ier 7 Living Image Software User s Manual Y Caliper Alternate method to delete one or more rows 1 Select the row s that you want to delete 2 Click and choose Selected from the drop down list To clear the sequence editor 1 Click X Remove 2 Choose All from the drop down list Acquire the Image The system is ready to acquire the image sequence after you set up the image sequence Sequence in the sequence editor 1 Initialize the IVIS System and confirm or wait for the CCD temperature to lock For more details see page 11 2 When you are ready to acquire the images click Acquire Se
201. is automatically determined by the software Alternatively you can used a loaded spectrum as the Initial guess Lock The lock option determines whether the spectrum is allowed to change If this option is chosen the spectrum of that component is not updated during unmixing Unimod Choose this option to apply the unimodality constraint Unimodality forces the spectrum to have only one peak one extremum however small magnitude extrema are allowed if they are less than the Unimod Tolerance value This tolerance value limits the rising slope of the second spectral peak For example 5 tolerance means that the increase in magnitude of the neighboring nodes cannot exceed 5 134 Ti Living Image Software User s Manual Y Caliper Table 9 3 Spectral unmixing options continued Option Description 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 LP 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
202. isplay in the l wj Living Image browser Click this button to open all images in a sequence Click this button to close all open images in the active sequence Editing an Image Sequence You can add individual images to a sequence or remove user specified images from a sequence Open the image sequence that you want to edit For details on how to open image data see page 47 2 Ifyou 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 45 Note Only individual images not an image sequence can be added to a sequence 3 In the image window click the Edit button ao Qi a amp hk Units Counts wo Apply to all A TLT20050624145507_SEQ gt Tool Palette QM Q R hk Photo Adjustment J E E J100 Color Scale Limits Auto Full Manual Individual Color Table Rainbow v Reverse C Logarithmic Scale 52 Living Image Software User s Manual i Y Ca POl ns 4 In the Edit Sequence box that appears choose the image s that you want to add or remove trom the sequence E Edit Sequence Sequence Clicks TLT20050624122348_001 TLT 200506241 22348_002 TLT 200506241 22348_003 TLT 200506241 22348_004 TLT 200506241 22348_005 TLT 200506241 22348_006 Images in the active sequence O Reactivate 00 Retire gt gt
203. ity photons mm3 just beneath the surface of each element of the mesh e Divides the interior of the subject into a solid mesh 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 photon density 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 or mesh from a structured light image Parallel laser lines are projected onto the subject to produce a structured light image Figure H 1 If the Structure option is chosen in the Control panel a structured light image is automatically acquired 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 247 H 3D Reconstruction of Light Sources 248 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
204. k to show the controls to adjust the fluorescence opacity and the color table display in the concentration plot Click anywhere in the window to hide the controls Show Labels Choose this option to display image labels on the concentration plot and composite image Th Living Image Software User s Manual Y Caliper 9 2 Spectral Unmixing Results Window Spectra Tab The results are displayed in the three tabs of the Spectral Unmixing Results window The spectrum plot shows the normalized spectra of the unmixed results You can edit the appearance of the spectrum plot using the tools in the spectral unmixing tool palette Spectrum tab Figure 9 1 A list of the spectra in the results Add remove a check mark to show hide the spectrum in the spectrum plot Click a spectrum row to display in the preview pane File Edit View Tools Acquisition Window Help a ey A amp R Units Photons O Apply to all Tool Palette F TLT20060406164950_SEQ Spectral Unmixing A Sequence Yiew Spectra Distribution Analyze Options Spectrum Results Spectrums 7 Xx Show Type Name Label Color SOL Unmixed Unmixed 1 SOL Unmixed Unmixed 2 1 00 Normalized Amplitude Normalize V Show Legend Spectrum Preview 1 00 Normalized Amplitude 0 50 0 0 720 760 800 840 Wavelength rar Spectrum Plot Figure 9 1 Spectral unmixing tools and spectrum plot Table
205. l Parameters tab Tool Palette gt ROI Tools gt Surface Topography FLIT 3D Reconstruction Analyze Params Properties Results Backgound Uniform Surface Sampling C NNLS Weighted Fit Restore Defaults gt 3D Tools v Y Ca PS cas Properties tab Tool Palette i Planar Spectral Imaging r Surface Topography i FLIT 3D Reconstruction Analyze Params Properties Results Tissue Properties Bladder l Internal medium index of refraction f 4 Plot Tissue Properties er 1 Wavelength nr gt 3D Tools gt Spectral Unmixing sid 3 Inthe Params tab confirm the parameter defaults or enter new values For more details on the parameters see Appendix H page 247 It is recommended that you start with the default parameter settings If necessary fine tune the settings In the parameters tab Reconstruct In the Analyze tab select the source locations to include in the analysis and click Figure 12 1 page 155 shows example 3D reconstruction results 157 12 3D Reconstruction of Sources 12 3 DLIT amp FLIT Results 158 The Results tab displays information about the photon density voxels and DLIT or FLIT algorithm parameters Tool Palette D Planar Spectral Imaging FLIT 3D Penondtiuetai lt DLIT 3D Reconstruction Analyze Params Properties Results FLIT Results FLIT_1 Loaded a Analyze Params
206. large values favor more shallow sources For more details see page 254 202 Ti i Living Image Software User s Manual Y Caliper Table B6 3D analysis preferences continued Item Description N Surface Limits The maximum number of surface intensity points to use in the reconstruction at a given wavelength The range is 200 to 800 and the default is 200 The time required for reconstruction is shortest for smaller values of N for example 200 However a large N value may give a more accurate result because more data are included in the fit Voxel Size Limits Voxels are the small cubes of space inside a subject each of which contains a light source much like a pixel in a 2D image The DLIT reconstruction begins with large voxels specified by the voxel size limit the length of a side of the voxel cube in mm At each Iteration the algorithm reduces the size of the voxel by a factor of two until the optimum solution is found The voxel size limits are a minimum of five and a maximum of 10 The default range is set to 6 9 mm A larger range of voxel limits ensures a more reliable solution but requires more computational time The default range of 6 9 is usually adequate to determine the optimum solution Voxel Size This is the step increment in voxel size stepping from the minimum voxel Increment size limit to the maximum voxel size limit For example if the voxel size limit ranges from 6 9 mm a voxel size increment 1 gi
207. lick the Sequence Editor Changing an Imaging Parameter 1 In the sequence editor double click the cell that you want to edit S panel _ 1 editor Imaging word WE 7 opty Photographe Settings e Germ Fao Exctation Emission Structure FOV Met Medium Bba 640 No amp Madi m Bba 640 No Medken Flick 640 No Macin 1 kct 40 No Madum 1 oe G40 Ne na l 1 he 8 Focus ine udiecthag terroer ature ED 2320 ntisize _ umber of Segments rech to al X Ramon A update C inset C Adi L For details on these functions see Table 3 5 2 Enter a new value in the cell or make a selection from the drop down list 3 To apply the new value to all of the cells in the same column click Apply to all 4 Click outside the cell to lose focus Editing a parameter using the control panel 1 In the sequence editor select the row that you want to modify 2 Inthe control panel choose new parameter values and or imaging mode 3 Click A Update Table 3 5 Sequence Editor 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 file m Displays a dialog box that enables you to save a sequence setup xsq Display Photographic Choose this option to include the photograph exposure time binning Settings and F Stop in the sequence editor Number of Segments Choose this optio
208. lines The line spacing and binning are automatically set to the optimal values determined by the FOV stage position and are not user modifiable If this option is chosen the system automatically acquires a photographic image followed by a luminescent image then coregisters the two images Turns on the lights located at the top of the imaging chamber 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 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 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 m
209. list To display the line profile 1 Open an image and in the Image Information tools click the Line Profile button AL A blue line appears on the image and the Line Profile window appears File Edit view To oe Qa amp Window Help Inal Units Counts vo Apply to all F Tool Palette cy A 11T20050624145507_006 wt EE E E Be laa 64 7 Eag units cm J mage Binning 8 Width 12 6cm Height 12 6 cm Max 22447 Image X 10197 4 012 cm Image Data 6 counts Crop Distance By i 0 00 0 00 0 00 0 00 Distance 0 00 gt ROI Tools gt Planar Spectral Imaging gt Surface Topography _ gt Point Source Fitting _ DLIT 3D Reconstruction Counts Color Bar Min 1122 Max 19546 iLine Profile Window Line Orientation Horizontal Width i a Position 90 L a amp x Min 0 00 x Max 12 55 a YMin 374e6 Y Max 5 17e6 5l Full Scale Logarithmic Scale TLT 200506241 22348 _003 Overlay 109 Photons s om 2 sr 3 00 2 00 1 00 0 00 0 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 the line over the image The blue line determines the pixel intensities that are plotted in the line profile graph The line profile is updated as you move the blue line
210. list in the Properties tab 4 Inthe Analyze tab select the acquisition wavelengths 560 660 nm 5 If necessary edit the minimum radiance associated with an acquisition wavelength or angle For more details on the minimum radiance see page 253 Note t is recommended that you only analyze images that have signal well above the noise 6 To edit the minimum radiance double click the entry and enter a new value Note he minimum radiance level can be observed by looking at individual images and adjusting the min level on the color bar in radiance units photons sec cm2 Tissue fierce Tool Palette E gt gt Planar Spectral Imaging fa gt gt Surface Topography gt Surface Topography DLIT 3D Reconstr DLIT 3D Reconstruction Analyze Params i Froperties Results Tissue Properties Muscle Internal medium index of refraction 14 po Source Spectrum Firefly z Tissue Properties 6 Wavelength ron gt 3D Tools 7 Tool Palette E gt gt Planar Spectral Imaging al gt gt Surface Topography si gt Surface Topography si E I T DLIT 3D Reconstruction Analyze Params Properties Results Sequence Ti 720050624722346 5EQ Source Sre Select Wave Filters Filter o 2 ege ir 1 70e 07 3D Tools 153 12 3D Reconstruction of Sources 7 Inthe Params tab confirm the parameter def
211. m 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 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 Click this button to return the image to the default display magnification Click this button to move a magnified image pan in the image window For more details see page 65 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 Click this button to hide or display the color scale min max information in the image window e mgee a m Photo 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 Gamma Click and move the slider left or right to adjust the gamma of a image displayed in overlay mode Alternatively enter a gamma value Gamma is related to image contrast Opacity Click and move the slider left or
212. mage 1LT200 0624145507_00 2 Click an ROI shape button and Select us crs csi ory o Auto 1 trom the drop down list The create tool appears on the image 3 Use the ring to move the create tool to the location for the ROI 4 Click Create on the ring tool The ROI appears on the image and the ROI label displays the intensity signal 5 To draw another ROI repeat step 3 to step 4 Note For information on how to save ROIs see page 104 89 6 Working With ROI Tools 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 OJ Square or Contour G and select Free Draw from the drop down list In this example the Contour shape was selected for the free draw method Note he ROI shapes that are available depend on the type of ROI selected File Edit View Tools Acquisition Window Help 2 H A amp R Units Counts oO Apply to all Tool Palette gt Image Adjust j gt Corrections Filtering gt Image Information ROI Tools oO e y ae Wj v TLT20050624145507_002 L Apply toSeque Autoal f ype Measuremer Auto 1 Save ROIs ame ROI_1_KS54 Delete Load Auto ROI Parameters hreshold o o Lower Limit 0 0 Max 10944 8000 Minimum Size 20 ROI 1 2 942e 05 gt S
213. mits DLIT result The kappa parameter is a parameter that is searched during a reconstruction to determine the best fit to the image data For more details see Kappa Limits DLIT page 254 N surface limits DLIT result The maximum number of surface intensity points to use in the reconstruction for each wavelength The range is 200 to 800 and the default is 200 The reconstruction time is shorter for smaller values of N for example 200 However larger values of N may give a more accurate result because more data are included in the fit Starting Voxel Size DLIT FLIT The length of the side of the voxel cube in mm units for the coarsest Initial grid size in the adaptive gridding scheme Voxel size limits DLIT result The starting voxel size range evaluated by the algorithm to determine the optimum solution Voxel size increment DLIT result The incremental change in voxel size evaluated at each iteration during the DLIT analysis Uniform Surface Sampling TRUE the option is chosen and the surface data for each wavelength is sampled spatially uniformly on the signal area FALSE the option is not chosen and the N brighter surface elements are used as data In the reconstruction NNLS Simplex Optimization TRUE the option is chosen and a non negative least Squares optimization algorithm is used in addition to the SIMPLEX algorithm to seek the optimum solution FALSE only the NNLS algorithm is used to seek the op
214. move over the image Table 5 9 Line Profile window Item Description Line Choose Vertical Horizontal or Free Hand from the drop down list to set the Orientation 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 71 5 Working With Data Making Image Measurements 72 Table 5 9 Line Profile window continued Item Description Position Line position pixels X Min Displays the minimum and maximum value of the x axis Use the arrows to X Max 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 Displays the minimum and maximum value of the y axis Use the amp arrows to Y Max 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 Max 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 Select this option to apply a log scale to the y axis Scale 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
215. n a single wavelength so that more information is available for the analysis Ideally chose wavelengths or source positions where the signal is well above zero not buried in the CCD noise and the optical property of the medium ug exhibits a large change The larger the difference in ep the higher the quality of information that the w S B Living Image Software User s Manual Y Caliper Parameters Tab wavelength data adds to the analysis The recommended wavelengths are 580 640 nm for tissue and 560 620 nm for the Xenogen XPM 2 tissue phantom Minimum Radiance The minimum radiance determines the lower radiance photons sec cm sr threshold of the data to be used in the DLIT FLIT analysis The software automatically computes a default minimum radiance value and this is the recommended starting point for an analysis If too much noise is included in the analysis increase the minimum radiance value An optimum minimum radiance value can be evaluated by viewing the image data in photon units photons sec cm sr and adjusting the color bar Min to be above the level of noise in the image Tool Palette Tool Palette Pa Planar Spectral Imaging ROI Tools C Surface Topography set gt Surface Topography 7 DLIT 3D Reconstruction FLIT 3D Reconstruction Analyze Params Properties Results Analyze Params Properties Results Angle Limit deg BO Angle Limit deg lower
216. n also manually adjust the scale or location of organs on the surface You can also import a custom organ atlas created from Open Inventor files iv brain cecum Bolon B sExterior eyesInterior Beart Bieum Figure 12 10 3D tools Organs tab 175 12 3D Reconstruction of Sources Displaying Organs 176 Table 12 7 Organ atlases Atlas Description Mouse Atlas Female Male or female in a dorsal or ventral orientation Atlas includes organs or Male only no skeleton CT Female or Male Male or female in dorsal ventral or lateral orientation Atlas includes organs and skeleton Table 12 8 3D tools Organs tab Item Description Organs Choose this option to enable the selection and display of organs on the surface Organ Choose the male or female organ database from the drop down list database Organs A list of the organs in the selected organ database Select the organ s that you want to display on the surface Reset Click to display the selected organs in their default positions 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 Update scene Click to display the selected organs on the surface 1 Inthe 3D Tools click the Organs tab Fia Ed Wina Tonks Aegidii Windy Hig 21 m ip LA pan k Unk Souris ae O Apy bs al Te coe theater 2 Confi
217. n to set the number of segments to acquire and the time delay between segments One segment the sequence specified in the sequence editor Delay Specifies a time delay between each segment acquisition Applies the selected cell value to all cells in the same column Apply to All ae Deletes the selected row from the sequence editor A Update Updates the selected row in the sequence editor with the acquisition a parameters in the control panel 29 3 Acquire an Image or Image Sequence Table 3 5 Sequence Editor Item Description F Inserts a row above the currently selected row using the information Insert from the control panel mm Adds a new row below the currently selected row using information from the control panel Adding or Deleting Images From a Sequence 1 Select the row s of interest and right click the sequence editor to view a shortcut menu of edit commands see Table 3 6 Imaging Wizard C Display Photographic Settings Mode Exposure Binning FStop Excitation Emission Structure FOY Height 1 ni Auto Medium 1 Block 500 No C 1 50 2 D E 3 Auto Medium 1 Block 560 No C 1 50 Select All 4 Auto Medium 1 Block 580 No 1 50 Delete rows s HD to Medium 1 Block 600 No c 1 50 Replace Row s 6 HD tuto Medium 1 Block 620 No c 1 50 Paste Row s C Number of Segments 1 Delay 0 0 min Apply to All sA Update Insert Add Table 3 6 Sequence editor shortcut menu edit commands Command Descr
218. nal 91 93 band gap 226 bandpass filter 225 binning 66 67 206 208 bioluminescence image 15 18 browse image data 45 46 browser 46 C Caliper Corporation technical support 3 cascade images 58 color table 211 colorize data 74 75 composite image 112 115 control panel 12 191 194 conventions 2 copy ROI measurements 107 correction filtering tools binning 66 cosmic correction 66 dark background subtraction 66 flat field correction 66 smoothing 67 cosmic correction 66 cosmic ray corrections 216 counts 213 crop box 73 D dark background subtraction 66 dark charge 218 dark current 217 data graphic image 211 scientific image 211 265 Index detection efficiency 205 detection sensitivity adjusting the lens aperture 205 exposure time 206 field of view 206 DICOM file 33 35 size limit 43 diffusion model 240 display modes 56 display units counts 213 efficiency 215 photons 214 DLIT troubleshooting 189 DLIT results 158 160 manage 187 drift correction 217 E edit image sequence 29 31 edit image sequence 52 53 efficiency 215 225 228 229 electronic background dark charge 218 dark current 217 drift 217 read bias 217 EM gain 40 export image data 35 exposure time 206 F f stop fluorescent imaging 228 field of view 206 filter bandpass 225 fluorescent 226 filter spectra 225 flat field correction 66 flat fielding 216 FLIT troubleshooting 189 FLIT results 158 160 manage 187 266 fluore
219. nal 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 211 The resulting image data comprise a two dimensional array of numbers each pixel contains the counts associated with the amount of light detected v i B Living Image Software User s Manual Y Caliper The IVIS Imaging Systems are equipped with a CCD that ranges from 1024x 1024 to 2048x 2048 pixels in size and thus have a high degree of spatial resolution At binning 1 each pixel is read and the image size number of pixels is equal to the physical number of CCD pixels Figure C 3 Binning 1 Binning 2 Binning 4 CCD pixel Signal 4 times larger Signal 16 times larger Spatial size doubled Spatial size quadrupled Figure C 3 A small segment of the CCD At binning 2 4 pixels are summed together at binning 4 16 pixels are summed At binning 2 four pixels that comprise a 2x 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
220. name for the results Name DLIT_4 f v gt 3D Tools gt Spectral Unmixing Figure 12 12 3D analysis results To save results 1 Inthe Results tab of the DLIT FLIT 3D reconstruction tools confirm the default file 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 results 1 In the Results tab make a selection from the Name drop down list 2 Click Load To copy user specified results 1 Inthe Results tab select the results of interest 2 Right click the results table and select Copy Selected from the shortcut menu that 187 12 3D Reconstruction of Sources appears To copy all results 1 In the Results tab right click the results table and select Copy All from the shortcut menu that appears All of the results table is copied to the system clipboard To export results 1 Inthe results tab right click the results table and select Export Results from the shortcut menu that appears 2 Inthe dialog box that appears choose a folder for the results enter a file name and click Save 188 Ti Living Image Software User s Manual Y Caliper 1 3 Troubleshooting Guide 13 1 DLIT FLIT Analysis Troubleshooting Table 13 1 DLIT FLIT analysis Issue Solution Large voxels gt 2 mm e The Ngata gt Nvoxels IS required for an overdetermined problem It is possible that there are
221. nential function of the product of the depth and the optical property u 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 1 240 Ti Living Image Software User s Manual Y Caliper The results of the fit are the total flux of the bioluminescence source S and the source depth d 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 u is a function of the absorption and reduced scattering coefficients Hef SHa U s U 2 Calculation of the function K in Equation 1 requires all three coefficients u4 u s and uep 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
222. nfirm the binning and f stop defaults or specify new settings for the photographic Image od If necessary click 7 Image Setup jn 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 22 10 When you are ready to acquire the image click Acquire During image acquisition the Acquire button becomes a Stop button To cancel the acquisition click Stop The image window appears Figure 3 1 If this is the first image of the session you are prompted to choose an autosave location Living Image 3 2 Do you wish to enable auto sawing of acquired data For this session Ne This can be changed anytime From the Acquisition menu 11 To specity a folder for autosaved data click Yes in the prompt and choose a folder in the dialog box that appears 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 12 In the Edit Image Labels box that appears enter information for the image label and click OK Figure 3 1 If you do not want to enter label information click Cancel _ a Living Image Software User s Manual Y Caliper E H zs Wo t i g hF inet I apiy bal Tool Perce tes cam pep Sever
223. ngth of the fluorescent sources 127 156 Living Image Software User s Manual W Caliper To set up a sequence using the imaging wizard 1 Click Sequence Setup in the IVIS acquisition control panel The Sequence Editor appears A IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter FS a ed Soos fon 7 d Field of view System Status Subject height 1 50 Sem Sequence Setup Focus use subject height v Temperature p Locked Sequence Editor 4 If necessary click Femever and select All to clear the Sequence Editor 5 Click Imaging Wizard Sequence Editor maga Mada Binhuminescenco Tavar To begin setting up a bioluminescence image sequence click here ER dle abcd SE A To begin setting up a fluorescence image sequence click here 6 Click Next Piuoeseewe ip a property in which uprertent molecules absorb light et one wavelength and dhase endl light of kiige wavelengths woresten reporters imhde genet encoded probrins dri well os yes are panei kes Moorescent images cen be obtained in either the Lnl ihunnineatin bop uninated or Trane Dhuimination bottom dhunineated a modes Cene net 25 3 Acquire an Image or Image Sequence 7 Double click the type of bioluminescence or fluorescence sequence that you want to acquire and step through the rest of the imaging wizard
224. nits Counts v Display Overlay vi oT Image Min 46 Max 4405 3000 Overlay i 11T20050624145507_001 DEAR Units Counts v Display Bias mo ij Image Min 437 Max 580 570 560 Counts Color Bar Min 515 Max 572 Bias 11720050624145507_001 DER Counts Display Height Map Yv i Image Min 1 Max 30 30 20 10 0 00 Counts Color Bar Min 0 Max 30 Height Map TLT20050624145507_001 Units Counts v Display Photograph v Photograph TLT20050624145507_001 Units Counts Display Structure ov Structure j 11120050624145507_001 Units Counts Display 3D view 63 3 20 0 15 8 Figure 5 6 Display modes for a single image The software coregisters the luminescent and photographic image to generate the overlay image v DAR j TLT20050624145507_001 Units Counts Display Luminescent Image Min 96 Max 2307 Max 4405 2000 3000 Luminescent DAR j TLT20050624145507_001 DAR Units Counts Y Display Reference g Image Min 210 Max 52477 5000 Counts Color Bar Min 268 Max 19865 Reference DER g o 57 5 Working With Data Tagging an Image Organizing Images 58 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
225. none_ Analyze gt Surface Topography _ DLIT 3D Reconstruction gt Spectral Unmixing 2 Inthe tool palette click Planar Spectral Imaging 121 8 Planar Spectral Image Analysis 3 In the Analyze tab select the emission filter Tool Palette wavelengths for the analysis gt Image Adjust sd It is recommended that you do not include a gt Image Information wavelength in the analysis if the signal is less than Srortols or equal to the autoluminescent background If autoluminescent background Is a concern you can analyze Properties Results create a background ROI and link it to the Sequence ILTZ0050624145507_SEQ Tissue Musde Source Airefiv measurement ROI prior to planar spectral analysis fselect Fitters For more details see Measuring Background i Corrected Signal page 91 I 4 Inthe ROI List drop down select All or a particular ROI 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 Measuring ROIs in an Image page 84 You only need to draw the ROI s on one image d the sequence The software copies the ROI s to a other images of the sequence during the analysis The ROI should include as much of the light emission from a single source as possible _ Point Source Fitting gt DLIT 3D Reconstruction 5 Choose the tissue properties Tool Palette a In the Propert
226. ns wide open at f 1 right lens closed down at 1 8 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 217 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 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 binning 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 dimensio
227. o all 222222222222222522222 2252222222 222228 2 2252222 222522 os c2ee gt ROI Tools Min 1 26e3 Max 9 76e5 Planar Spectral Imaging r SOPS TEI Analyze Properties Results p sec em 2 sr Spectral Results Unsaved ROI Depth mm Total Flux phot s ROl1 2 326 0 191 3 01e6 3 87e5 Vecceevcedsu svevecauespeuevscvsveevecuvesevecbectwadeseeupeccssevsevetecsvecend _ gt Plot Linear Fit Plot Intensity Save Results Color Bar Min 6 36e4 Max 6 76e5 Name Splm_2 z vj gt Surface Topography 7 DLIT E Fie Edit view Tools Window Help 10 X SAAT A amp RP Units Photons C Appi toal HHHH AAA Photons Displey Overlay sv Info a gt Image Adjust gt Qe gt Corrections Filtering gt Image Information Image Min 1 09e3 Planar Spectral Imaging bia ys aa go Analyze Properties Results D j o v Spectral Results ROI 2 9 938e 05 ROI Depth mm Total Flux phot s pale 2 426 0 199 2 42e6 3 23e5 Sbooedvevedu svedeVavedpucvecevsveuvacsesdevesveteverassUpeceetevsucedscsceseed tiasa gt Fane Save Results Name Splm_2 z Min 6 32e4 Delete Load Max 6 41e5 Figure G 5 Planar spectral analysis results Top Dorsal view of the left lung bottom ventral view of the left lung Ti i Liv
228. ocrystals 700 or 800 nm 2 Select Tools gt Colorize sequence name _SEQ on the menu bar The software renders each luminescent or fluorescent image in color and combines them into a single image 74 Th Living Image Software User s Manual Y Caliper 3 To access controls for manually i HX20070420121444_SEQ adjusting the color rendition click Sequence View Colorized View Colorize 2 R colorize color Camera NIR _ Log Scale Real Color emue Adjust T Color E Filter Range 660 a20 A Table 5 12 Colorize tools Item Description Colorize 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 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 Color Camera VIS Regular camera setup that mainly renders color in the visible range Itis similar to the color response of a commercial digital camera NIR fluorophores appear dark red to invisible using the
229. 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 I umination intensity Prior to instrument delivery Xenogen Corporation 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 10 2 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 cm 215 D Image Data Display amp Measurement 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 3 0 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
230. on filter for your application Note The excitation filter selection automatically sets the emission filter position 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 bioluminescent imaging the Open position no filter is automatically selected by default Choose this option to automatically acquire a photographic image 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 Gamma controls see Image Layout Window page 62 Choose this option to take 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
231. on if the settings are for the Properties tab in the FLIT or DLIT tools Planar Spectral Choose this option if the settings are for Properties tab in the Planar Imaging Spectral Imaging tools B 6 3D Analysis i Preferences Tool Palette General User Theme Tissue Properties 3D Analysis gt Image Adjust System IVIS 200 v gt ROI Tools Angle Limit dea Uniform Surface Sampling gt Planar Spectral Imaging C NNLS Simplex Optimization gt Surface Topography Kappa Limits F NNLS weighted Fit _ Point Source Fitting N Surface Limits DLIT 3D Reconstruction voxel Size Limits Analyze Params Properties Results Voxel Size Increment Angle Limit deg 70 o lower upper N Surface Limits Yoxel Size Limits Reto Deak Voxel Size Increment 1 Reconstruction Method TPCB Uniform Surface Sampling C NNLS Simplex Optimization C NNLS Weighted Fit Restore Defaults gt Spectral Unmixing Figure B 8 Set 3D analysis preferences left for the DLIT reconstruction tools right Table B6 3D analysis preferences Item Description system Select the IVIS Imaging System from the drop down list Angle Limit deg The angle between the object surface normal and the optical axis For more details see page 253 Kappa Limits Kappa x is a parameter that is searched during a reconstruction to determine the best fit to the image data Small values of kappa tend to favor deeper sources while
232. on is low w S B Living Image Software User s Manual Y Caliper D 2 Quantifying Image Data Counts Select Counts Photons or Efficiency for the image data The Living Image software can quantify and display scientific image data using three types of measurements Figure D 1 e Counts e Photons e Efficiency for fluorescent images only Data Choose This to Display Recommended For Display Counts An uncalibrated measurement of the Image acquisition to ensure that the photons incident on the CCD camera camera settings are property adjusted Photons A calibrated measurement of the photon ROI measurements on bioluminescent emission from the subject images Efficiency A fluorescence emission image ROI measurements on fluorescent normalized to the incident excitation images intensity radiance of the subject illumination intensity 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 setting
233. on light above the stage or transillumination excitation light below the stage e Analyze bioluminescence images when more than one reporter is used in the same animal model Use the imaging wizard to setup the image sequence required for spectral unmixing For more details on the imaging wizard see page 24 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 and emission filters is sufficiently large for example gt 40 nm so that the excitation light does not leak through the emission filter where it can be detected by the CCD 127 9 Spectral Unmixing 9 1 Spectral Unmixing 1 Load the image sequence In this example 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 2 Inthe Analyze tab of the Spectral Unmixing tools put a check mark next to the emission wavelengths that you want to include in the analysis File Edit View Tools Acquisition Window Help a A H ia amp R Uns Photons v C apply to all te Tool Palette 4 TLT20060406164950_SEQ See E aren Excitation wavelength uns Pons El ese sto J A a om Options Spectrum Results Select W
234. or Measuring Note 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 Alternately 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 a 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 117 7 Image Math 7 3 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 do this To coregister multiple images 1 Acquire an image sequence using the appropriate filters for each reporter Alternately create a sequence from images acquired during different sessions For more details see page 53 2 Open the image sequence ARW20050826124002 SEQ ek Sequence View Note To view all Units Efficieney 4 Use Saved Colors a gga images in the sequence click the Display All button to open each Image overlay mode In a separate Image window 3 Open one of the images
235. ore details on the calibrated FOV positions A E see Table A 3 page 194 Click to move the stage to a position for cleaning the imaging chamber below the stage Only available on the IVIS 200 and Spectrum imaging systems Click to move the stage from the cleaning position back to the home position _ Living Image Software User s Manual Y Caliper Table A 1 IVIS acquisition control panel continued Item Description 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 how to install the XFO 24 lens see the XFOV 24 Lens Instructions IMPORTANT ALERT If you remove the XFO 24 lens from the system be sure to remove the check mark from the XFOV 24 check box Subject height Sets the position of the focal plane of the lens CCD system by adjusting the stage position The subject cm 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 CU IMPORTANT ee ALERT The IVIS System has a protection system to prevent instrument damage however always pay close attention to subject height particularly on the IVIS Imaging System 200 Series For example it is possible
236. ormalized For more details see Quantifying Image Data page 213 If the image is displayed in photons 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 215 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 7 To eliminate the excitation light as a variable from the measurement the data can be displayed in terms of efficiency Figure F 8 v r Y Ca PS cas Living Image Software User s Manual Yo Intensity ome FOV 10 FOV 15 FOV 20 ome fV 25 Width cm Figure F 7 Illumination profiles for different FOVs on an IVIS Imaging System 100 Series measured from the center of the FOV To enable a more jae ARYW20050826124002 001 quantitative Units Efficiency Display Overlay whem comparison of fluorescent signals choose Efficiency e Min 4 43e 5 Max 5 07e 4 Efficiency Color Bar Min 6 86e 5 Max 4 69e
237. ory loaded configuration If you modify a factory loaded configuration save it to a new name To delete a custom table configuration 1 Select the configuration from the User Lists drop down and click Delete 2 Note Factory loaded table configurations cannot be deleted To export the table 1 Click Export 2 In the dialog box that appears select a folder and enter a name for the Tile txt then click Save j ROI Measurements EES a ROI Measurements Image Number Image Layer Subject Subject Labi Subject ID Bkg ROI Total Count Avg Counts A TLT20050624145507_006 ROIS Overlay L ba L Dy 8 522e 05 1 272e 04 Customized Selections Copy Measurements Types Image Attributes ROI Dimensions Counts v none none w Selected To copy the table to the system clipboard e Selected rows Select the rows of interest and click Selected Alternatively select the rows then right click the table and choose Copy Selected on the shortcut menu e All rows Click All Alternately right click the table and select Copy All on the shortcut menu Ti Living Image Software User s Manual Y Caliper F Image Math Using Image Math to Create a New Image naaa e 00 eee eae 112 Subtracting Tissue Autofluorescence o oo eee ee ee es 115 Overlaying Multiplelmages 0 0 0000 eee ee ee ee 118 The Living Image software provides tools that enable you to mathematically combine two images
238. oth Save Results Name SURFACE_2 _ l x Point Source Fitting SS Analysis Params Properties Results Model T ype Transillumination Fluorescence v ngle Limit deg 70 Spatial Fiter 01 e Parameters starting values x 5128 Mes 18151 mm 0140 mm 20628 _ e mm 50 def F yield N mm 2 0 0 z MuaEx 1 cm 0 05 MusEx 1 em 14 28 Jef Restore Defaults Mask No mask w Statistics Weighting LMFtin a MusEmf 1 cm 11 80 ef a gt FLIT 3D Reconstruction f Min 72 buc 2544 84 Mre 45 5 If you want to fix a parameter starting value click the unlocked icon el so that it becomes a closed lock 6 If you want to construct the source only in a region of interest make a selection from the Mask drop down list 7 Confirm the angle limit and spatial filter defaults or enter new values 8 To specify different starting values for the optical properties a Click the Properties tab b Make a selection from the Tissue Properties drop down list c Confirm the internal medium index of refraction or enter a new value 9 In the Params tab click LM Fitting The source appears on the mesh and the Results tab displays the point source fitting results 147 11 Point Source Fitting 11 3 Checking the Point Source Fitting Results 1 In the Results tab click Photon Density Maps 2 Select the image trom the Image sources drop down list
239. otons at the pixel location of the mouse pointer Crop Distance The x y pixel coordinates at the upper left corner of the crop 73 tool OR The x y pixel coordinates at the A end of the distance measurement cursor 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 measurement cursor The width and height of the image crop tool OR Ax Ay from the A to B end of the distance measurement tool Distance The length of the diagonal from corner A to corner B in the image crop tool OR The length of the distance measurement cursor 68 ier 7 Living Image Software User s Manual Y Caliper Use the tools to make measurements in an image and 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 69 associated intensity the image and the intensity counts or photons at that location Histogram Histogram of pixel intensities in an image 70 Line profile Plots a line graph of intensity data at each pixel along a 71 user specified horizontal or vertical line in the image Viewing X Y Coordinates amp Intensity Data 1 In the Image Information tools choose Cm or Pixels from the Units drop down list 2 Put the mouse pointer over the location of interest in the image The x y coordinates and in
240. ou to save the voxels from the active data In Open Inventor format iv File gt Print Displays the Print box File Print Preview ch Displays the Print Preview box that shows what will be printed File 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 Customize tab File Exit Closes the Living Image software 261 J Menu Commands Tool Bar amp Shortcuts Table J 1 Menu bar commands and toolbar buttons continued Toolbar Button Menu Bar Command Description Edit Copy Edit Image Labels Edit Preferences View Tool Bar View Status Bar View Activity Window View Tool Palette View Activity Window View Image Information View ROI Properties View ROI Measurements View Image Layout Window View Acquisition Control Panel Tools gt Image Math for Tools Image Overlay for Tools Colorize xx_SEQ Tools gt Transillumination Overview Acquisition gt Background Replace Acquisition Background View Available Dark Charge Acquisition gt Background Clear Available Dark Charge Acquisition gt Background Measure Acquisition Fluorescent Background Add or Replace Fluorescent Background Acquisition Fluorescent Background Measure Fluorescent Background Acquisition Fluoresc
241. ox 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 216 Adaptive FL Opens the Photo Mask Setup box that enables you to set the photo Background mask for adaptive fluorescent background subtraction For more Subtraction details on adaptive fluorescent background subtraction see Appendix F page 235 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 5 9 For more details on binning see Appendix C page 206 66 Y Caliper Living Image Software User s Manual feSciences Table 5 6 Tool palette Corrections Filtering tools continued Tool Description Smoothing Computes the average signal of the specified number of pixels and replaces the original signal with the average signal Figure 5 9 Smoothing removes signal noise without changing pixel size Note This type of smoothing is defined differently from the smoothing performed in the Living Image software Click this button to return the binning or smoothing to the previous setting and update the image Binning at acquisition 8 no smoothing Binning 2 smoothing 5x5 Figure 5 9 Example of binning and smoothing image data 5 9 Image Information Tools
242. pass 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 5 shows filter transmission curves of a more realistic nature Because the filters are not ideal some leakage undesirable light not blocked by the filter but detected by the camera may occur outside the bandpass region The materials used in filter construction may also cause the filters to autofluoresce 225 F Fluorescent Imaging Fluorescent Filters and Imaging Wavelengths 226 Band Gap 100 gt Excitation a Filter 1 0 mi lransmission 3 ro 0 001 Wavelength nm Figure F 4 Typical excitation and emission spectra for a fluorescent compound The graph shows two idealized bandpass filters that are appropriate for this fluorescent compound Separation 20 nm Excitation Optical Density Wavelength Figure F 5 Typical attenuation curves for excitation and emission filters In Figure F 5 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 IVIS Imaging Systems transmission in the bandpass region is about 0 7 OD 0 15 and blocking outside of the
243. pears when kinetic acquisition begins and plots the Graph maximum intensity signal in each frame The graph provides a convenient way to look for signal trends or select particular frames for viewing Kinetic Acquisition Control Panel Se Display Photograph v 14 frames sec Acquisition Settings Luminescent v Overlay Ima Dynamic Range mage maar Min 111 14 bit B5 Max 15366 Exposure Time msecs 35 S 12000 pinning 10000 4 F Stop 8000 1 6000 EM Gain 100 4000 Excitation Filter Block Emission Filter Open FL Lamp Level Color Bar High Min 111 Photograph Light Level Max 13488 40 7 a 5 2000 Counts C Accumulate Color Scale Auto Minimum Madmum counts 111 Maximum File Size 110 9 MB 6 8 Time secs Time secs 17 05 e Click a point in the graph to view the corresponding image frame e Put the mouse pointer over the graph to view a tooltip that shows the frame number and time e Right click the graph to view the available shortcut menu of graph display options Figure 4 3 Maximum vs time graph 41 4 Acquire Kinetic Data 4 2 Viewing amp Editing Data Kinetic Acquisition window After stopping acquisition you can view the data in the Kinetic Acquisition window 1 To start the playback click the button After playback starts the button changes to m stp To stop the playback click the button To view a particular frame do one of th
244. perform a test acquisition 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 2 Start the acquisition click the button 3 Ifthe photographic image is saturated stop the acquisition click the button and reduce the photograph light level Ti i Living Image Software User s Manual Y Caliper _ CAUTION Extended acquisition of saturated images can shorten the life of the CCD and should be avoided 4 Restart the acquisition If necessary repeat step 3 and step 4 Table 4 1 Kinetic acquisition settings Item Description Acquisition Settings Select the type of data to acquire luminescent or fluorescent from Luminescent v Z Overlay E this drop down list Choose the Overlay option to acquire Dynamic Range photographic images 14 bit v Exposure Time msecs 35 z Binning 4 v F Stop E 1 v EM Gain E 100 v Excitation Filter Block Emission Filter Open v FL Lamp Level High Photograph Light Level 40 7 I Color Scale Auto Minimum 125 Maximum File Size 110 9 MB Dynamic Range 14 bit If this option is chosen the signal intensities range from O to 16384 counts per pixel 16 bit If this option is chosen t
245. quence in the control panel Thumbnail images appear as the images are acquired During acquisition the Acquire Sequence button becomes a Stop button To cancel the acquisition click Stop images wizard ca E C Cela Photog antec Settings Height POUR ine ain h Fie Edit View Tools Acquisition Window Help SAHURA R ee r o ToalRalette aP TLT20050624145507_SEQ TER gt Image Adjust Tool palette gt ROL Tools priate gt Planar Spectral Imaging Units Counts v C Use Saved Colors gt Surface Topography gt DLIT 3D Reconstruction gt Spectral Unmixing Sequence View Thumbnail images 31 3 Acquire an Image or Image Sequence 32 Living Image 3 2 Do you wish to enable auto saving of acquired data For this session Sr This can be changed anytime From the Acquisition menu 3 If this is the first acquisition of the session you are prompted to choose an autosave location 4 To specify a folder for autosaved data click Yes in the prompt and choose a folder in the dialog box that appears All sequences 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 5 Inthe Edit Image Labels box that appears enter information for the image label and click OK Figure 3 1 If you do not want to enter label information click Cancel
246. quences see page 24 You can also set up the sequence manually see page 26 22 Ti Living Image Software User s Manual Y Caliper Table 3 3 Application specific image sequences Analysis IVIS Imaging System Lumina 100 Series 200 Series Spectrum Kinetic Planar spectral image analysis Optional Optional J J Optional Computes the total flux and average depth of a bioluminescent source below the surface Display multiple fluorescent or bioluminescent A A A A J reporters Uses the Image Overlay function to display multiple luminescent or fluorescent images on one photographic image Subtract tissue autofluorescence using blue shifted J J J J J background filters Uses the image math feature to subtract a background image from the primary image Point Source Fitting J J J A A Estimates the optical properties of tissue the location and power of a point source or the fluorescent yield of fluorophores Spectral unmixing Optional Optional J J Optional Removes tissue autofluorescence from a fluorescence image DLIT Analysis d J Reconstructs the surface topography of the subject and the brightness and 3D location of bioluminescent sources FLIT Analysis J J Reconstructs the surface topography of the subject and brightness and 3D location of fluorescent sources Optional with premium filters 3 Acquire an Image or Image Sequence Setting Up a Sequence Th
247. ra background acquired with the camera shutter closed See Appendix E page 217 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 that must be determined for quantitative image analysis Saturation Map Displays image regions that saturated the CCD digitizer in red ROI measurements should not be made on saturated regions ROI measurements made on image regions that do not contain saturated pixels are accurate unless the image is badly saturated Structure A structured light image of parallel laser lines scanned across the subject Available in the IVIS Imaging System 200 Series and IVIS Spectrum The surface topography of the subject is determined from the structured light image Reference A structured light image of a white plate that is acquired and stored on disk prior to instrument installation 3D View A three dimensional rendering of the subject For more details see Appendix H page 247 Export Opens the Export Active View As Image box so that the active image data can be exported bmp jpg png tiff or postscript format Info Click to display or hide information about the image in the image window Living Image Software User s Manual i 11720050624145507_001 DER U
248. re click the arrow and select Auto IVIS Acquisition Control Panel ER Lum INESCENCE Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter imaging settings Imaging settings a E Photog raph IC Field of View System Status imaging settings Idle Acquire eam imaging settings Subject height 1 50 cm Sequence Setup Figure 2 5 IVIS acquisition control panel auto exposure selected Structured light NOTE The options available in the IVIS acquisition control panel depend on the selected imaging mode the imaging system and the filter wheel or lens option that are installed 13 2 Getting Started This page intentionally blank lt a i Living Image Software User s Manual Y Caliper 3 Acquire an Image or Image Sequence Acquire a Bioluminescence Image anaana aa es 15 Acquire a Fluorescence Image With Epi lllumination n anono a 18 Acquire a Fluorescence Image With Transillumination a nononono aaa 19 Acquire an Image Sequence nnana 0 eee te es 22 Manually Setting the Focus 2 42 4 44 e644 eee eS oS EHS OSE HE RHE 34 Manually Saving Image Data naana 34 Exporting Image Data 1 dassos 35 The IVIS Imaging System is ready for image acquisition after the system is initialized and the CCD camera reaches operating demand temperature locked 3 1 Acquire a Bioluminescence Image 1 Start the Living Image software double click the icon on the desktop
249. re 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 illuminated to help determine the source location 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 1 IMPORTANT OOO ALERT Handle the Xenogen High Reflectance Hemisphere by its black base plate while wearing cotton gloves provided by Xenogen 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 Xenogen 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 220 Another source of background
250. rking With ROI Tools 6 1 About ROIs ADO ROR co ob 4 heed rrira iiaa aa aa 81 Sa Mea APEERE EEEREN EENT E EREEREER eee ehbaeneenes 82 Measuring ROIs in an Image naaa ee es 84 Measuring Background Corrected Signal n a aoaaa ee eee eee es 91 Measuring ROls in Kinetic Data 2 0 a e 94 RANI Ge os 6 4 5 E ee CO we OR SO OS 97 Managing the ROI Measurements Table 0 000 eee eee ees 106 A region of interest ROI is a user specified area in an image Figure 6 1 The ROI tools enable you to create three types of ROIs measurement average background or subject ROI Table 6 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 6 1 For more details see Managing the ROI Measurements Table page 106 File Edit View Tools Acquisition Window Help eQaa RSW Units Counts O Apply to all Tool Palette amp TLT20050624145507_006 l gt Image Adjust seo ETT Units Counts Display Overl v info gt Corrections Filtering sli Coun Repay Overlay Info a gt Image Information 7 ROI Tools D i HE Yy x Image Min 48 Max 22447 CI Apply to Sequence ype Measurement ROI Save ROIs ame ROI_2_K5A Delete Load Auto ROI Parameters
251. rm that the surface is in the perspective view click the button or press the R key 3 Choose the Render Organs option Living Image Software User s Manual Y Caliper 4 Select an organ atlas All of the organs in the selected atlas appear on the surface 5 To co register the digital organs and the surface a Click the Fit organs to the mouse button i b On the drop down list that appears click a button to choose an option Rigid registration Performs linear transformation but keeps the shape of the atlas surface A Full registration Performs linear transformation and volume deformation NOTE 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 the A or A tool to automatically fit the organs For more details on manual registration see below 6 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 do not select Skin in the Organs list 7 To clear all organs from the surface click the Deselect All button CE To remove a specific organ remove the check mark next to the organ name 8 Todisplay a specific organ s choose the organ name To display all organs on the surface click the Select All button NOTE If you manually change the location orientation or s
252. roperties 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 bioluminescent light sources in a subject For more details on DLIT analysis see Chapter 12 page 151 and Appendix H page 247 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 bioluminescent sources located deeper inside Information about the depth of the bioluminescent 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 bioluminescent 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
253. ror associated with high radiance measurements Click to begin the point source fitting Make a selection from this drop down list to specify starting values for the parameters other than the defaults Note Selecting a tissue property automatically updates MuaEm MusEm MuaEx and MusEx in the Params tab The internal medium index of the tissue selected from the Tissue Properties drop down list You can also enter a user specitied value Absorption coefficient at the emission wavelength Reduced scattering coefficient at the excitation wavelength Effective attenuation coefficient Muett 3Mua Mua Mus Diffusion coefficient Diff Mua Mus 3 145 11 Point Source Fitting Table 11 1 Tool palette point source fitting continued Item Description X location of the source Y location of the source Z location of the source Starting ChiSqure Ending ChiSqure 11 2 Point Source Fitting X coordinate of the source location Y coordinate of the source location Z coordinate of the source location Error between the measured and simulated photon density at the start of the analysis Error between the measured and simulated photon density at the end of the analysis Point source fitting is performed separately on each image in a sequence 1 Open the image sequence that you want to analyze DAGI Edit view Tools Window Help A X ENO E HH HHHH Sequence View SEO SESS AES ae Units Coun
254. s A TLT20060406164950_SEQ Sequence View Spectra Distribution PCA Biplot PCA Variance Figure 9 5 PCA explained variance 137 9 Spectral Unmixing This page intentionally blank 138 X Caliper Living Image Software User s Manual feSciences 1 0 Generating a Surface Topography Generate the Surface Topography n nonono 00 00 e ee eee eee eee 139 Managing Surfaces on ok eee eee DERE AEH ESSE Cee ESE OGRE EH ES 142 A surface topography surface is a reconstruction of the animal surface derived from structured light images For additional details on the surface topography see page 247 A surface is required for point source fitting and 3D reconstruction of bioluminescent sources DLIT analysis or fluorescent sources FLIT analysis inside a subject 10 1 Generate the Surface Topography 1 Load the image sequence that you want to Tool Palette analyze For details on how to load a sequence Rottools ___ _ e see page 45 2 Inthe tool palette click Surface Topography Surface Reconstruction Structure Light TLT20050624145507_001 3 Click Reconstruct poang The tomography analysis box appears Figure 10 1 E Low Me Loss Recovery Save Results Name SURFACE_1 wt Surface Smoothing Dra regen of imta ectang to proceed Crop box PLT MT _ OO TTS Poe a Mask purple Figure 10 1 Tomography analysis box 4 Draw a crop box that includes a
255. s aoaaa a a a a a 152 3D Reconstruction of Fluorescent Sources aooaa a a 156 DLIT amp FLIT Results 0 a 158 Si Sa eee E eh EE eh oe ee E hoe eee EE r E EE 164 3D Tools Mesh Tab 00 0 a 170 3D Tools Volume TaD 4c ee ek Dae SAE DE wos DESDE DSe DASH HR ESOS 172 3D Tools Organs Tab 2 0 ee 175 3D TOGIS ANIMNGUON TaD coda ce she ba rrinin AEG HES EERE EOS KES 182 Managing DLIT FLIT Results 0 0 0 cee ee 187 The Living Image software includes algorithms for 3D reconstruction of bioluminescent or fluorescent sources tomographic analysis e Diffuse Tomography DLIT algorithm For tomographic analysis of bioluminescent sources Analyzes a bioluminescent image sequence obtained on the IVIS Imaging System 200 or IVIS Spectrum e Fluorescent Tomography FLIT algorithm For tomographic analysis of fluorescent sources Analyzes a fluorescent image sequence obtained on the I VIS Spectrum imaging system The steps to perform 3D reconstruction of bioluminescent fluorescent sources include 1 Acquire an image sequence 2 Generate the surface topography surface of the subject 3 Set the user modifiable DLIT FLIT algorithm parameters for example analysis wavelengths source spectrum and tissue properties and reconstruct the position geometry and strength of the luminescent sources For more details on the DLIT FLIT algorithm see Appendix H page 247 151 12 3D Reconstruction of
256. s Cy5 5 Background 580 610 Uses same as Cy5 5 Cy5 5 Alexa Fluor 660 Alexa Fluor 680 Name Excitation Passband nm Emission Passband nm Dyes amp Passband GFP 445 490 GFP EGFP FITC DsRed 500 550 DsRed2 1 PKH26 CellTracker Orange Cy5 5 615 665 Cy5 5 Alexa Fluor 660 Alexa Fluor 680 ICG 710 760 Indocyanine green ICG GFP Background 410 440 GFP EGFP FITC DsRed Background 460 490 DsRed2 1 PKH26 CellTracker Orange Uses same as Cy5 6 UsessameasICG ICG Background 665 695 Uses same as ICG Indocyanine green ICG F 3 Working with Fluorescent Samples Tissue Optics Effects 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 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 excitation 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 6 The excitation light also causes the tissue to autofluoresce The amount of autofluorescence depends on the intensity and wavelength of the excitation source and th
257. s e ROI ROIs applied to the sequence are available in the drop down list Select an ROI to analyze just the area in the ROI This option is recommended for well plates or dark objects where it is hard to achieve a good mask from the photograph 5 In the tool palette click Unmix Spectra The spectral unmixing results appear The Distribution tab shows a photon density map of each unmixed result and a composite image that includes all of the fluorescent signals each displayed in a different color TLT20060406164950_SEQ Sequence View Spectra Distribution Image Adjust Show Labels Unmixed 1 Autofluorescence Unmixed 2 Composite Unmixed 1 Unmixed 2 Fluorophore signal 129 9 Spectral Unmixing 130 6 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 Pi KSA20001014141047_0 Tage Adast Corrections Filtering Image Inbortnel ion ROI Tools C Sequence Views sprata Distribution Corganitr Image adst E Show Labets To view the composite image separately double click the composite image The Composite tab displays the composite image This enables you to export print or copy the composite image to the system clipboard Item in the Description Concentration Plot Clic
258. s 1 Right click the results table and select Export Results from the shortcut menu that appears 2 Inthe dialog box that appears choose a folder for the results enter a file name txt and click Save Tool Palette ROI Depth tmm Total Flux phot s ROIZ 2 341 0 269 2 66e7 5 18e6 Iil Plot Linear Fit Plot Intensity Epot Save Results Splm_2 wt gt Surface Topography gt Point Source Fitting gt BLIT 3D Reconstruction Tool Palette Analyze Properties Results ROI Depth imm Total Flux phot s ROIZ 2 341 0 269 2 66e7 5 18e6 lt ii gt Plot Linear Fit Plot Intensity Save Results Mame Spin_2 wt gt Surface Topography gt Point Source Fitting gt BLIT 3D Reconstruction Ti i Living Image Software User s Manual Y Caliper 9 Spectral Unmixing Speca WWI 6 gs hw Bes 44 OS OHH OS 6 eS EES Ee eK ERE eee 128 Spectral Unmixing Results Window 1 0 2 es 131 Spectral Unmixing Parameters 2 0 0 0 eee te es 132 Spectral Unmixing Options a ee ee 134 The Living Image software uses spectral unmixing to distinguish the spectral signatures of different fluorescent or bioluminescent 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 excitati
259. s 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 SILT e00S0b 2414550 04 Le Coui Dede Dieta we i irf a In counts mode the ROI measurements include 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 Figure D 2 Image window and ROI Measurements table counts mode 213 D Image Data Display amp Measurement Photons 214 When image data is displayed in photons the photon emission from the subject or radiance is displayed in photons sec cm2 sr Counts are a relative measure of the photons incident on the CCD camera and photons are 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 Surface Radiance Tissue gt Q Solid Angle units of steradians Figure D 3 Isotropi
260. s Copy Measurements Types Image Attributes _ ROI Dimensions z Counts v _none_ w _none_ v Selected Refresh 1 To reorder the columns drag a column header left or right in the table 2 Tochange the measurement units make a selection from the Measurement Types drop down list 3 To include image information in the ROI table make a selection from the Image Attributes drop down list 4 To include ROI dimensions in the table select units Pixels or cm from the ROI Dimensions drop down list Living Image Software User s Manual v Y Ca Be cas To create amp save a custom table configuration 1 In the ROI Measurements table click Configure The Configure Measurements box appears Configure Measurements User Lists Customized Unsavec v Name Available Items Analysis Comment Analysis User ID Angle Selected Items Total Counts Avg Counts Stdey Counts Add gt Click to reorder the available items in Animal Model Animal Number Animal Strain Max Counts ROI Pixels Move Down Areatccd Pixels xcipixels Ycipixels widthipixels Heightipixels User ID Sequence ID Date and Time Binning Exposure Field of View ascending or descending alphabetical order Ss Area cm2 Avg Dark Charge Counts Avg Efficiency Avg Fluorescent Bkg Counts Avg Radiance p s cm2fsr Cell Line Comment Commenti Commentz Cosmic Corrected
261. s i oi ffo 4 l roo Tool aa palette abe Color bar i T mip Pls x Sanh me iis ce i i Looe i a 7 H Tii In the color bar check the image min and max to determine whether the signal of interest is above the noise level and below CCD saturation For 16 bit imaging a signal ranging from 600 60 000 counts is recommended for 14 bit imaging 600 16 000 counts are recommended If the signal level is unacceptable adjust the exposure time or binning level For more details on the image window see Table 3 1 page 18 Edit Image Labels UserID kS LabelSet PANTE Check any 5 Fields For display User Group Experiment Edit the label information here Comment1 Comment2 C Time Point C Animal Number C Animal Strain C Animal Model O Sex C view C Cell Line C Reporter C Treatment Fi Luc Injection Time C IACUC Number Apply To Sequence Edit Image Labels box Figure 3 1 Image window amp Edit Image Labels box 17 3 Acquire an Image or Image Sequence Table 3 1 Image window Item Description Units Select the measurement units for the image display counts photons or efficiency from this drop down list Display Select the image type for example overlay that you want to display from this drop
262. s 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 221 E Luminescent Background Sources amp Corrections 222 J _ Photograph Luminesce Photograph Luminescent nt overlay overlay Figure E 2 Background light emission Background light emission from a female white furred Swiss Webster left and a female nude Nu nu mouse right 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 Xenogen technical support a i Living Image Software User s Manual Y Caliper Appendix F Fluorescent Imaging Description and Theory of Operation a a a eee eee 223 Fiter SpE s 424 6 8 848 SEES E SHES HEADER A HEH EDR ERED 225 Working with Fluorescent Samples 00 200 eee eens 227 Image Data Display 2 a 228 Fluorescent Background 4486446684668 68 He we Dw ew es 229 Subtracting Instrument Fluorescent Background 235 Adaptive Background Subtraction 0 0000 ee eee eee 235 Subtracting Tissue Autofluorescence Using Background Filters 236 F 1 Description and Theory of Operation System Components The IVIS Imaging System 200 Series and IVIS Lumina offer built in fl
263. s the dimensions of the image window Restore Defaults Click to apply the default settings Apply Individual Color Choose this option to apply a separate color scale to each Scale for Sequences thumbnail of a sequence If this option is not chosen all of the thumbnails are displayed using the same color scale Show Transillumination Choose this option to display a cross hair at each transillumination Locations location when you load transillumination data When you mouse over across 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 chosen the tool palette includes the point source fitting tools and the image window includes the Colorized View tab Show Activity Window on A drop down list of options for when to display the activity log Figure B 2 Save Settings 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 a in the image window to view the current folder path setting Figure B 2 196 ad Living Image Software User s Manual Y Caliper Table B1 General preferences continued Item Description Window Size amp Applies the ac
264. s v Display overlay a nea ROI 3 BKG 2 9 709e 04 Image Ti Min 64 Max 44260 4 BKG 1 1 042e 03 P s a 2 i oe f S p gt k H gt 2 w at b ROT 2 BKG 1 4 742e 05 5 re Rotate ROI 1 BKG 1 2 358e 06 4 Copy ROI Copy All ROIs Duplicate ROI Counts Hide ROI Tag Color Bar Delete ROI Min 2213 Delete All ROIs Max 36749 Ppa a Lock Position ROI Properties Ex ROI BKG 2 ROI Label BKG 2 BkgROI SubjROI Info Use as BKG for Future ROIs in JJH20050630142125_006 Entire sequence C Lock Position xc pix 198 13953 ci pix 132 95681 Angle deg 0 0000 C Lock Size width pix 23 12292 Heightt pix 21 92691 Line Size 2 ue coor BE C 93 6 Working With ROI Tools 6 5 Measuring ROls in Kinetic Data Quick Guide Drawing a Kinetic ROI 94 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 a result kinetic ROIs are continuously displayed during kinetic data playback You can draw a kinetic ROI using any of the methods or shapes in Table 6 1 page 82 NOTE Large kinetic data sets may require more time to create plot and measure the ROIs because the software first appli
265. s with large voxels specified by the voxel size limit the length of a side of the voxel cube in mm At each iteration the algorithm reduces the size of the voxel by a factor of two until the optimum solution is found The voxel size limits are a minimum of five and a maximum of 10 The default range is set to 6 9 mm A larger range of voxel limits ensures a more reliable solution but requires more computational time Voxel Size Increment DLIT This is the step increment in voxel size stepping from the minimum voxel size limit to the maximum voxel size limit For example if the voxel size limit ranges from 6 9 mm a voxel size increment 1 gives four starting voxel sizes 6 7 8 and 9 mm The default increment of 1 mm is usually adequate however smaller increments can be used if you want to sample finer voxel sizes Smaller increments will significantly increase the time required for reconstruction Ti i Living Image Software User s Manual Y Caliper Background FLIT Choose this option to take the background fluorescence for example autofluorescence or non specific probe in circulation into account Background fluorescence and fluorophore emission contribute to the photon density signal at the surface The background fluorescence signal is modelled in order to isolate the signal due to the fluorophore only where an average homogenous tissue background fluorescence yield is determined empirically Background fluores
266. scence adaptive background subtraction 235 reconstruct 3D sources 156 157 fluorescence efficiency 228 229 fluorescence image epi illumination 18 19 fluorescence image trans illumination 19 22 fluorescence imaging components 223 225 fluorescent filters 226 fluorescent imaging efficiency 225 f stop 228 normalization 225 focus manually 34 FOV settings 194 G graphic image data 211 H High Reflectance Hemisphere 219 histogram 70 image adjusting appearance 63 cascade 58 correct or filter 66 67 correcting filtering tools 66 67 histogram 70 image window 55 56 information 59 label 61 line profile 71 magnify or pan 65 measurements 72 pixel data 69 tag 58 thumbnails 51 tile 58 image analysis tools 8 image data browse 45 46 colorize 74 75 export 35 open 47 49 save manually 34 image information 67 68 Living Image Software User s Manual image layout window 62 63 image math 112 115 image overlay tool 118 120 image sequence 51 53 acquire 31 32 analyses 8 application specific 23 create from individual images 53 54 edit 29 31 52 53 imaging wizard 24 26 manual setup 26 28 image window 17 3D perspective 169 display modes 56 image window single image 55 56 imaging amp image analysis example workflow 7 overview 5 8 imaging wizard 24 26 index of refraction 159 information about an image 59 infusion pump 257 infusion syringe injection system control panel
267. se as BKG for future ROIs in 2 To set ROI position enter new Xc pix and Yc pix TLT20050624145507 006 values in the ROI Properties box Entire sequence 3 To rotate the ROI clockwise enter the degrees in the Angle deg box and click outside the box C Lock Position 4 To lock the current ROI position choose the Lock Position option Xe pix 120 47356 e pix 109 36069 Note The RO position cannot be changed until the Angle deg 0 0000 Lock Position option Is cleared C Lock Size Width pix 17 49013 4 Height pix 17 04815 3 Position of the ROI selected in the image Line Size 2 4 ure coor I C Living Image Software User s Manual ae y Caliper Editing ROI There are two ways to resize a circle or square ROI Dimensions 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 j 11120050624145507_006 Units Counts Display Overlay w 1 Select the ROI and place the pointer over a handle mi on the ROI 2 When the pointer becomes a E arrow drag the handle To resize an ROI using the ROI Properties box di ROI Properties ER 1 Double click the ROI in the image RoI ekai ss The ROI Properties box appears and displays the Ro label sks
268. 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 and structured light image e Emission filter 2 Luminescent image Table 12 1 Recommended image sequence for DLIT analysis Image Type Emission Filter Options Photographic J Select the Reuse Photographs option in the control panel Structured light J Luminescent J J The binning level must be the same for all of the luminescent images After the surface is generated the 3D reconstruction of the light sources can proceed Figure 12 1 shows example results For more information on the DLIT algorithm and user modifiable parameters see Appendix H page 247 Living Image Software User s Manual Y Ca li Re as 1 In the tool palette click DLIT 3D Reconstruction 2 In the Properties tab make a selection from the Tissue Properties and Source Spectrum drop down lists Muscle is usually the best choice of tissue properties for general in vivo reconstructions Note The internal medium index of refraction is automatically entered when you select a tissue as 3 If you want to view the Tissue Properties u Mor Us Or Source Spectrum for the tissue and light source selected above make a selection from the Plot drop down
269. simulated 160 photon radiance 214 photons 214 pixel 211 pixel data 69 planar spectral analysis optimizing precision 245 planar spectral image analysis 121 123 planar spectral imaging 239 245 diffusion model 240 intensity graph 125 linear fit graph 125 luciferase spectrum 241 optical properties 241 results 124 tools 123 124 point source fitting 143 147 results 148 149 tools 143 146 preferences 195 203 pseudocolor image 211 R radiance photon 214 radiance units 214 read bias 217 reconstruct 3D fluorescent sources 156 157 reconstruct 3D luminescent sources 152 155 reduced Chi2 159 ROI 81 automatically draw 88 89 background corrected signal 91 93 delete 105 edit dimensions 101 edit position 101 free draw 90 managing 97 268 manually draw 86 87 measurement ROI free draw 90 measurement ROIs 84 89 Measurements table 86 move 100 move or edit label 103 quick guide 84 ROI line 102 save 104 subject 90 tools 82 83 ROI kinetic quick guide 94 95 ROI Measurements table 106 108 configure 108 110 copy or export 110 ROI properties 97 99 ROI types average background 82 91 measurement 82 subject 82 S save data manually 34 ROI 104 kinetic data 44 43 scientific image data 211 segment 29 smoothing 67 208 source spectrum 160 spectral imaging See planar spectral imaging spectral unmixing 127 130 options 134 135 parameters 132 133 PCA biplot 136 PCA explained varianc
270. software provides preset key frames or you can specify the 3D views for the key frames 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 Click to create a new key frame from the current 3D view Click to update the selected key frame to the current 3D view Click to delete a selected or all key frames from the key frame box Click to move a selected key frame up in the key frame box Click to move the selected key frame down in the key frame box The total time of the animation sequence Click to view the animation sequence defined by the current key frames and animation parameters Displays a dialog box that enables you to save the current animation to a movie mov mp4 or avi Displays a dialog box that enables you to open an animation setup xml Displays a dialog box that enables you to save the current key frames and animation parameters to an animation setup xml 183 12 3D Reconstruction of Sources Viewing a Preset Animation 184 To view a preset animation 1 2 File Open the DLIT FLIT results
271. specified 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 To remove a tag 1 Position the pointer over the tag 2 Right click the image and select Remove Tag on the shortcut menu 3 Toremove all tags right click the image and select Remove All Tags on the shortcut menu To move a tag 1 Position the 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 Aline between the pixel and the tag identifies the location associated with the tag When multiple image windows are open you can organize them in a cascade or tile arrangement i a m tka a oe To tile the open 5 image windows choose Window gt Tile on the menu bar ce m m jm i Living Image Software User s Manual Y Caliper e amp ww je a UM S202 amp OS moam Dawns To organize the image windows in a cascade choose Window Cascade on the menu bar 5 5 Viewing Image Information At acquisition the software captures image information that includes all of the text information that is associated with every image for example camera parameters and user labels Click Info to display the label set information and acquisition information for the image Flo Ei Viwe Tob Aqhikn Wede Help Sat AG WM iunsicons O aptos
272. t light sources includes e A surface mesh 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 A 1 Table A 1 IVIS System filters for bioluminescence amp fluorescence tomography IVIS Imaging System Filters Bandwidth nm 200 Series 6 emission filters 550 670 nm 20 Spectrum 10 excitation filters 415 760 nm 20 18 emission filters 490 850 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 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 is 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 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 vox
273. te 1 Open a sequence acquired in fluorescence transillumination mode 2 Select Tools gt Transillumination Overview for xx SEQ on the menu bar The overview appears wo xy lad R Ey 3 W Lints Carts Apply toal v ILIT 2D Reconstruction Sequence view Transillumination overview v Y Ca POT n Living Image Software User s Manual 5 12 Viewing amp Editing 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 File Edit view Tools Window Help oN H la amp gr Untsi Photons O Apply to all j DA20081107162612 SE Units Photons Display Overlay a i a G eee Photo Adjustment Brightness i Gamma J Opacity Color Scale 7 38e9 Max 5 43e9 Color Scale Limits Auto Full Manual Individual 6 0 Color Table YellowHot i C Logarithmic Scale C Reverse gt Corrections Filtering gt Image Information 40 x10 gt ROI Tools Current image 2 0 pisecjem zjsr Color Bar Min 5 84e8 Max 7 38e9 Current image number top slider position To select a particular image enter a new number or move the top slider I 26 Bl om C Accumulate m T m a E Ma End frame image in the selected data Start frame image in the m Use the bottom sliders to select a range of data for viewing or export range
274. te commands The specified ROIs are deleted from the image Note his does not delete ROIs saved to the system global save Tool Palette E Image Adjust sd Image Adjust gt Corrections Filtering gt Image Information ROI Tools Oo r Y X I C Apply to Sequence r ype Measurement ROL wt Save ROIS ame ROT 1 E54 Auto ROI Parameters hreshold F s Preview Use Bkg Offset Replace ROIs To permanently remove ROls from the system Save Load SavelLoad Auto ROT Parameters Select the ROI s that you want to delete from the f drop down list of saved ROIs vette C e eee 2 Click Delete _ 105 6 Working With ROI Tools 6 7 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 213 To view the ROI Measurements table click the button Alternately select View gt ROI Measurements on the menu bar Tool Palette Column headers in the table include ROI information ROI measurements and dimensions and information about the gt Corrections Filtering image recorded at acquisition gt Image Information ROI Tools oO oy C Apply to Sequence ype Measurement ROI Save ROIs ame ROI_1_KS5A P ROI Measurements
275. tensity Vs Lambda E E Fine faa Intensity vs Lambda Plot for ROI 1 600 Wavelength nrm The 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 To export graph data 1 Click the Export Data button H 2 Inthe dialog box that appears select a directory for the data and enter a file name CSV The data can be opened in a spread sheet application such as Microsoft Excel 125 8 Planar Spectral Image Analysis 8 4 Managing Planar Spectral Imaging Results 126 To save results 1 Select the results of interest Splm_xx from the Name drop down list 2 Click Save The planar spectral imaging results are saved with the image To view results 1 Select the results of interest 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 of interest and select Copy Selected 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 select Copy All from the shortcut menu that appears All of the results table is copied to the system clipboard To Export Result
276. tensity data are displayed in the tool palette Note The information is updated when you change the pointer position File Edit View Tools Acquisition Window Help a H A amp R Uns Counts v C Apply to all a tn An _ a Tool Palette _ amp 117 20050624145507_006 Sl Units Counts Display Overlay v i ws foni Boer pere B ee f lan Of fr P E Units cm Image ee e A Image Binning 8 Width 12 6cm Height 12 6 cm Min 48 Image X Y 5 878 7 181 om Max 22447 Image Data 22447 counts Crop Distance Bi 0 00 0 00 por 0 00 0 00 Distance 0 00 Counts Color Bar Min 1122 Max 19546 69 5 Working With Data Viewing an Image The image histogram plots a frequency distribution of the pixel intensities in an image Histogram The software sorts the intensities into groups or bins x axis and plots the number of pixels per bin y axis Tool Palette L gt Image Adjust si Image Adjust Hia lO 7 E g gl Units cm lt Image Binning 2 To display the image histogram 1 Open an image 2 In the Image Information tools click the Image Histogram button las Width 12 6 cm Height 12 6 cm Image X Y 2 778 11 424 cm Image Data 2 counts j Histogram Window Ful MinBin 1122 MaxBin 19546 Bins 512 Fey amp TLT20050624145507_006 Overlay Note By default the Auto min max range of t
277. terior view Living Image Software User s Manual fl Preferences General User Acquisition Theme Tissue Properties 3D Analysis Auto Exposure Camera Settings Luminescent Fluorescent Auto Exposure Preferences First Preference Second Preference Third Preference Target Max Count Luminescent 600 Exposure Time YW F Stop v None v Fluorescent 4000 Range values Exp Time sec Binning 60 Max Restore Defaults Chk on Duos tuton to proceed aE F Sequence ser oe Start the Living Image software In the control panel Confirm the default user preferences or specify that appears initialize the IVIS imaging system new preferences A IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop EM Gain Excitation Filter Emission Filter Loo fee ream vp vor vis a 7 Field of view D System Status Idle Acquire 12 5 cm Subject height 1 50 cm Sequence Setup Specify acquisition settings for a single image image Acquire the image s or kinetic sequence sequence or kinetic sequence p s Jab amp l mee ii D me hE ie k i eLAta Aa Ww o OC tenance Yew ieee eet TF ae Carte Ue Pore Saved a gi a awa cE Piana bainan TE im d Eh ai Tool eg S palette View the image s or kinetic data Analyze an ima
278. the IVIS imaging system acquires a photographic and fluorescence image that are used to generate an overlay image Table 2 1 Images used to generate an overlay image Image Type Description Photographic A short exposure of the subject illuminated by the lights located at the top of the imaging chamber Figure 2 2 The photographic image is displayed as a grayscale image Bioluminescence A longer exposure of the subject taken in darkness to capture low level bioluminescence emission The bioluminescence image is displayed in pseudocolor that represents intensity Figure 2 1 For more details on bioluminescence image data see Appendix D page 211 Fluorescence An exposure of the subject illuminated by filtered light The target fluorophore emission is captured and focused on the CCD camera Fluorescence image data can be displayed in units of counts or photons absolute calibrated or in terms of efficiency calibrated normalized For more details on fluorescence image data see Appendix F page 223 Figure 2 3 shows an example workflow on an IVIS Imaging System 2 Getting Started i Photographic image Bioluminescent image Overlay image Figure 2 1 Example bioluminescence overlay image The Living Image software automatically coregisters the photographic and bioluminescence images to generate the overlay image Illumination LEDs Camera lens opening Sample stage Figure 2 2 IVIS Imaging System 100 Series in
279. the ceiling of the imaging chamber Figure F 1 The reflectors provide a Ti Living Image Software User s Manual Y Caliper F 2 Filter Spectra 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 215 The emission 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 bioluminescent imaging IVIS Imaging System Number of Emission Filter Number of Available Wheel Positions Fluorescence Filters 200 Series 24 two levels each with 12 22 60 mm diameter positions Lumina 8 7 100 or 50 6 5 75 mm diameter 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 4 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 band
280. timum solution NNLS Weighted Fit TRUE the option is chosen and the DLIT or FLIT algorithm weights the wavelength data inversely proportional to its intensity in the NNLS reconstruction FALSE the option is not chosen Min Radiance The wavelength image data minimum radiance photons sec cm 2 sec to use in the DLIT or FLIT analysis Index of Refraction The internal medium index of refraction that is associated with the user selected tissue 159 12 3D Reconstruction of Sources Viewing Photon Density 160 Table 12 3 3D reconstruction results continued Item Description Tissue Properties The user specified tissue in which the sources should be located Source Spectrum The emission spectrum of the type of bioluminescent source Photon Density Maps Simulated The photon density computed trom DLIT FLIT source solutions which best fit the measured photon density see page 161 Measured The photon density determined from the image measurements of surface radiance Wavelength The wavelength of the photon density map in the active image Source Image The image number of the transillumination source image Photon Density Maps Click to open the Photon Density Maps window Save Results Name The default name for the active DLIT or FLIT results Delete Click to delete the selected DLIT or FLIT results Load Click to load the selected DLIT or FLIT results Save Click to s
281. tion parameters For more details on the acquisition parameters see Table 4 1 page 39 37 4 Acquire Kinetic Data Kinetic Acquisition Control Panel Display 13 frames sec After acquisition choose the type of data to display Acquisition Settings Luminescent Overlay Dynamic Range 14 bit x 35 Exposure Time msecs 5 Binning 4 v F Stop 2 v EM Gain 50 v Select the type data to acquire luminescent or fluorescent To adjust a setting using 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 Excitation Filter Block Emission Filter Open FL Lamp Level High Photograph Light Level 51 6 gt 4 C Accumulate Play Color Scale Auto Minimum 0 Maximum No data available 0 File Size Nia Figure 4 2 Kinetic Acquisition window Accumulated Signal 38 5 Click the button to start acquisition After acquisition begins the button changes to a m stp The Maximum vs Time graph plots the maximum signal in each data frame For more details on the graph see page 41 6 To stop acquisition click the button The Accumulate option enables you to view increasing signals in real time If you plan to accumulate signals it is recommended that you
282. tive image window size and position settings to Position subsequently opened image data Most Recently Used Applies the active image window size and position settings to Dataset History subsequently opened image data Display ROI Label As Measurement Photons Select the type of measurement in photons to show in the ROI label Counts Select the type of measurement in counts to show in the ROI label Some of the general preferences specify how the main application window is organized Tool palette E W DE a ee ee suas A h iscan D a a peers bi ae Activity window hidden by default Figure B 2 Main application window 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 of the window and release 197 B Preferences B 2 User Preferences Preferences General User Acquisition Theme Tissue Properties 30 Analysis User Setting s Existing User ID New User ID Delete User ID Preferences Defaulks Label Set Menogen Universal Edit User Label Choices Figure B 3 User preferences Table B2 User preferences Item Description User s Settings Existing UserID 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
283. to a movie ile 7A In the image window click the 4 button Figure 5 12 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 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 5 Working With Data Exporting an Image 1 To select an image move the frame slider or enter a frame number in the spin box from a Kinetic Data Set E DA2008110716261 Unis Photors Display Overlay kio Export Graphics button ofa Accumulate 7 231 a H Extract Extract Acoundated Ina Extract Krete Stream Spin box Figure 5 13 Image window selecting an image for export 2 Click Extract and choose Extract Current Image Anew 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 appears select a destination folder enter a file name select a file type and click Save 80 ier 7 Living Image Software User s Manual Y Caliper 6 Wo
284. to create a new image The primary use of image math is to subtract tissue autofluorescence background from signal Living Image Tool Use This Tool To See Page Image Math Mathematically combine add multiply subtract or divide 112 two user specified images Image Math Remove autofluorescence from a fluorescent image 115 Image Overlay Coregister multiple fluorescent or luminescent images on the 118 same photographic image to view multiple reporters in a single image To perform image math open an image sequence see page 47 or a group of images see Creating an Image Sequence from Individual Images page 53 111 7 Image Math 7 1 Using Image Math to Create a New Image To create a new image using image math 1 Load an image sequence File Edit view Tools Acquisition Window Help ao A H A amp R nts ficiency v C Apply to all Tool Palette xj j 11720060510114512 SEO gt Image Adjust d Sequence View 7 ROI Tools its Efficiency C Use Saved Col oon ay Units Efficiency Z Use Saved Colors ype Measurement ROI Save ROIs lame ROL_1_KSA Delete Load Save Auto ROI Parameters hreshold J os Lower Limit E 0 0 Minimum Size 20_ S Preview Use Bkg Offset Replace ROIs Min 6 55e 6 Max 1 24e 4 92 Restore Defaults Save f Load gt Spectral Unmixing Min 7 84e 7 Max 1 50e 5 112 ad Living Image
285. to display the associated 3D view and time stamp _ J Frames Per Second position in the time scale 0 100 at 222a 6e which the frame occurs in the animation Perspective Figure 12 11 Animation tab 182 Living Image Software User s Manual n Y Ca Ber ce Table 12 9 3D tools Animation tab Item Description The time stamp of a key frame in the animation on a time scale of 0 100 Time Scale Presets Key frame Preset Key Frame Factor H HR S A Gag Total Duration Play Record Animation Setup Load Save 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 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 A drop down list of predefined animation setups 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
286. to recognize the different types of autofluorescence The following examples illustrate sources of autofluorescence including microplates other materials and animal tissue 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 9 shows autofluorescence originating from four different plastic microplates The images were taken using a GFP filter set excitation 445 490nm emission 515 575nm 60 405 White polystyrene Clear polypropylene a pipeedemw rer me i Ca i am e in i Rees A z Clear polystyrene Black polystyrene Figure F 9 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 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 10 The specular reflection is an optical illumination autofluorescence signal reflecting from the microplate surface and is not dependent on the microplate material ad Living Image Software User s Manual Y Caliper Specular Reflection Specular Reflection Spe
287. tools Figure 5 8 to subtract background or apply corrections to the image data For more details on sources of background see Appendix E page 217 You can also apply smoothing and soft binning to the image data For more information on binning and smoothing see Appendix C page 205 Tool Palette gt Image Adjust Corrections Filtering E Read Bias Subtraction and Flat Field Flat Field Correction Correction are default mandatory aera eater corrections In photons mode In counts Pae ar an mode these corrections can be cleared Binning Smoothing None Figure 5 8 Tool palette Corrections Filtering tools Table 5 6 Tool palette Corrections Filtering tools Tool Description Read Bias Subtraction Select this check box to subtract dark background from the image Dark Charge data lf a dark charge image is available for the imaging conditions the Subtraction dark 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 217 Note In photons mode dark background subtraction is a mandatory default In counts mode the check box can be cleared 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 216 Note In photons mode flat field correction is a mandatory default In counts mode the check b
288. top 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 a e Living Image Software User s Manual Y Caliper _ 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 infusion when acquisition stops even though the pump may not be stopped 868 e amp 8 iy HE Tas Be w Man ots l Time secs 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 209 IVIS Syringe Injection System This page intentionally blank 260 Ti Living Image Software User s Manual Ww Caliper Appendix J Menu Commands Tool Bar amp Shortcuts ae AA i A amp hk Figure J 1 Living Image toolbar Table J 1 Menu bar commands and toolbar buttons Menu Bar Command Toolbar Description Button a Lair File Open Displays the Open box so that you can select and open an image data Tile Displays the Browse For Folder box so that you can select and an im
289. ts C Use Previously Saved Colors 7 Transillumination Location Mi gt Surface Topography B e EN Analysis Params Properties Results Sequence CX200702227839972_ SEQ Tissue Muscle Source MA Select Single Image Source Image EswL EmWL MinRadiance Gam 720 2 44e 07 6 2 675 720 8 70e 06 675 720 2 82e 06 EG 4 675 720 2 82e 06 FE rs gt FLIT 3D Reconstruction Min 926 Muc 51284 92 Mae 18151 Iim 132 Min 72 Mime 45 2 In the Analysis tab select an image in the sequence 3 In Surface Topography tools generate or load a surface For more details on generating the surface see page 139 Note t is recommended that you use the smoothing tool to generate a good quality surface 146 ad Living Image Software User s Manual Y Caliper 4 Click the Params tab The default starting values for the source location power and tissue optical properties are displayed Note The software automatically selects the correct model type for the image data D File Edit View Tools Window Help Xx ene axa Sequence View oImage Adiust O Units Counts gt Use Previously Saved Colors Transillumination Location A sg Surface Topography B man Surface Reconstruction Structure Light CK20070222183912_001 Path Averaging Size Auto Smoothing Level Low x Restore Surface Smoothing Loss Recovery Height v Smo
290. ubes v Threshold Intensity 1 00e 4 Color Table BlackRed 3 x C Reverse C Logarithmic Scale Source Voxel Measurement Vox e Fluorescence Yield 6 572 5 A mm zZ J 22 6 m l liim l volume 0 2441 mn F d ata Center of Mass 4 1667 16 7667 12 1333 Host Organ skin Export voxels Center Of Mass E Coronal sagittal amp transaxial views Figure 12 9 Example 3D reconstruction FLIT results 3 Repeat step 2 to display data for other voxels The voxel data is updated 4 To clear the voxel data click any where in the 3D view window To measure the depth of a source s 1 Click the Measure Voxels button Ea 2 Click a voxel or draw a box around a group of voxels 3 Click Center of Mass The Coronal Sagittal and Transaxial views display the intersection through the 173 12 3D Reconstruction of Sources voxels center of mass A Click the Measurement Cursor button The Sagittal and Transaxial views show the distance between the voxel s and the dorsal or ventral surface Figure 12 9 5 To measure another distance drag each end of the measurement cursor to anew position The distance measurement is updated Viewing X Y 1 In the Coronal Sagittal or Transaxial Coordinates windowpane click the position of interest The x y coordinates mm of the position are displayed 2 Ifyou drag the mouse cursor the coord
291. uch as the light emitting diodes LEDs of electronic equipment some materials contain phosphorescent compounds af B Living Image Software User s Manual Y Caliper _ Do not place equipment that contains LEDs in the imaging chamber Phosphorescence is a physical process 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 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 1
292. uded in the ROI if the pixel intensity is greater than the threshold a user specified percentage of the peak pixel intensity Draw line segments that define the ROI 90 Quick Guide Drawinga These steps provide a quick guide on how to apply a measurement ROI to an image or Measurement ROI on image sequence For more details about measurement ROIs see page 84 an Image or Image Sequence 1 Open an image or image sequence and click ROI Tools in the tool palette Ti Living Image Software User s Manual Y Caliper 2 In the ROI tools select Measurement ROI from the Type drop down list 3 Click the Contour button For an image or image sequence select Auto All from the drop down list For kinetic data select Kinetic ROI The software automatically draws measurement ROIs on the image The ROI label shows the total intensity in the ROI If you are working with a sequence open an image to view the intensity label For more details see page 84 j 11120050624145507_006 Sle aa Tool Palette oO lw Apply to Sequence Image Min 48 Max 22447 ype Measurement ROL ree Measurement ROI ame overage Bkg ROT Subject ROI Delete mn Toa ROI 1 1 788e 06 Auto ROI Parameters hreshald aooo 110 il 4 f itis necessary to adjust the ROI boundaries change any of the auto ROI parameters use the slider or 4 arrows Ror 2 5 172e 05 Counts Color Bar Min 1122 Max 19546 e Threshold
293. ult position and size click Reset and the w7 Dutton 9 To turn off the transform tool click the button Circle line is thicker when selected y Lise Ta kato aO Use X Y or Z keys to restrict scaling to only one aris Scaling On Z 179 12 3D Reconstruction of Sources 180 To check the organ and surface alignment 1 Check the organ position in the Coronal Sagittal and Transaxial windowpanes Cseaunce view E Dve 2 Inthe 3D View tab click in the windowpane with the surface Bottom Front Back Left Right dl B Living Image Software User s Manual Y Caliper Importing an Organ You can import an organ atlas Open Inventor format one organ per iv derived from Atlas MRI or CT scans NOTE The imported atlas must include a skin file named skin iv To import an organ atlas 1 Open the DLIT results that are associated with the organ atlas 2 Select File Import Organ Atlas on the menu bar E Import Organ Atlas Organ Files Select Skin Mesh Generate Mesh Co efhicients Organ Atlas Mame Add Organ Files Save Organ Atlas 3 In the Import Organ Atlas box that appears click Add Organ Files 4 Inthe next dialog box that appears select all of the Open Inventor files that you want to include in the atlas one iv per organ and click Open 5 Inthe Select Skin Mesh drop down list select the skin organ file which must have the Tile name skin iv
294. ults Figure 9 2 Tool Palette lt Spectral Unmixing E Analyze Options Spectrum Results Spectral Unmixing Results Item Value Mumber of Iterations 16 Number of Components 2 Number of Wwavelenghts 7 Number of Samples 2700 Lack of Fit P4 3 50637 Lack of Fit E P 3 79595 Divergence counter 0 Maximum Iterations 200 Denoise PTA Medium Normalization Equal Heightidefaulk Non negativity method nnls Weighting mode None Flot Spectra Plot Distribution Save Results Name SPUM_4 Delete Load Figure 9 2 Tool palette Spectral unmixing tools Results tab 132 Living Image Software User s Manual n Y Ca Ber ce Table 9 2 Spectral unmixing tools Results tab Item Description Number of Iterations The number of iterations that the algorithm used Number of Components Number of Wavelengths Number of Samples Lack of Fit PCA Lack of Fit EXP Divergence Counter Maximum Iterations Denoise PCA Normalization Non negativity Method Weighting Mode Column Weighting Mode Row Weighting Mode Plot Spectra Plot Distribution The number of components unmixed The number of wavelength pairs used in the analysis The number of pixel samples used in the analysis The fitting residue compared to the data filtered by principal component analysis The fitting residue compared to the experimental data The number of divergences that occurred The maximum number o
295. uorescence imaging capability as standard equipment The IVIS 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 change between bioluminescent and fluorescent imaging applications Figure F 1 For more details see the VIS Imaging System 200 Series System Manual the IVIS Lumina System Manual or the XFO 6 or XFO 12 Fluorescence Option Manual Emission d iS AOR filter wheel imaging features Excitation filter wheel Excitation Emission Filter Wheel Filter Wheel Light source Assembly not visible Reflectors IVIS Lumina IVIS Imaging System 200 Series IVIS Imaging System 50 or 100 Series Figure F 1 Fluorescent imaging hardware 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 223 F Fluorescent Imaging 224 combination provides high emission throughout the 400 950 nm wavelength range Figure F 2 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 at these wavelengths The Living Image software controls the illumination intensity level off low or high The illumination intensity at the low setting is approximately 18 that of the high setting Quartz
296. urface Topography f f A Counts gt Point Source Fitting WE it Color Bar gt DLIT 3D Reconstruction 4 Min 547 gt Spectral Unmixing k P Max 8622 Preview Use Bkg Offset Replace ROIs Restore Defaults Save Load gt Planar Spectral Imaging 3 If you selected or OJ 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 Drawing a Subject ROI 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 91 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 GJ button 3 Select Auto All 90 Ti i Living Image Software User s Manual Y Caliper _ To manually draw a subject ROI 1 Select Subject ROI from the Type drop down list 2 Click the O button and select 1 3 Position the subject ROI so that it includes the measurement ROI s and the associated average background ROI 6 4 Measuring Background Corrected Signal If a subject has significant autoluminescence or autofluorescence you
297. ve 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 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 me o Living Image Software User s Manual Y Caliper Center pixel value the mean value of the nine pixels in the 3x3 neighborhood Figure C 4 3x3 pixel neighborhood 209 C Detection Sensitivity This page intentionally blank 210 Ti i Living Image Software User s Manual Ww Caliper Appendix D Image Data Display amp Measurement D 1 Image Data Scientific Image Data Graphic Image Data Pseudocolor Images Mae ela 4 64 6 oe eS ee EO ey Sw ye ee Se EE 211 Quantifying Image Data 2 ee 213 Fat Fe 6 4 a oe ee bow eA ORDERED Oe Se HE DHSS 216 Cosmic Ray Corrections 1 ee a 216 Scientific image data is a two dimensional array of numbers Ea
298. ves four starting voxel sizes 6 7 8 and 9 mm The default increment of 1 mm is usually adequate however smaller increments can be used if you want to sample finer voxel sizes Smaller increments will significantly increase the time required for reconstruction Uniform Surface If this option is chosen the surface data for each wavelength will be Sampling sampled spatially uniformly on the signal area If this option is not chosen the maximum N surface elements will be sampled for the data This means that the N brightest surface elements will be used as data in the reconstruction Typically non uniform sampling is recommended if there is a single bright source while uniform sampling is preferred if there are several scattered sources NNLS Simplex If this option is not chosen the software uses a linear programming Optimization algorithm to seek the solution Simplex solution If this option is chosen the software also applies a non negative least squares optimization algorithm at each iteration to provide a better solution for source power The Simplex solution is more robust but tends to underestimate the source flux in each voxel Therefore the NNLS Simplex option is recommended NNLS Weighted Fit Choose this option to weight the wavelength data proportionally to Its intensity in the NNLS reconstruction This option is especially useful if the intensity of longer wavelength data is orders of magnitude greater than the
299. ving Image Software User s Manual Y Caliper Creating a Custom Animation Table 12 10 Preset animations Name Choose This Animation Setup to Spin CW Rotate the 3D reconstruction clockwise Spin CCW Rotate the 3D reconstruction counterclockwise Zoom In Magnify the 3D reconstruction Fade In Increase opacity from 0 100 Fade Out Decrease opacity from 100 0 To create an animation you must specify a custom animation setup or edit an existing setup 1 Open the 3D results of interest 2 Confirm that the 3D view shows the properties of interest for example position or scale of the 3D reconstruction organs voxels surface or photon density maps a A W A amp R Units Counts C Apply to all Tool Palette gt ROI Tools gt Surface Topography gt FLIT 3D Reconstruction 3D Tools kh rA Q F os A Lae m Mesh Volume Organs Animation Preset Animations Presets Spin CW on Current Axis v Frame Factor 1 Animation Setup Total Duration secs 5 N mm 2 Source Intensit4 3 Inthe 3D tools click the Animation tab 4 If necessary clear the key frame box click the button and select Delete All 5 To capture the first key frame click the f button The first key frame is added to the key frame box 6 Tocapture the next key frame adjust the 3D view to show the properties of interest and click th
300. ving Image browser see page 47 For details on these items see Table 5 2 Du t a a R UntsiCouws Appt toal Pi TLT20050624145507_SE0 es A BMBQ4 isis Choose Individual to Photo Adbustmert apply a separate color p J scale to each ee thumbnail in a sequence Figure 5 3 Image window sequence view If DLIT or FLIT analysis results are loaded click the 3D View tab to display the 3D reconstruction of the luminescent sources IVIS Imaging System 200 or Spectrum only For more details on 3D reconstruction see page 151 Pe Cee Ww Teck Atqaetion Widow Hp eA AG mcos D ape e y J Render Proton Derety Map Angy Sansk ed Wavetetin 580 f rechi Intonaty Color Tabie f es A T Figure 5 4 Image window 3D view 51 5 Working With Data Table 5 2 Image window sequence view tab Item Description Units Choose counts photons or efficiency for the image data from the drop down list For more details on counts photons or efficiency see Appendix D page 213 Choose this option to display an image using the color table that was last applied to the image data Click to show or hide the sequence information Use Saved Colors Info br Click this button to open the Edit Sequence window that enables you to K add or remove images from the active sequence For more details on editing a sequence see page 52 Click this button to acquire a preview of the sequence for d
301. w unsaved analysis results Splm_xx Deletes the selected results Opens the selected results Saves the analysis results results name appears in the Name drop down list dl Living Image Software User s Manual Y Caliper 8 3 Viewing amp Exporting Graphical Results To view a graph of the results gt Image Adjust 1 In the Results tab select an ROI SS gt Image Information 2 Click Plot Intensity or Plot Linear Fit gt ROI Tools Planar Spectral Imaging Analyze Properties Results Spectral Results Unsaved ROI Depth mm Total Flux phot s ROIZ 2 341 0 269 2 66e7 5 18e6 Pl Plot of Linear Fit Results Log Noma Flux K Linear Fit Plot for ROI 1 Plot Linear Fit Plot Intensity Save Results gt Name SpIm_2 v gt Surface Topography _ Point Source Fitting 12 10 gt DLIT 3D Reconstruction mueff cm 1 The 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 Ue 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 Pj Plot of In
302. 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 1 211 D Image Data Display amp Measurement A 1L120050624145507_ 004 em Units Counts Display Photograph A TLT20050624145507_ 004 Units Counts Display Overlay v Image Min 49 Max 58989 50000 Image Min 96 Max 2307 2000 40000 30000 20000 10000 Counts Counts Color Bar Min 4050 Max 51182 Color Bar Min 96 Max 2033 A photographic image is a gray scale pseudoimage In Overlay display mode a pseudocolor image of the luminescent data is overlaid on a photographic image Color table associated with the data Figure D 1 Example pseudoimages Overlays 212 A pseudocolor scheme is typically used to display the numerical contents of scienti
303. wizard see page 24 ee Living Image Software User s Manual Y Caliper 2 2 Starting the Living Image Software 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 C Programs Xenogen Living Image 3 2 Macintosh Applications Xenogen LivingImage 3 2 NOTE 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 menu button and select All Programs gt Living Image Alternatively click the Living Image software desktop icon F Macintosh Users Click the Living Image software desktop icon or run the software from the application folder The main window appears select a user ID from the drop pe fo m ua mi reb down list or enter a new User ID 48 gt 0a ye up to three letters and click OK The control panel appears if the workstation controls the IVIS Imaging System see next page 2 Getting Started Menu bar for more details see Appendix J page 261 Living Image 3 2 File Edit View Tools Acquisition window Help s AH
304. you initialize the system 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 stage temperature on the IVIS imaging system is 37 C but may be set to a temperature from 25 40 C 2 Getting Started E IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop EM Gain Excitation Filter Emission Filter Field of view b M Temperature Status Demand Measured Acquire 125 lem camera temp 90 G go cir 1 10 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 Mig oystem is initialized but CCD camera temperature is out of range E System is initialized and CCD camera is at or within acceptable range of the demand temperature and locked The system is ready for imaging Figure 2 4 IVIS acquisition control panel NOTE The items in the IVIS System control panel depend on the particular IVIS Imaging System and the imaging mode selected luminescent or fluorescent Image Setup or Sequence Setup mode For more details on the control panel

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