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1. To save results Tool Palette a To copy selected results 1 Select the results of interest CE ae 1 Right click the results row of LMFIT_xx from the Name drop Rates interest and select Copy Selected down list Analysis Params Properties Results from the shortcut menu that RER E appears Starting Chi 2 8 58e 09 2 Click Save Ending Chi 2 7 03e 08 The selected results are copied to he point source fitting results are Point Source Fitting Resulte the system clipboard saved with the IMAage Parsmeters Fitted value i Mua Em 0143er To copy all results To view results oe Let 1 In the Results tab right click the 1 Select the results of interest from Diti 0 049 em results table and select Copy All the Name drop down list Total source flux 2 22e 1 photons from the shortcut menu that 1 location of the source 0 161 lem LE a eS SE aT le appears 2 Click Load etene oe All of the results table is copied to Savarese the system clipboard To delete results Name LMFIT13 O O 1 Select the results that you want to aise delete from the Name drop down E ELT D Reconstr list SE R 2 Click Delete Figure 8 3 Saving and point source fitting results 98 XENOGEN Living Image Software User s Manual Ufe Changing 9 Spectral Unmixing Performing Spectral Unmixing a ee ues 99 Spectral Unmixing Results Window 02 2 88 101
2. 5 photons sec Perspective Source Intensity 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 0 144 Living Image Software Users Manual XENOGEN ee ite Changing Life Changing Creating a Custom To create an animation you must specify a custom animation setup or edit an Animation existing setup For more details on editing a setup see Figure 10 31 To create a custom animation Fie Edt wiw Tools window Help JEK 1 Confirm that the 3D view _ In the 3D tools click the bia If necessary clear the key To capture the first key frame SAA h la amp Open the 3D results of SA RGN EEEE Sequence View interest E Planar Spectral Imagina JE Surface Topography 2 i shows the properties of interest for example position or scale of the 3D reconstruction organs Mesh Volume Organs Animation h h Preset Animations VOXxe S mes i or p oton Presets Spin Cw on XAsis density maps Frame Factor 1 aj Animation Setup Animation tab frame box click the button and select Delete All Frames Per Second ic al 0 Total Duration secs 5 photons sec Perspective Source
3. Living Image Software User s Manual XENOGEN Life Changing Life Changing 10 7 Managing Results To save results 1 In the Results tab of the DLIT FLIT 3D reconstruction tools confirm the default file name or enter anew 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 In the Results tab select the results of interest 2 Right click the results table and select Copy Selected from the shortcut menu that appears To copy all results Tool Palette ea gt Surface Topography Y BLIT 30 Reconstruction Analyze Params Properties Results S BLIT Results DLIT SpecinlD Lich Loaded Value A 5 a2e 1 0 kep Total source flus phot Final size Number of sources Reduced Chie Starting veize best Kappa best Nurt best l uh aurt sarnnles f Fhotor Density Maps e Save H esults Name DLIT_3 Delete Load Default name for the 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 In the results tab right click the results table and select Export Results from the shortcut menu that ap
4. Red 123 E E Sat Green 130 Define Custom Colors gt gt Val Blue iL 89 User defined custom colors Figure 5 10 Changing the appearance of the ROI 68 Living Image Software User s Manual XENOGEN ee _ Lfe changing Life Changing Moving or Editing the ROI Label g ILIAMISUbeT ASS oe eS To edit an ROI label 1 Double click the ROI Alternatively right Unde Counts Daiplau O veiliy abel read wre click the ROI Ctrk click for Macintosh pS ea users and select Properties from the Image Number TLT20050624145507 004 s shortcut menu Rol rene The ROI Properties box appears 2 Edit the name in the ROI Label box and click Done V Lock Position ef pix 1106 0000 TLTZD050624145507_004 Unite Counts P Display Overlay Finke ey Yo pid 164 0000 Angle deg 0 0000 Lock Size Width pig 19 0000 To move an ROI label 1 Place the pointer over the ROI label Line Size 2 When the it becomes a f drag the Line Color label 3 Click to release the label at the new lacatian Figure 5 11 Moving or editing an ROI label 69 Saving ROIs 70 5 Working With Region of Interest ROI Tools You can save ROIs with an image and you can also save ROIs to the system global save ROIs
5. If it is necessary to introduce suspect materials into the imaging chamber screen the materials by imaging them Acquire an image of the material alone using the same settings for example FOV and exposure time that will be used to image the sample to determine if the material is visible in the luminescent image Microplates white black or clear plastic can be screened this way Screen all three types with a test image White plates appear extremely bright by IVIS Imaging System standards and interfere with measurements Black or clear plastic microplates do not phosphoresce making them better choices The Xenogen High Reflectance Hemisphere provides a more definitive way to determine the presence of an undesirable light source Figure C 1 It is a small white hemisphere that is coated with a non phosphorescent material A long exposure image of the hemisphere should produce a luminescent image in which the hemisphere is not visible 161 C Luminescent Background Sources amp Corrections Figure C 1 Xenogen High Reflectance Hemisphere and a plastic marker pen Left Photographic image Right Photograph with luminescent overlay The hemisphere is illuminated by phosphorescence emitted from the pen If any part of the hemisphere exhibits what appears to be luminescent emission it is actually the light reflected from a source illuminating the hemisphere Observe the side of the hemisphere that is illuminated to help determi
6. 2 xx Window gt etc Window gt Other Windows gt Browser Window Help User Guide Help About Living Image 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 k Click this button then click an item in the user interface to display information about the item 201 a G Menu Commands amp Tool Bar This page intentionally blank 202 XENOGEN Living Image Software User s Manual Ufe Changing Appendix H User Preferences Viewing User Preferences Managing User IDs You can manage user IDs and specify defaults for some preferences that are associated with the user ID selected at the start of a new session User modifiable preferences include the e Appearance of the application window Figure H 1 e Image color settings e Folder path setting for exporting image data e Number of recently viewed data sets to include in the Recent Files menu e Default tissue properties and DLIT algorithm parameters e Host and user settings for the optional Biotrue Collaborative Data Management System H 1 Viewing User Preferences To view the user modifiable settings select Edit Preferences on the menu bar If you have not yet logged on to the syst
7. C Logarithmic Scale EConections 7Filtering gt Image Information gt ROI Tools Planar Spectral Imaging gt Surface Topography Figure 4 4 Tool palette XENOGEN Living Image Software User s Manual as Chanainn Undocking the Tool Palette To change the position of the tool palette in the application window 1 Position the pointer over the tool palette title bar so that it becomes a 2 Drag the tool palette to the new position in the main window and click Note lo undock the tool palette drag it a distance greater than Its width 3 To dock the tool palette to the left or right of the image window drag the palette to the right or left of the main window and release Click here to drag and undock the tool palette File Edit view Tools Window JE K i amp AW Units Counts 4 7 Apply to all EEEE U Counts Display Overlay zino e ps gt Corrections Filtering gt Image Information gt ROI Tools image gt Planar Spectral Imaging BES gt Surface Topography 3000 2000 1000 Counts Color Bar Min 220 Max 3432 Figure 4 5 Tool palette docked top and undocked bottom The tool palette can be docked at the left or right side of the main window 35 a 4 Working With Images 4 3 Working With an Image Sequence An image or image sequence is displayed in an image window Multiple image windows can be open at the same time The opti
8. The Surface Topography and DLIT 3D Reconstruction Tools are displayed 3 If you want to smooth the surface confirm the default surface generation options and surface smoothing parameters or enter new values For more details see Table 10 3 4 If you want to save the results confirm the default name for the results or enter anew name 5 Click Create The tomography analysis box appears For the IVIS 200 or IVIS Spectrum the procedure continues in Figure 10 3 For the IVIS 3D the procedure continues in Figure 10 4 Figure 10 2 Reconstruct the mesh Table 10 3 Surface topography options amp parameters Tool Palette Image Adjust gt Planar Spectral maging lt Surface Topography 5 uface Generation Options Structure Light TLT 20050624 22346 O01 Surface Smoothing Parameters Smoothing Level Lo Restore Loss Recovery Height Smooth Save Results Mame SURFACE_1 W Delete Load Ovenante gt DLIT 3D Reconstruction Item Description Surface Generation Options Structured Light A drop down list of structured light images in the sequence Select one for use in reconstructing the mesh Photo Back Projection This option is only available for data obtained on an IVIS imaging System 3D Series If this option is chosen photographic image data is also used to reconstruct the mesh Surface Smoothing Parameters Smoothing Level The amount of smoothing to apply to a re
9. e Diffuse Tomography DLIT algorithm For tomographic analysis of bioluminescent sources Analyzes a bioluminescent image sequence obtained on the IVIS Imaging System 200 or 3D Series e Fluorescent Tomography FLIT algorithm For tomographic analysis of fluorescent sources Analyzes a fluorescent image sequence obtained on the IVIS Spectrum imaging system 10 1 3D Reconstruction of Bioluminescent Sources General Considerations The steps to perform 3D reconstruction of bioluminescent sources include e Acquire an image sequence IVIS Imaging System 200 Series amp IVIS Spectrum Acquire a photographic and a structured light image and two or more bioluminescent images at different wavelengths 560 to 660 nm Table 10 1 Or IVIS Imaging System 3D Series Acquire photographic structured light and luminescent images at multiple angles 0 315 degrees and multiple wavelengths Figure 10 1 e Generate the surface topography mesh of the subject e Specify the user modifiable DLIT algorithm parameters for example analysis wavelengths source spectrum and tissue properties e Reconstruct the position geometry and strength of the luminescent sources For more details on the DLIT algorithm see 3D Reconstruction of Light Sources page 189 Animal Requirements The best surface topography reconstruction is obtained from nude mice Furred mice especially those that are symptomatically stressed do not refl
10. Cmn dS gt Corrections Filtering B E Image Information gt ROI Tools Min 1 26e3 Max 9 76e5 Planar Spectral Imaging p sec em 2 st Analyze Properties Results a Spectral Results Unsaved ROI 1 1 250e 06 600 ROIL Depth mm Total Flux phot s RO 2 326 0 191 3 01e6 3 87e5 400 x107 200 lt gt Plot Linear Fit Save Results Color Bar N J5 im 2 z Min 6 36e4 igh aS Max 6 76e5 Delete Load gt Surface Topography DLIT a File Edit Yiew Tools Window Help JAX gaH m A amp R Unts Photons sC Apply toal eeaeee Units Photons Display Overlay M Finto a LtmoseAdwst E gt Corrections Filtering gt ROI Tools Image Min 1 09e3 Planar Spectral Imaging Mak 6 5765 Analyze Properties Results Spectral Results a ROI Depth mm Total Flux phot s ROI 2 2 426 0 199 12 4286 7 3 2365 40 x10 3 0 2 0 lt ll I 1 0 Plot Linear Fit p sec cm 2 st Save Results Color Bar Name Splm_2 Min 6 32e4 gt Safata Topography DLIT Figure E 5 Planar spectral analysis results Top Dorsal view of the left lung bottom ventral view of the left lung XENOGEN ee _Life Changing Life Changing Living Ilmage Software Users Manual E 5 Optimizing the Precision of Planar Spectral Analysis The accuracy of the planar spectral
11. Restore Defaults e Uniform Surface Sampling NNLS Simplex Optimization C NALS Weighted Fit Restore Defaults i gt 3D Tools gt 3D Tools i Figure F 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 Series or IVIS Spectrum and 60 for the VIS Imaging System 3D Series 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 195 a F 3D Reconstruction of Light Sources 196 Kappa Limits Kappa Kk 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 large values favor more shallow sources The limits
12. gt Surface Topography gt FLIT 3D Reconstruction Spectral Unmixing Edit Image Labels UselD KSA LabelSet xenogen Universal Check any 5 fields IV User De MI 77 M Group E W V Experiment ooo V Commenti F a A I Comment2 E I Time Point p e e M AnimalNumber o Animal Strain at Animal Model i seo Sex H IM T View ot T Cell Line ie I Reporter E I Treatment F M LuclectionTime xf ACUC Number T Appl To Sequence Erca 2 Delay 0 0 min Applyto All X Remove A Update Ci inser aaa HAK 28 MaF 4098 29 Image window Figure 3 13 Acquiring a transillumination fluorescence image sequence on the IVIS Spectrum continued from Figure 3 12 26 Living Ilmage Software Users Manual XENOGEN gt Life Changing Working In the Sequence In the sequence editor you can add or delete an image to the sequence or edit Editor a parameter value Figure 3 15 To specify an image acquisition sequence it may be convenient to edit an existing sequence setup xsq and save the setup to a new name IVIS Acquisition Control Panel x Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter SM Luminescent fio pl sec v Medium j x lock 10 Medium 1 Block 1 Medium 1l Block 1 Medium 1 Block 1 Medium Block ezo c 150 o0
13. spectral Results Unsaved ROI Depth mr Total Flux phot AHT h riei eres es ri i HLA H 2026 1 85e5 rT La eer ine Ato LEE soir seen Cn fe F E TA A r TEAT T E n e EE E TEA AAE RSIS ar oa fa BCR Intensity vs Lambda Plot for ROI 1 1 00 Normalized Fh aa E e u gt Save R esults Name Splm_3 sE Delete Load 600 620 Wavelength roi pe Stee Toners 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 toa csv A Plot of Linear Fit Results EER 1 Click the Export Data button Hig Linear Fit Plot for ROI 1 2 In the dialog box that Log Norm Fhir tR 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 12 10 mutt crm 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 uopA 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 e
14. File Export DICOM File Export 3D Mesh File Export 3D Volume File gt Print File gt Print Preview File gt Recent Files File Exit Edit Copy H BS La amp e Toolbar Description Button pe Displays the Open box so that you can select and open an image data file a oe i a Fa a Displays the Browse For Folder box so that you can select and an image data folder The selected folder is displayed in the Living Image browser a Opens the Biotrue CDMS Browser Note The browser is only available if the system includes the Biotrue CDMS option Saves overwrites the active image data 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 Opens a dialog box that enables you to import an organ atlas atlas Opens a dialog box that enables you to import dcm image data that can be viewed in the Living Image software 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 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 Opens the Browse for Folder dialog box that enables you to export the active image data to DICOM format dcm
15. Living Image Software Manual XENOGEN a ife Changing Life Changing XENOGEN Life Changing Y Ca PET rs Living Imagee Software User s Manual Version 3 0 2002 2007 Xenogen Corporation All rights reserved PN 122444 Xenogen Corporation 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 xenogen 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 XENOGEN fe zs Life Changing Living Image Software User s Manual Contents 1 WOEIGOMC 2 34 ee bos dirinin sapiri adri ripi EGE D A 1 1 1 What s New In the Living Image Software 0 0 2 200 l 2 Getting Started 2 2 2 en 5 2 1 Starting the Living Image Software ooo e a e 5 2 2 Basic Living Image Software Tasks 2 0 6 2 3 Living Image Help 2
16. 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 D 9 shows autofluorescence originating from four different plastic microplates The images were taken using a GFP filter set excitation 445 490nm emission 515 575nm White polystyrene Clear polypropylene p sec om 2 sr Clear polystyrene Black polystyrene Figure D 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 Exo 4sec Two types of autofluorescent effects may occur Overall glow of the material Usually indicates the presence of autofluorescence XENOGEN a ene etl Living Image Software User s Manual tite Changing Hot spots Indicates a specular reflection of the illumination source Figure D 10 The specular reflection is an optical illumination autofluorescence signal reflecting from the microplate surface and is not dependent on the microplate material Specular Reflection Specular Reflection Specular Reflection Specular Reflection Figure D 10 Specular reflection The four symmetric hot spots on this black polystyrene well plate illustrate the specular reflection of the illumination source Imaging parameters
17. iw oR amp we ee Oh eA RA EO EH 34 Working With an Image Sequence 025 2805 36 Creating an Image Sequence from Individuallmages 38 Working With a Single Image 0 005 ee eens 39 Viewing Image Information 0 0 0 0 eee eee ees 43 Adjusting Image Appearance a ee eee ee es 45 Correcting or Filtering Image Data 5008 46 Image Information Tools a eee ee ee ee ee 48 Image Layout Window a a ee a 54 The Living Image Browser provides a convenient way to browse image data view information about the data and open a single image or an image sequence Figure 4 1 You can also navigate to an image of interest and open it without the browser see page 34 Browse For Folder E Xenogen 2 G Images for Katherines amp BWR20040830123508_5E iG CK20031215150449_5EQ E Gy CK20031215152405_SEQ H G CK20050330172433_SEQ E GQ CK20050418112426_SEQ E Ga 20040819143946_SEQ 3 0c20050120183340_SEQ i eee M gji ee ie ey a Click a column header to sort the entries in ascending or descending alphanumeric order Figure 4 1 Browsing image data using the Living Image browser 31 a 4 Working With Images Opening Image Data With the Living Image Browser 32 Item in the Living Image Browser Function Label Set Image information column headers displayed in the Living Image browser Add to List
18. 12 55 Y Min 3 7466 Y Max 5 17e6 Full Scale Logarithmic Scale line on the image to TLT20050624122348_003 Overlay update the line graph 10 Photons s em 2 1 3 00 2 00 1 00 0 00 Figure 4 22 Line profile plots intensity y axis at each pixel x axis from a user specified line in the image Item in the Line Description Profile Window Line Orientation Choose Vertical or Horizontal from the drop down list to set the orientation of the line in the image window Width Sets the line width Position Line position pixels X Min Displays the minimum and maximum value of the x axis Use the arrows to change the x axis X Max 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 52 XENOGEN meee ee Life Changing Living Image Software User s Manual Item in the Line Description Profile Window Y Min Displays the minimum and maximum value of the y axis Use the arrows to change the y axis Y Max 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 If Logarithmic Scale S
19. 2000 7000 1500 1500 OOD 1000 500 500 Photograph Luminesce Photograph Luminescent nt overlay overlay Figure C 2 Background light emission Background light emission from a female white furred Swiss Webster left and a female nude Nu nu mouse right 163 a C Luminescent Background Sources amp Corrections 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 164 Living Image Software User s Manual XENOGEN eT Life changing Life Changing Appendix D Fluorescent Imaging Description and Theory of Operation 22 004 165 Filter SPECIA 458k ob eR ORS ae Rae a 167 Working with Fluorescent Samples 288 169 Image Data Display 2 a a 170 Fluorescent Background a ee ee ee 172 Subtracting Instrument Fluorescent Background 177 Subtracting Tissue Autofluorescence Using Background Filters 178 D 1 Description and Theory of Operation System Components The IVIS Imaging System 200 Series and IVIS Lumina offer built in fluorescence imaging capability as standard equipment The IVIS Imaging System 3D 100 or 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
20. Drop down list of the absorption and scattering properties of various tissues Tissue index of refraction 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 ues and reduced scattering coefficient u s Click to display the spectrum of the selected bioluminescent source intensity versus wavelength normalized to one Name of the analyzed ROI Estimated depth of the point source Estimated total photon flux from the point source Click to display a graph of normalized intensity versus the effective attenuation coefficient LLaz 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 Click to display a graph of normalized intensity versus wavelength Intensity is normalized by the selected source spectrum and filter transmission properties Default name for the analysis results Deletes the selected results Opens the selected results Saves the analysis results results name appears in the Name drop down list 87 a 7 Planar Spectral Image Analysis 7 3 Performing Planar Spectral Image Analysis To perform planar spectral image analysis 1 Open the image sequence that you want to analyze File Edit view Tools Window Help JA
21. 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 D 9 and Figure D 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 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 D 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 leakage and low level autofluorescence from inside the IVIS System imaging chamber The ring like structure is a typical background autofluorescence leakage pattern The image represents the minimum background level that a fluorophore signa
22. Opens a dialog box that enables you to save the 3D mesh of the active data in Open Inventor format iv Opens a dialog box that enables you to save the voxels from the active data in Open Inventor format iv Displays the Print box Displays the Print Preview box that shows what will be printed 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 Closes the Living Image software Copies the active image window to the system clipboard a J E E E E a m a 199 a G Menu Commands amp Tool Bar Menu Bar Command Toolbar Description Button Edit Image Labels Opens the Edit Image Labels dialog box that enables you to edit the label set information for the active data Edit gt Preferences View gt Tool Bar View Status Bar 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 View Activity Window Displays the Activity window at the bottom of the main application window The Activity window shows a log of the system activity View Tool Palette View Activity Window 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 th
23. ROIs that you want to add to the image or the grid ROI dimensions The specified type shape and number of ROls are added to the image File Edit View Tools Window Help Se QA a amp Units Counts C Apply to all gt ImageAdust O E g Y Xx C Apply to Sequence Type Measurement AOI v Threshold a a Save ROls AFET E Name ROI2 KSA v Delete Load gt Planar Spectral Imaging pP Pu _ RO 1 1 004e 06 ROI 2 2 554e 05 ROI handle Note The grid shape is useful for drawing a grid of ROIs on an image of a microplate plate 5 To adjust the ROI position a Place the mouse pointer over the ROI When the pointer becomes a click the ROI b Drag the ROI to the location of interest 6 To adjust the ROI dimensions a Place the mouse pointer over the ROI When the pointer becomes a S click the ROI b Place the mouse pointer over an ROI handle so that it becomes a Drag the handle to resize the ROI Note You can also change the ROI position or size using the adjustment controls in the ROI Properties box see Changing the ROI Position page 66 and Editing ROI Dimensions page 67 A ROI Measurements OX 7 To compute the Click Number Rol Image Layer Total Counts Ava Counts Stdev Counts Min Counts Max Count ntens ty n th e R O TLT 20050616134836 _001 ROI Overlay 3 1fe 04 _ 9 770e
24. Select the number of spectral components to unmix the number of fluorophores 1 For example if the image data includes one fluorophore then there are two components to unmix the fluorophore signal and the tissue autofluorescence oy Mask Photo Unmix Spectra Components 2 j Photo Mask Setup Make a selection from the Mask drop down list e All The entire image is analyzed e Photo Opens the Photo Mask Setup window The purple mask specifies the area for the analysis 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 Use the Threshold slider or He arrows to adjust the mask so that it matches the underlying photograph of the subject as closely as possible without including any area outside the subject image Click Set Image Adjust b Click Unmix Spectra The spectral unmixing results appear Unmixed 1 Autofluorescence Composite Unmixed 1 Unmixed 2 Figure 9 1 Removing tissue autofluorescence by spectral unmixing 100 lenga Adus Units Photons w C Use Previously Saved Colors 7 Transillumination Location 1 Show Labels Photograph Image MAECEN hammers Photo Mask Setup window PCA Biplat PCA Explained Yariance a S Unmixed 2 Fluorophore signal Living Ilmage Software Users Manual XENOGEN ee a Canaan Life Chan
25. This row specifies settings for the photographic image XENOGEN E te Eaz Life Changing Living Ilmage Software Users Manual Table 3 1 General controls for all IVIS Systems Item in the Control Panel Description Luminescent Fluorescent Exposure time Binning f stop Excitation Filter Emission Filter Photographic Auto Fluor Lamp Level Overlay Lights On 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
26. amp Number Comment 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 ROI measurements and other Column headers in the are reordered data types column headers ROI Measurements table that can be added to the ROI _ To save the table configuration enteraname in Measurements table the Name box and click Save Note You cannot overwrite a factory loaded configuration If you modity a factory loaded configuration save it to anew name To delete a custom table configuration 1 Select the configuration from the User Lists drop down list and click Delete Note Factory loaded table configurations cannot be deleted Figure 5 15 Configuring the ROI Measurements table 74 XENOGEN Living Image Software User s Manual te changing Copying or Exporting the ROI Measurements Table i ROI Measurements DER Click Number Total Counts Image Layer Avg Counts Min Counts May Cou TLT 200506241 45507_001 i0 2 601e 02 44066 03 TLT200506241 45507 004 4 Customized Selections Copy coe Types ki E e To export the table To copy 1 Click Export e Selected rows in the table select the rows of interest and 2 In the dialog box that appears click Selected enter a name for the table and click Save e All rows in the table click All The information is saved to a text The information is copied to fi
27. 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 me arrows or enter a new value in the box 9 Click Finish The mesh and 3D tools appear For more details on the tools see page 127 Fie Edt View Tools Window Help EA SAAN DRAN HER Sequence View L 3D View S Surface Generation Options Structure Light CK200506241 51158_001 Photo Back Projection Surface Smoothing Parameters Smoothing Level Low v Loss Recover Height Save Results Name SURFACE_3 alh pas e eo DLIT 3D Reconstruction Analyze Params Properties Results Sequence CX20050624757158_SEQ Tissue Muscle Source Freh Select Wave Filters Filter Views Minimum Radiance EE gt 3D Tools Perspective Figure 10 3 IVIS Generating the surface topography IVIS 200 Series or IVIS Spectrum continued from Figure 10 2 113 _ 10 3D Reconstruction of Sources IVIS Imaging System 3D Series Generating the surface topography mesh continued from Figure 10 2 Wi View Surface Topography Analysis Crop box Vaid exon region Phaghtrason oft 02 05 oe 6 Confirm that the default crop box includes the subject and a small margin around the subject If necessary resize the crop box 7 Click Next to display the nex
28. requires more computational time The default range of 6 9 is usually adequate to determine the optimum solution This is the step increment in voxel size stepping from the minimum voxel size limit Uniform Surface Sampling 208 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 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 XENOGEN Living Image Software User s Manual ee ee Preferences DLIT Params Tab Description NNLS Optimization If NNLS optimization option is not chosen the software uses a linear programming 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
29. results IE Surface Topography DLIT A Plot of Linear Fit Results r DOR p AFlot of Intensity Vs Lambda T Linear Fit Plot for ROI 1 Intensity vs Lambda Plot for ROI 1 Log Norm Flux K 1 00 Normalized Flux au 10 00 pe 9 50 9 00 8 50 13 16 14 12 10 g 6 4 600 620 mueff crn 1 Wavelength nrn Figure E 4 Planar spectral analysis results 185 a E Planar Spectral Imaging 186 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 E 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 File Edit view Image Window Help eA X gaku amp A amp K o Units Photons o Apply to all peee aetna Units Photons Display Overlay vln
30. 0 0200 160 Background Light From the Sample 58 162 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 D page 172 C 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 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
31. 2 Key Frame 3 Key Frame 4 Key Frame 5 Key Frame 6 Frames Per Second 10 oe Total Duration secs 5 Sa bera 5 BEN Fag AE i photons sec Perspective Source Intensity Living Ilmage Software Users Manual XENOGEN ee Life nae Life Changing Item in the Animation Tab Description Time Scale Presets Key frame Preset Key Frame Factor Animation Setup Load Save The time stamp of a key frame in the animation on a time scale of 0 100 For example if the animation is 10 sec long and includes five key frames Key frame 1 Time stamp 0 first frame of the animation Key frame 2 Time stamp 25 frame occurs at 2nd sec of animation Key frame 3 Time stamp 50 frame occurs at 5th sec of animation Key frame 4 Time stamp 75 frame occurs at 8th sec of animation Key frame 5 Time stamp 100 last frame of the animation A drop down list of predetined 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 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 anima
32. 3D source 1s reconstructed or when you open saved results View voxel data 130 Rotate or move the 3D Image in the image window 132 Select a drawing or lighting style for the mesh 133 Change the view perspective 135 a a coronal sagittal or transaxial plane through the 3D image and view the resulting cross section 136 slice Make measurements in the coronal sagittal or transaxial section 136 Display a 3D diagram of an organ s on the 3D reconstruction 137 Click to select a tool to work with the mesh or 3D results perspective view only to select a mesh drawing style to select a shading style for the mesh Fie Edit View Tools Window Help e gt lt ibe sas Le Jaaa aaa aaa 13335 al Ei Sequence Wien EN 3D view en Planar Spectral Imaging E Surface Topography 5 gt DLIT 3D Reconstruction v 3D Tools C4 DH Eg BOL Mesh Volume Organs Render Mesh Opacity CURE i T V Render Photon Density Map bel Animation v a oo Intensity ee 2 92e 5 q Color Table Foro ay Reverse C Logarithmic Scale Slice Coronal Ha Sagittal Trangaxial oP y photons sec Perspective Source Intensity Figure 10 13 3D tools and DLIT results in the 3D view window 127 10 3D Reconstruction of Sources 3D Tools Description Image Tools S op A A drop down list of tools for viewing and working with
33. 4 separate layout areas in the window A different image can be pasted into each layout area fas 2 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 fm ae E 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 55 a 4 Working With Images This page intentionally blank 56 Living Ilmage Software Users Manual XENOGEN eee ite aan Life Changing 5 Working With Region of Interest ROI Tools 5 1 About ROIs Table 5 1 ROI types Description About ROls ROI Tools Managing ROls Drawing Measurement ROls Drawing an ROI Using the Free Draw Method Drawing an ROI Using the Free Draw Method Managing the ROI Measurements Table A region of interest ROD is a user specified area in an image The ROI tools enable you to create three types of ROIs Table 5 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 5 1 For more details see Managing the ROI Measurements Table page 72 See Figure 5 1 for
34. 575 GFP EGFP FAG 575 650 DsRed2 1 PKH26 CellTracker Orange 695 770 Cy5 5 Alexa Fluor 660 Alexa Fluor 680 810 875 Indocyanine green ICG Uses same as GFP Grr EGFP FITC Uses same as DsRed DsRed2 1 PKH26 CellTracker Orange Uses same as Cy5 5 Cy5 5 Alexa Fluor 660 Alexa Fluor 680 Uses same as ICG Indocyanine green ICG D 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 D 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 the type of tissue Autofluorescence can occur throughout the animal but is strongest at the surface where the excitation light is strongest 169 a D Fluorescent Imaging Specifying Si
35. Analysis j Single View Surface Topography Analysis Crop image Threshold image Crop image Threshold image Draw a region of interest Use left and right rectangle to proceed lt arrow keys to nudge the threshold value to generate a mask image CK20070117110905_001 CK20070117110905_001 Threshold a Valid crop region 7 Click Next to display the mask The mask is a purple overlay on the subject image that defines the area of interest for the surface topography reconstruction The mask should match the underlying photograph of the subject as closely as possible without including any area outside the subject image 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 ate e Click the arrows or enter a new value in the box 9 Click Finish A Fie Edit view Tools Window Help ie amp 10 The mesh and 3D tools appear saanu e Raw For more details on the tools see t 263 f Sequence view 30 View page 127 Surface Genetation Options Structure Light CK20050624151158_001 Photo Back Projection Surface Smoothing Parameters Smoothing Level Loss Recovery Height Save Results Name SURFACE_3 all
36. Color data ounts Min and Max gt Corrections Filtering Color Bay Nin 397 ROI Tools Max 18712 gt Surface Topography Figure 4 16 Tool palette image adjust tools Image Adjust Tool 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 Figure 4 11 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 Saar So 2 45 _ 4 Working With Images Image Adjust Tool Description Photo Adjustment Brightness Click and move the slider left or right to adjust the brightness of an image displayed in overlay or photograph mode
37. Intensity click the Fh button The first key frame is added to the key frame box To capture the next key frame adjust the 3D view to show the properties of interest and click the 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 the key frame sequence see Figure 10 31 Note 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 Confirm the defaults for FPS frames per second and Total Duration length of animation or enter new values Note 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 Th Click Record 2 In the 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 In the dialog box that appears select a directory and enter a file name xml Figure 10 30 Creating an animation 145 a 10 3D Reconstruction of Sources Editing amp Saving an Animation Setup To open an animation setup 1 To select a predefined setup ma
38. Label ROIT j ROI Double ROlLabel R02 subject ROls in the Rotvabel Roi Bkg RO SubjRO Info click to edit BkgROl SubjRO Info selected image BkgRO SubjRO Info Image Number Image Number Shape Circle TLT20050624122348_003 Selected image CK20031215152405_001 l sa RE D aT Information none ubjec 7 O about the ISt O selected ROI average Label Subject 2 background Lock Position ROls in the Lock Position C Lock Pasition Xe pix 120 0000 Image ef em 7 8750 lI Enter ID and Xe om 8 1289 label information lt i here for the subject ROI selected above Yet pis Yet em 4 4100 Ye cm 4 5000 Angle deg Angle dea 0 0000 C Lock Size Width cm 1 8000 Lock Size width cm 1 5750 Lock Size Width pis Height pix 16 0000 Height cm 1 2600 Height cm 11 8000 4 Line Size 2 Line Size 2 E Done Subj ROI tab Line Size 2 gt a A Yv is Done Bkg ROI tab measurement ROI selected in the image Line Color Line Color Line Color Done Info tab Figure 5 7 ROI properties Items in the ROI Description Properties Box 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 ROI The Subject ROI tab shows a drop down list of
39. Preview Tissue Properties Source Spectrum Saye as default settings for C DLIT C Spectral Imaging Wavelength un Tool Palette Tissue Properties Muscle w z Internal medium index of refraction 1 4 gt Image Adjust E E Planar Spectral Imaging t Y Surface Topography DLIT Analyze Params Properties Results Source Spectrum Firefly Display Tissue Properties Source Spectrum Normalized amp riplitude These preference settings specify the defaults for the Properties tab in the Surface Topography DLIT tools or the Planar Spectral Imaging tools depending on the option DLIT or Spectral Imaging selected in the Preference box ne a 600 Wavelength rr Figure H 4 Preferences Tissue Properties tab left and Surface topography DLIT tools Properties tab right Preferences Tissue Properties Tab Description Tissue Properties Choose a default tissue type that is most representative of the area of interest Source Spectrum Choose the default luminescent source Index of Refraction The software automatically sets the internal medium index of refraction based on the selection in the Tissue Properties list Display Preview Tissue Properties Choose this option to display a graph of the absorption coefficient u effective attenuation coefficient Ue and reduced scattering coefficient u s or usp Source Spectrum Choose t
40. Select Tools lmage Overlay for xx_SEQ on the menu bar The image overlay window appears and shows the first photographic image in the SEQUENCE Sequence ARW20050826124002_SEQ continued in Figure 6 4 Be ie AR W 200508261 24002_001 ARW20050826124002_002 i jlmage Overlay for Sequence Photographic images in the sequence i Fluorescent Images f ARW 20050826124002 O01 ARW20050826124002 002 Fluorescent or luminescent images In the sequence C Edit Layer Properties Opacity Image overlay window Figure 6 3 Coregistering multiple images continued in Figure 6 4 82 XENOGEN Living Image Software User s Manual tte Changing To coregister images continued from Figure 6 3 Select white or black for the Set the number of color scales 4 Select a photographic image in the upper box low end of the color scale displayed per column 5 To select all of the fluorescent or luminescent PTASIE images in the lower box click the a button Sequence ARW20050826124002_SEQ Color Scale Type White Levels Color Scale Per Column 3 B Photograph Images Alternatively to select images of interest do ARw 20050826124002_001 ARW200508261 24002_002 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 Fluorescent Images To select adjacent images in the list Dress and ARW20
41. Spectral Unmixing Parameters 0 0005 eee eee 105 Spectral Unmixing Options 0 00 eee ee ee es 106 Spectral unmixing removes tissue autofluorescence from fluorescence image data Spectral unmixing can be applied to images acquired using epi illumination excitation light above the stage or transillumination excitation light below the stage 9 1 Performing Spectral Unmixing It is recommended that the image sequence include several images acquired using the fluorophore excitation filter as well as several emission filters NOTE Make sure that the band gap between the excitation and emission filters is sufficiently large so that the excitation light does not leak through the emission filter where it can be detected by the CCD Figure 9 1 shows how to perform spectral unmixing 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 a 9 Spectral Unmixing File Edt View Tools Window Help ig Qakd R amp R 1 Load the image sequence aaa An To perform spectral unmixing x Sequence View gt gt Planar Spectral Imaging 7 Spectral Unmixing Analyze Options Spectrum Results 2 In the Analyze tab select the emission wavelengths that you want to include in the analysis Select Wavelengths 675 Excitation Emission wavelength wavelengths of the sequence SEAR
42. XENOGEN eee Ba Life Changing The Living Image software controls image acquisition on the IVIS Imaging System and provides tools for optimizing image display and analyzing image data 1 1 What s New In the Living Image Software The Living Image 3 0 software provides many new and improved features for all IVIS Systems Table 1 1 lists the new Living Image software features and their availability on the different VIS Imaging Systems Table 1 1 Living Image software New Features Spectral unmixing tools eliminate autofluorescence Fluorescence tomography algorithm FLIT enables 3D reconstruction of fluorescent sources Comprehensive image analysis and display tools that are organized in a convenient tool palette New visualization tools including an opacity slider min max color bar sliders zoom and panning tools Ability to rebin increase or decrease the pixel size in an image and smooth images Extensive selection of ROI region of interest tools for selecting and quantifying features or exporting data to other applications such as Microsoft Excel Circle rectangle contour and free draw ROI shapes are available in manual or auto modes New image analysis features line profiles histograms distance measurement and pixel measurement Export image data in DICOM format for viewing with other DICOM readers Improved algorithms for fluorescent background correction and new Image Math tools automate
43. acquires images from different angles at the same FOV Living Ilmage Software Users Manual XENOGEN eT Life Changing 3 5 Acquiring Images To acquire an image or image sequence 1 Start the Living Image software double click the f icon on the desktop 2 Initialize the IVIS System and confirm or wait for the CCD temperature to lock For more details see Chapter 2 page 5 Systems equipped with thermoelectric cooling may require several minutes to reach the demand temperature 3 Place the anesthetized subject s in the imaging chamber and close the door For details on anesthetizing animals see the Xenogen XGI 8 Gas Anesthesia System User Manual 4 Acquire the image or image sequence Table 3 6 shows where to find details on image acquisition for your VIS system and imaging mode Table 3 6 Image acquisition Imaging Mode IVIS Imaging System See Page Lumina 100 200 Spectru 3D Series Series m Series Bioluminescent or fluorescent image J J J J 18 19 Bioluminescent or fluorescent image J 20 21 Bioluminescent image sequence J J J J J 23 24 Fluorescent image sequence reflectance mode Fluorescent image sequence transillumination mode J 118 17 3 Initializing the System amp Acquiring Images To acquire an image on the IVIS Lumina IVIS Spectrum IVIS Imaging System 100 Series or 200 Series 1 For luminescent imaging a Choose the Luminescent option b Conf
44. and Figure 10 9 page 120 2 Set the FLIT algorithm parameters and reconstruct Figure 10 6 page 116 the position geometry and strength of the fluorescent sources To reconstruct the surface topography mesh en Palette 1 Load the image sequence that you want to analyze F 2 In the tool palette click Surface Topography Sfae Gereon poms Structure Light CeK2007017 71710905 007 The Surface Topography and FLIT 3D Create Reconstruction tools are displayed cese Surface Smoothing Pararieters 3 Confirm the default surface generation options Smoothing Level Low s estore and surface smoothing parameters or enter new values For more details see Table 10 6 page 121 Loss Recovery Height Snooty Save Results Name SURFACE_1 Delete Load Save 4 Confirm the default name for the results or enter anew name EFT 3D Reconstuction 5 Click Create gt Spectral Unmivng The tomography analysis box appears Single View Surface Topography Analysis 6 Draw a crop box that includes a one cm margin Gopinage around the subject if possible Crop image continued in Figure 10 9 Draw a region of interest rectangle to proceed CK20070117110905_001 Valid crop region Figure 10 8 Reconstructing the mesh 119 _ 10 3D Reconstruction of Sources To reconstruct the surface topography mesh continued from Figure 10 8 j Single View Surface Topography
45. and fluorescent imaging applications Figure D 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 RE t filter wheel imaging features Excitation filter wheel Excitation Emission Filter Wheel Filter Wheel Light source Assembly not visible IVIS Lumina IVIS Imaging System 200 Series IVIS Imaging System 50 or 100 Series Figure D 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 combination provides high emission throughout the 400 165 a D Fluorescent Imaging 166 950 nm wavelength range Figure D 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 Halogen Lamps with Dichroic Reflectors IR Cut Off EJV Lamp 3400 K 10 ome FKE Lamp 3200 K Relative Spectral Radiance 400 600 800 1000 1200 1400 1600 1800 Wavelength nanometers Figure D 2 Relative spectral radiance output for the quartz halogen lamp with di
46. background fluorescence subtraction New overlay tool that enables you to overlay two or more images using different color representations for displaying data from multiple reporters New acquisition sequence editor that is highly intuitive and supports in place editing of sequence parameters New universal label set helps you label images consistently and in a manner that is compatible with database search engines See Page J9 118 34 45 47 61 48 29 19 82 Zo 24 S S S S S S S IVIS Imaging System m im MMM KARAKARA MMi S S S S S S S S S S 1 Welcome Table 1 1 Living Image software New Features Faster more robust stop feature for terminating image acquisition when you want to change imaging parameters and start over Biotrue Collaborative Data Management System an optional web based system for storing managing and sharing data Improved planar spectral imaging feature for estimating depth of a luminescent source based on spectral information Structured light analysis algorithm for determining the surface shape of the subject animal The Diffuse Luminescent Imaging Tomography DLIT analysis algorithm for quantifying the intensity and location of bioluminescent sources in 3D space A tissue database that contains wavelength dependent absorption and scattering properties A spectral database that includes emis
47. 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 A 3 Surface Radiance Tissue gt Q Solid Angle units of steradians Figure A 3 Isotropic 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 ohotons 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 1s displayed in photons mode the units change to photons sec cm2 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 b
48. colors of the selected color table Logarithmic Choose this option to apply a log scale to the relationship between numerical data and the color Scale range in the color table A log scale increases the range of meaningful numerical data that can be displayed 4 8 Correcting or Filtering Image Data The Corrections Filtering tools enable you to subtract background or apply corrections to the image data For more details on sources of background see Appendix C page 159 You can also apply smoothing and soft binning to the image data For more information on binning and smoothing see Appendix B page 155 m To view the tools click Corrections Filtering in the tool palette Figure 4 17 46 XENOGEN em eee Living Image Software User s Manual Ufa Changing ina Palette _ Ea ImageAdust O z Corrections Filtering EA F Read Bias Subtraction Read Bias Subtraction and Flat Field Correction are mandatory defaults in Flat Field Correction photons mode In counts F Cosmic Correction mode these corrections can be cleared Binning ef elf Smoothing E Ringe jane 5 gt Image Information gt ROI Tools gt Surface Topography Figure 4 17 Tool palette Corrections Filtering tools Corrections Filtering Description Tool Read Bias Subtraction Select this check box to subtract dark background from the image data For more details on background see Appendix C page 15
49. density threshold ohotons mm 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 ni 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 the slider to change the position of the coronal sagittal or transaxial plane through the mesh The intersection of the plane and subject slice is shown in the coronal sagittal and transaxial views in the 3D view window Figure 10 21 Volume Tab Volume Choose this option to display the voxels Min The minimum voxel intensity photons sec Max The maximum voxel intensity ohotons sec Render voxels A drop down list of shapes for voxel display as Threshold Choose this option to apply a minimum threshold intensity to the voxel display Intensity Use the Intensity slider the arrows or enter a value in the box to set the minimum threshold intensity ail Color Table Specifies the color table for the voxel intensity scale Reverse Choose this option to revers
50. 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 171 a D Fluorescent Imaging D 5 Fluorescent Background Autofluorescence 172 Autofluorescence is a fluorescent signal that originates from substances other than the fluorophore of interest and is a source of background Almost every substance emits some level of 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 VIS Imaging Systems are carefully chosen to minimize autofluorescence Pure fused silica is used for all transmissive optics and fiber optics to reduce autofluorescence However trace background emissions exist and set a lower limit for fluorescence detection To distinguish real signals from background emission it is important to recognize the different types of autofluorescence The following examples illustrate sources of autofluorescence including microplates other materials and animal tissue Microplate Autofluorescence
51. field at any f stop and FOV setting For more details on manual focusing see page 28 Generally f 1 is recommended for low light luminescent images and f 2 or f 4 is recommended for brighter luminescent or fluorescent images narrow focus m i _ deepfocus Figure B 2 Lens f stop positions Left lens 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 C page 159 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 im
52. 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 30000 Color Min 2000 i 45000 Figure D 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 Exp 4sec Reference standard TR 613 Red is available through Precision Dynamics Co http www pdcorp com healthcare frs html 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 D 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 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
53. is added to the active sequence and the click number is added to the Sequence Clicks windowpane Close 6 When you are done editing the sequence click Close Tha BARIIANARAAR nnntanta AKA iinNataN ann Figure 4 7 Adding or removing images from a sequence 37 a 4 Working With Images 4 4 Creating an Image Sequence from Individual Images To create a sequence from images EX Living Image Browser acquired during different sessions Click Number UserID User Group Experiment Comment Time Point JJH20050630142125_001 1 In the Living Image Browser browse JJH20050630142719_002 WJJH20050630142719 004 for the images of interest For more E cat Tose lest andi eg details on browsing see Figure 4 1 page 31 Note In the browser an image that Is a member of a sequence cannot be selected for grouping For example these images cannot be selected for grouping Therefore browse for individual images which may or may not be part of a sequence not image Sequences File Edit Yiew Tools Window Help ees 2 In the browser select the images SAA RRG e that you want to group together E Sequence View Ta 3DView l y J p 9 sii Units Counts C Use Previously Saved Colors 4 Se sF eTo select adjacent images in the e Planar Spectral imapin seq e Suface rovoganhr 7 DUT browser press and hold the Shift ee key while you click the first and last
54. is considered to contain a point light source at its center The index i enumerates the set of voxels S is the value of the strength 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 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 j surface element is the sum of the contributions from all the voxels Pi XG H 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 to be computed very rapidly Once the Green s functions Giz are known the goal is to solve Equation for the source strength S in each voxel The DLIT algorithm attempts to minimize 2 Equation 2 while requiring that the source strength in each voxel is positive Equation 3 2 l 2 X Fgh Pj Wys 2 i S 20 3 A combination of methods called Simplex and Non Negative Leas
55. 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 binning is often acceptable for in vivo images where light emission is diffuse For more details on binning see Appendix B page 156 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 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 stoo 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 t 16 to produce the sharpest image and a luminescent image is taken with a large aperture f 1 to maximize sensitivity For more details on t stop see Appendix B page 155 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 For systems equipped with spectral imaging
56. organ list Note To return the organs to the default orientation and scale click Reset 138 JEX HEA f Sequence View E 3D View gt Surface To Surface o E Transform tool Use Tab key i ibe SOE E EERE EETA rege azkan to restrict scaling to only one axis Pa 5 Scaling On 1 Circle line is thicker when selected 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 Jo return the organ drawing to the default position and size click Reset and the ee button 9 To turn off the transform tool click the button XENOGEN Living Image Software User s Manual ee E To check the organ position on the mesh Gi seqmncavaw E Dv 1 Check the organ position in the Coronal Sagittal and Transaxial windowpanes 2 In the 3D View tab click in the windowpane with the mesh 3 Press the V key or the button to display alternative views of the mesh Bottom Front Back Figure 10 25 Checking organ and mesh alignment This example shows alternative views of the skin that has been aligned on the mesh 139 a 10 3D Reconstruction of Sources Importing an Organ Atlas You can import an organ atlas Open Inventor format one organ per iv derived from MRI or CT scans NOTE The imported atlas must include a skin file named skin iv To import an organ atlas 1 Open the DLI
57. 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 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 intensity of shorter wavelength data Biotrue Preferences Preferences General User 3D View Tissue Properties DLIT Params Biotrue Host biotrue xenogen com User Name users Password seseeses Use proxy Ox tones Figure H 6 Preferences Biotrue tab These preference settings specify how the workstation connects to the optional Biotrue Collaborative Data Management System Preferences Blotrue Tab Description Host Location of the server on the network User Name Your user name Password Your password Use Proxy Choose this option if you connect to the Internet through a proxy server Proxy Server The name of the proxy server Proxy Port The port used by the proxy server If you use a proxy server check with your system administrator for the appropriate proxy server name and port information 209 210 _ H User Preferences H 2 Managing User IDs The settings specified in the Preferences box are associated with the user ID selected at the start
58. selected for in vivo image data because hot pixels can significantly affect an ROI measurement Cosmic ray correction is not recommended when imaging very small objects such as individual cells An individual cell may only light up one or two pixels and can sometimes be misinterpreted as a cosmic ray In this case clear the Cosmic Correction option in the Corrections Filtering tools to avoid filtering out single cell images Living Ilmage Software Users Manual XENOGEN a Camaag Life Changing Appendix B Detection Sensitivity CCD Detection Efficiency 2 0 0 0 2 eee eee ee es 155 BINAINO wow om wt ee ew oh amp oe amp SR Bede A Ae R He 156 SMIOUWING 2 essi ated ee ee ADR wee 4d eS eG 158 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 B 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 t
59. so you can identify and eliminate problematic materials If a background source is phosphorescent and the phosphorescent lifetime is relatively short you can try keeping the sample in the dark for a long period before imaging to reduce background light emission Occasionally there is no way to eliminate the natural light emission of the sample The natural light emission associated with living animals autoluminescence is a major area of interest in in vivo 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 XENOGEN eT _Life Changing Living Image Software User s Manual tite Changing 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 housed in Background emission levels were not reduced A phosphorescent component the dark 12 hours prior to imaging in mouse fur or skin is not the source of light emission Test 2 White furred animals were shaved No increase or decrease in background emission levels prior to imaging Test 3 Alfalfa known to be An alfalfa free diet reduced background emission slightly but not significantly phosphorescent was eliminated from the animal diet The sources of autoluminescence are not yet fully understood No external sources have been prov
60. that are saved to the system are available in the ROI Tools and can be applied to other images File Edit view Tools Window Help JAK i gt ey a Ld A amp he i Units Counts io Apply to all 5 Units Counts Display Overlay v ino gt ee A gt Image Adjust sid Min 40 O 0 9 X Max 11711 C Apply to Sequence Type Measurement ROI v Threshold F 25 gj 8000 Save ROls Name ROI_1_KSA v EA gt Planar Spectral Imaging RO 1 7 845e 05 4000 RO 2 2 903e 04 2000 Counts Color Bar Min 763 Max 9596 To save the ROI measurements with the image 1 Close the image Note If you are working with an image from an open sequence first close the image then close the sequence Click Yes in the confirmation message that appears The ROI measurements are saved to the AnalyzedClickInfo txt file associated with the image To save ROls to the system 1 In the Name drop down list confirm the default name or enter a new name for the RO s 2 Click Save The RO s from the image are saved to the system and can be selected from the Name drop down list to apply to other images To load ROIs on an image 1 Open the image of interest 2 In the ROI tools make a selection from the Name drop down list and click Load Note If you load ROI s onto an image then draw additional ROIs the Save button changes to Overwrite If you w
61. the Coarse Normal cosse isto 16 Up Update Field of View D Jv Normal M em Bef Fine Light Levet ho af ore or Fine option 3 Click Up or Down to move the stage and change the focus 4 If necessary select another f stop setting from the drop down list and adjust the light level using the E arrows 5 Click Update to apply the settings The resulting focal plane cm above the stage is automatically entered in the Subject height box 6 Click OK when the image is focused Figure 3 16 Manual focusing procedure 28 XENOGEN ee etal Living Image Software User s Manual Ute Changing 3 7 Saving or Exporting Image Data 7 File Edit view Tools Window Help JI x SARAH S RaR eeaeee eaS ESSES SSES SSS E Sequence View CEY E E Pianar Spectrai megng J 2Surface Topography gt DLIT 3D Reconstruction Units Counts C Use Previously Saved Colors To save the image data 1 Click the Save button f l Alternatively select File gt Save on the menu bar 2 In the 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 To export the image data to a graphic file for example bmp 1 Click the Export Graphics button mg 2 In the dialog box that appears select a directory choose a file type and enter a fi
62. the mesh or DLIT results For more details page 132 Select 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 136 Select to zoom in or out on the image use a click and drag operation Select a to move the subject in the window use a click and drag operation Select a to rotate the subject around the x y or z axis use a click and drag operation Drawing Styles PAN A h A drop down list of drawing styles for the mesh for examples see Figure 10 13 page 127 The Surface face drawing style a is the default Point cloud Wire frame Surface face Wire frame and surface face Shading Styles A drop down list of shading styles for the mesh for examples see page 133 The Reflect smooth surface face shading style is the default Smooth face Smooth surface face Reflect surface face Reflect smooth surface face Ai E 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 10 20 k Select this tool from the drop down list to display the perspective view Click to show or hide measurement cursors in the coronal sagittal or transaxial views Clic
63. the settings for the photographic image and choose the Reuse option 5 In the control panel select the Transillumination option and click Setup The Transillumination Setup window appears 6 In the Transillumination Setup window click the locations of interest Specifies the locations for transillumination and image acquisition jo m E E E Yi 7 In the sequence editor click E Add The acquisition parameters are added to the table Each row in the table specifies one image in the sequence Transillumination setup window 8 If you want to save the sequence setup information xsq a In the sequence editor click the Save button f l b In the dialog box that appears select a directory for the file enter a Tile name and click Save continued in Figure 3 13 Figure 3 12 Acquiring a transillumination fluorescence image sequence on the IVIS Spectrum Procedure continues in Figure 3 13 25 3 Initializing the System amp Acquiring Images To acquire a fluorescence image sequence in transillumination mode on the IVIS Spectrum continued from Figure 3 12 9 To acquire the images click Acquire Sequence in the control panel During image acquisition the Acquire Sequence button becomes a Stop button To cancel the acquisition click Stop 10 VWhen all of the images are acquired and displayed in the image window confirm that the signal of interest is above the noise lev
64. tissue optical property values to choose from E 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 E 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 E 4 An Example of Planar Spectral Imaging Melanoma cells were injected intravenously into the tail vein of nude mice After 13 days metastases developed in the lungs kidney and hind limb bone An image sequence was acquired on the IVIS Imaging System 200 Series using filters at six wavelengths from 560 to 660 nm in 20 nm intervals 183 E Planar Spectral Imaging 184 NOTE When using the 560 nm and 580 nm band pass filters tissue optics result in
65. 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 445 109 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
66. x a Fluorescent YM Photograph ie Medium fe x e Structure IV Overlay F biotic Alignment Grid aid System Statu Field of View C v i 2 Acquire Sevice ST om Subject height fj 50 cm Sequence Setup Focus f use subje Initialize TVIS system Click the temperature box to display the demand and measured temperatures of the CCD camera and stage System Status Temperature box color indicates Demand Measured a ea System not initialized Stage Temp 350 g H w oe System is initialized but CCD camera Temperature NN Locked temperature is out of range Fa 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 3 2 Checking the system temperature For luminescent imaging the system acquires two images e A photographic image a short exposure of the subject illuminated by the lights located at the top of the imaging chamber Figure 3 3 The photographic image is displayed as a grayscale image e A luminescent image a longer exposure of the subject taken in darkness to capture low level luminescence emission The luminescent image is displayed in pseudocolor that represents intensity The Living Image software automatically coregisters the luminescent and photographic image to generate an overlay image Figure 3 4 Living Ilmage Software Users Manual XE
67. 0 15000 10000 5000 Counts Color Bar Min 797 Max 20932 Loading Sequence C Documents and Settings Administrator My Documents enogen S ample Data TLT 20040401145348_SEQ Sequencelnfo txt Loading Dataset C Program Files enogen L 3 0 10 02 2006 TLT_M20040401145348_002 ClickInfo txt No Background image found Loading Dataset C Program Files enogen LI 3 0 10 02 2006 TLT_M20040401145348_002 ClickI nfo txt No Background image found Loading Dataset C Program Files Xenogen Ll 3 0 10 02 2006 TLT M2004040N45348 002 ClickInfo txt ie a Window x L Figure H 1 Preferences General tab top and main application window bottom The main application window conta ins the image window tool palette and activity window Preferences General Tab Description Startup Defaults Show Activity Window Displays the activity window at the bottom of the application window Dock Tool Panel Sets the position of the tool palette in the application window Choose left or right Window Size Specifies the dimensions of the main application window Width Height Sets the dimensions of the image window Default Click to apply the default settings Preserve Settings Color Applies the color settings of the active image data to subsequently opened image data Folder Path Sets the default folder path to the current folder path setting Click the Export button in the imag
68. 0 Fis 40 FOV 15 20 ome fV J0 ome FOV 25 Width cm Figure A 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 of fluorescent signals When you select efficiency for the image data Figure A 2 the software normalizes the fluorescent emission image to a reference image and computes Efficiency Radiance of the subject lumination 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 to 102 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 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 Th
69. 0 7 8x10 7 1x107 photons s cm sr F photons s cm2 sr Signal Background 7 8 Signal Background 7500 Min detectable cells 3 8x10 Min detectable cells 400 Figure D 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 3x106 cells in an 11 week old male Nu nu mouse XENOGEN Living Image Software User s Manual Uife Changing 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 79 D 6 Subtracting Instrument Fluorescent Background The fluorescence instrumentation on an IVIS Imaging System is carefully designed to minimize autofluorescence and background caused by instrumentation However a residual background may be detected by the highly sensitive CCD camera Autofluorescence of the system optics or the experimental setup or residual light leakage through the filters can contribute to autofluorescence background The Living Image software can measure and subtract the background from a fluorescence image Fluorescent background subtraction is similar to the dark charge bias subtraction that is implemented in luminescent mode However fluorescent background changes day to day depending on the experimental setup Therefore fluorescent background is not measured dur
70. 0 Max 2033 Max 51182 A photographic image is a gray scale pSseudoimage In Overlay display mode a pseudocolor image of the luminescent data is overlaid on a photographic image Color table associated with the data Figure A 1 Example pseudoimages Overlays 150 A pseudocolor scheme is typically used to display the numerical contents of scientific image data like the luminescent or fluorescent images acquired on an IVIS Imaging System The pseudocolor scheme makes it easy to see areas of bright light emission The amount of light emission can be quantified using measurement ROIs For more details Drawing Measurement ROIs page 59 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
71. 000 10000 soo UselD KSA LabelSet penyrernnmyere Check any 5 fields Counts T re at E Max 20932 V Experiment V Commenti t I Comment2 Sadia p 8 Confirm that the signal of interest is above the s _ _ _ noise level and below CCD saturation Check the M ArimatWodel ee image min and max at the top of the color bar M Sex po aua T A signal of interest greater than 600 counts and less I View a cy than 60 000 counts is recommended If the signal Tet bine x level is unacceptable adjust the exposure time or Bi zj binning level I Treatment F Luc Injection Time Inthe Edit Image Labels box that appears enter the IACUC Numb A i moii et M image information and click OK If you do not want Er e to enter label information click Cancel Cancel Note For details on how to save or export the image Edit Image Labels box data see Figure 3 17 page 29 Figure 3 9 Acquiring an image on the IVIS Imaging System 3D Series continued from Figure 3 8 21 a 3 Initializing the System amp Acquiring Images Image Applications that Analyze an Image Sequence Some types of image analysis require an image sequence Table 3 7 Figure 3 10 shows how to acquire an image sequence You can also create a sequence by grouping together images that were acquired during different sessions For more details see Chapter 4 page 38 Table 3 7 Analyses performed on an image sequen
72. 01__ 9 934e 01 ___ 1 300e 01_ 4 410e 02 TLT20050616134836 001 AOL2 Overlay 6 383e 05 _ 1 620103 13 0766103 11 1006101 71 7386104 click the Measure TLT20050616134836 001 ROIS Overlay iS 30e 02 _ 5 208e 01 9 132e 00 2 200e 01 2 600e 01 button Y TLT20050616134836 002 R0I1 Overlay 1 121e 05 23 514e 02 3 171e 02 3 000e 00 1 284e 03 7 s The ROI label Customized Selections Copy j d iS 0 lays th e Measurements Types Click Attributes ROI Dimensions J Som anf intensity and the Counts none _hone_ Reftesh Configure Export Close Figure 5 2 Manually drawing measurement ROIs 60 ROI Measurements table appears aeo Units Counts Display Overlay 115 X E Image Min 40 Max 11711 8000 6000 4000 2000 Counts Color Bar Min 1103 Max 9596 Living Image Software User s Manual XENOGEN ee _Life changing Life Changing Drawing a Measurement ROI Using the Auto ROI Method The Living Image software can automatically draw measurement ROIs on an image To identify ROIs the software e Locates the peak pixel intensities in the image e Searches the neighborhood around the peak pixel and includes a pixel in the ROI if the pixel intensity is greater than the threshold a user specified percentage of the peak pixel intensity To draw measurement ROls using the Auto ROI method 1 Open the image o
73. 0508261 24002 002 hold the Shift key while YOu click the first and gt last Image in the selection Note In the overlay that is generated the signal in each image is assigned a different color table SRD The photographic image is at the bottom of the pas pers stack and the last fluorescent or luminescent oo image selected from the list is at the top of the stack I BlackBlue To reorder the images in the list 6 To remove all fluorescent or luminescent images 1 Choose the Edit Layer Properties option from the overlay click the B button 2 Select an image 7 To change the display of a fluorescent or luminescent image in the overlay 3 Click the 4 or arrows 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 8 To copy the overlay image to the system clipboard click the Copy button Ea 9 To export the overlay image to a graphic file click the Export button Cr Figure 6 4 Creating an overlay image of multiple reporters The image overlay window displays two different fluorescent signals on one photographic image 83 a 6 Performing Image Math Operations This page intentionally blank 84 XENOGEN Living Image Software User s Manual a 7 Planar Spectral Image Analysis Image Sequence Requirement
74. 20 0 0 0 eaaa Sa aaka 7 3 Initializing the System amp Acquiring Images 9 3 1 Initializing the IVIS Imaging System 2 2 2 00 0000 eee 9 39 Ilmarine Basics gc 2 28 eh ee bea ebe eee REE REDS HERE EEE ES 10 3 6 Manual Focusing s 282 eis 4 ebooh44 00 G4 4266 6 oe boo He Bo S 28 4 Working With Images 0 0 00 00 cee eee 31 4 1 Opening Image Data oon aaa a a 31 m2 The TOOLPAICUC 242224846 h ae REGED AY GEE SPREE eR ee eS os 34 4 6 Viewing Image Information 0 0 00 eee ee ee 43 4 8 Correcting or Filtering Image Data 0 0 0 2 0 2 0 000088 46 4 10 Image Layout Window a aooaa 54 5 Working With Region of Interest ROI Tools 57 Dsl ADOUUROIS sibs Soe RES eh nirani oe oe heaga aaa 57 3 INOUTOOIS 24a ee eee bee eee a eee hee ee bea eee eee Ge 58 5 3 Drawing Measurement ROIs 2 0 2 a 59 5 5 Measuring Background Corrected Signal 2 2 ee ee ee ee 62 5 6 Managing ROIs 2 2 a 64 6 Performing Image Math Operations 77 6 1 Using Image Math to Create a New Image 000 78 6 2 Subtracting Tissue Autofluorescence 2 ee 79 6 3 Overlaying Multiple Images nono noaa a ee 82 7 Planar Spectral Image Analysis 0 85 7 1 Image Sequence Requirements for Planar Spectral Image Analysis 85 7 2 Planar Spectral Imaging Tools aoaaa ee 86 8 Point Source Fit
75. 29 exposure time 156 F f stop fluorescent imaging 170 field of view 156 filter bandpass 167 fluorescent 169 filter spectra 167 flat field correction 47 flat fielding 153 FLIT results 123 125 fluorescence reconstruct 3D sources 118 121 fluorescence efficiency 170 171 fluorescence imaging components 165 167 fluorescence transillumination 18 fluorescent filters 169 fluorescent imaging efficiency 167 f stop 170 normalization 167 FOV settings 16 G graphic image data 149 grouping images into a sequence 38 H High Reflectance Hemisphere 161 histogram 50 image 212 adjusting appearance 45 cascade 42 correcting filtering tools 46 47 histogram 50 information 43 label information 44 line profile 52 luminescent 10 measurements 53 opening 34 overlay 10 photographic 10 pixel data 50 tag 42 thumbnails 36 tile 42 image acquisition image sequence 23 24 single image 18 20 IVIS 3D 20 21 image data browsing 31 save 29 image information 48 49 image layout window 54 55 image math 78 79 image overlay tool 82 83 image sequence create from individual images 38 edit 37 image sequence acquisition 23 24 image window 36 3D perspective 135 display modes 39 index of refraction 124 information about an image 43 initialization See system initialization instrument fluorescent background background instrument fluorescent background 177 IVIS Imaging System fluorescenc
76. 8 179 well plate 172 173 autoluminescence 62 average background ROI 57 62 B background fluorescent 172 178 light on sample 160 162 tissue autofluorescence 178 179 background light from sample 162 164 on sample 160 162 background corrected signal 62 63 band gap 168 bandpass filter 167 binning 47 156 158 bioluminescent sources 3D reconstruction 115 125 browsing for data 31 XENOGEN e Life ile Life Changing C cascade images 42 color table 149 composite image 78 79 control panel 12 16 conventions 3 copy ROI measurements 73 correction filtering tools binning 47 cosmic correction 47 dark background subtraction 47 flat field correction 47 smoothing 47 cosmic correction 47 cosmic ray corrections 154 counts definition 151 crop box 54 D dark background subtraction 47 dark charge 160 dark current 159 data graphic image 149 scientific image 149 detection efficiency 155 detection sensitivity adjusting the lens aperture 155 exposure time 156 field of view 156 DICOM files 29 DICOM format 29 diffusion model 182 display modes 39 display units counts 151 efficiency 152 photons 152 displaying organs 137 139 DLIT results 123 125 drift correction 159 211 a Index E edit image sequence 37 efficiency 152 167 170 171 electronic background dark charge 160 dark current 159 drift 159 read bias 159 export image data DICOM format 29 graphic file 29 exporting to DICOM
77. 8 undock 35 toolbar 199 total source flux 123 transillumination 18 U undock tool palette 35 uniform surface sampling 124 197 units See display units user IDs 210 user preferences 203 210 V voxel 130 131 192 size increment 196 size increments 124 size limits 124 196 vsize final 123 starting 123 W well plate autofluorescence 172 173 X Xenogen Corporation technical support 4
78. 9 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 A page 153 Note In photons mode flat field correction is a mandatory default In counts mode the check box can be cleared Cosmic Correction Select this check box to correct image data for cosmic rays or other ionizing radiation that interact with the CCD For more details on cosmic correction see Appendix A page 154 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 4 18 For more details on binning see Appendix B page 156 Smoothing Computes the average signal of the specified number of pixels and replaces the original signal with the average signal Figure 4 18 Smoothing removes signal noise without changing pixel size Note This type of smoothing is defined differently from the smoothing performed in the Living Image 2 5 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 4 18 Example of binning and smoothing image data 47 a 4 Working With Images 4 9 Image Inform
79. ALSE only the NNLS algorithm is used to seek the optimum 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 cm2 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 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 source solutions which best fit the measured photon density Measured The photon density determined from the luminescent 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 save the active DLIT or FLIT results 124 XENOGEN Living Image Software User s Manual ee ee Item
80. 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 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 not displayed 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 Color Table A yy Click the drop down arrow to select a color table for the image data For more details on color Rainbow kd L tables see Pseudocolor Images page 149 Reverse Choose this option to reverse the min and max
81. Ax H Units Counts Display Overlay l Adjust L Image TLT20050624122348_001 gt Corrections Filtering Fri Jun 24 2005 12 24 53 gt Image Information Em Filter 560 f Bin MJ8 FOV 12 6 f2 1s ROI Tools Camera IVIS 200 Beta IL SIE20EEV gt Surface Topography jlmage Information Sequences CK20050624151158 SEQ Images CK20050624151158_002 Em VT gy Series Male Nn nu Experiment DOB 02 28 05 Label Two traser beads Scruff Comment Dorsal Section Label User Label Name Set luminescent image photographic image enogen Default luminescent TIF photograph TIF 5 Value Friday June 24 2005 JA X Max 28314 15000 10000 5000 Counts Color Bar Min 397 Max 18712 List of images in the selected sequence or open single images if Individual Images is selected from the Sequences drop down list 43 _ 4 Working With Images Editing Image Label Information 44 You can edit user label information after image acquisition A TL1T20050624122348_001 Units Counts Display Overlay A Click TLT20050624122348 001 Series Male Nn nu Fri Jun 24 2005 Experiment DOB 02 28 05 Em Filter 560 Label Two traser beads Scruff Bin M 8 FOV 12 6 f2 1s Comment Dorsal Camera IVIS 200 Beta Il SIB20EEVY Analysis Comment test 1 Open the image ot int
82. Delete Load Save Z DLIT 3D Reconstruction Analyze Params Properties Results Sequence CX2O0050629757758_SEQ Tissue Muscle Source Fres Select Wave Filters Filter Views Minimum Radiance eiTe Perspective Figure 10 9 Reconstructing the mesh continued from Figure 10 8 120 XENOGEN Living Ilmage Software Users Manual nn Life Changing Table 10 6 Surface topography options amp parameters Item Description Surface Generation Options Structure Light Photo Back Projection Surface Smoothing Parameters Smoothing Level Restore Loss Recovery Smooth Save Result Name Delete Load Save A drop down list of structured light images in the sequence Select one for use in reconstructing the mesh This option is only available for data obtained on an IVIS imaging System 3D Series If this option is chosen photographic image data is also used to reconstruct the mesh The amount of smoothing to apply to a reconstructed mesh Removes smoothing that was applied to a mesh Make a selection from the drop down list None Volume Height Restores the type of data selected in the Loss Recovery drop down list volume height or none that was removed by the smoothing process and applies the selected smoothing level The name of the mesh Click to delete the mesh selected from the Name drop down list Click to load the mesh s
83. Figure 3 10 24 XENOGEN an Cumaeg Living Image Software User s Manual Tp a To acquire a fluorescence image sequence in transillumination mode on the IVIS Spectrum 1 Click Sequence Setup in the control panel to operate in sequence acquisition mode The sequence editor appears j IVIS Acquisition Control Panel mixi Imaging Mode Exposure Time Binning F Stop Excitation Alter Emission Filter e Display Photographic Settings a EE Mesum zio e CE E ee eee caesarean Eo 1 Medium 2 605 se No ic 1 50 6410 7638 Structure 1 Medium 2 605 680 High No C 1 50 6410 7638 4 V Overlay I Lights JY Alignment Grid 1 Medium 2 605 700 High No Ic 1 50 6410 7638 1 Medium 2 605 720 High No 1 50 6410 7638 Field of View C z ieee eee Service 13 4 cm kis ET E Subject height 1 50 cm Image Setup F gt Focus use subject height Temperature I Locked Initialize IVIS System Delay 0 0 Han Applyto Al X Remove A Update 1 Insert oma L EAE Sequence Editor 2 If necessary click Femve and select All to clear the table 3 In the control panel specify the settings for the fluorescence image exposure time binning F Stop excitation filter emission filter For more details on image acquisition settings see Figure 3 6 page 18 4 Specify
84. Frame Factor 1 Confirm that the 3D view shows the properties of interest for example organs voxels mesh or photon density maps In the 3D Tools click the Animation tab Animation Setup Time Scale J 4 lf necessary clear the key frame rria box click the button and Kaie Ja select Delete All cin s teed nS a 5 To view a preset animation gt Fey Recod make a selection from the auclictaes naa a Presets drop down list See Sie Table 10 7 for a description of the preset animations A list of the key frames appears 6 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 Click Record 2 In the dialog box that appears choose a directory and enter a Tile name mov mp4 avi and click Save Figure 10 29 Viewing a preset animation Table 10 7 Preset animations FRRRRRERRHERRRRRRERRERRRRRRRERRRI X i l gt Planar Spectral Imaging E gt Surface Topography The software provides several predefined animations Figure 10 29 or you can create your own For more details on creating an animations see Figure 10 30 JA K Sequence View 3DView
85. IS Lumina IVIS Spectrum IVIS Imaging System 100 Series or 200 Series Procedure continues in Figure 3 7 18 Living Ilmage Software Users Manual XENOGEN eae tie Life Changing To acquire an image on the IVIS Lumina IVIS Spectrum IVIS Imaging System 100 Series or 200 Series cont from Figure 3 6 Click Info in the image window to display the label set information After image acquisition is Fie Edit view Tools Window Help EA K completed the image window i a E ee oe Fi ag A ele lt Units Counts Apply to all displays the overlay image a aD a LA m The tool palette and Edit a Units Counts Display Overlay zro iy Image Labels box appear C Click TLT20050624122348_002 Series Male Nn nu Corrections F une Fri Jun 24 2005 Experiment DOB 02 28 05 z Em Filter 580 Label Two traser beads Seriff a Bin M S FOY 12 6 fe 13 Comment Dorsal Tool ore Camera VIS 200 Beta l SIB20EEV Image Min 39 Max 47071 ane UselD KS4 LabelSet pyannneennsren 20000 Check any 5 fields iV User nn 10000 V Group V Experiment V Commenti x Counts M Commente zi Color Bar Time Point 7 Min 235 Max 38915 Animal Number ce M AnmalStan xf J Animal Model i e SM Sex ls M View io T Cell Line p oe M Reporter e n Treatment a Lue Injection Time i gt a
86. IVIS Imaging System 200 Series select the tissue acquisition wavelengths 560 660 nm 6 If you want to view the tissue properties Ma Wes Usp Or source spectrum for the tissue and light source selected above make a selection from the Plot drop down list in the Properties tab 4 For images acquired on the IVIS Imaging System 3D series select the acquisition filters 550 585 nm 585 620 nm and 620 700 nm All view angles are selected by default for each acquisition filter Figure 10 10 Reconstructing 3D fluorescent sources 122 Planar Spectral Imaging gt Surface Topography Tool Palette E Planar Spectral Imaging O Analyze Params Properties Results Sequence COSA TTo SER Tissue sfnascte Source A ESAE Select Image Sources Source EswL EmWwL MinRadiani 640 T20 2 66e 06 B40 Yel 2 obe 016 B40 fell 2 66e 06 B40 720 2 86e 06 640 feQ 2 8Bbe 06 B40 pn 2 66e 06 640 feQ 2 6be 06 B40 720 4 93e 06 640 feu l16e 07 Reconstruct gt 3D Tools gt Spectral Unmixing H a H i Ho i T 0g i J 7 In the Analyze tab select the source locations to include in the analysis and click Reconstruct Figure 10 7 page 117 shows example 3D reconstruction results Living Ilmage Software Users Manual XENOGEN e ite Life Changing 10 4 DLIT amp FLIT Results The Results tab displays information a
87. NOGEN ee _Life changing Life Changing For IVIS Imaging Systems with fluorescent imaging capability a photographic and fluorescent image can be acquired and coregistered For more information on fluorescent imaging see Appendix D page 165 Illumination LEDs Opening for camera lens Sample stage Figure 3 3 IVIS Imaging System 100 Series interior view i Photographic image Luminescent image Overlay image Figure 3 4 Image types The software automatically coregisters the luminescent and photographic images to produce the overlay image 11 a 3 Initializing the System amp Acquiring Images 3 4 About the Control Panel IVIS Lumina and IVIS Imaging System 100 Series IVIS Spectrum and IVIS Imaging System 200 Series IVIS Imaging System 3D Series Figure 3 5 Control panel 12 The IVIS System control panel provides the image acquisition functions Figure 39 NOTE 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 f TWIS Acqursition Control Panel F Step Excitation Alter Emission Rilter 2 Lf xd Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter Luminescent 00 2 sec Medium z z foen z This row specifies settings for the re luminescent or L A A an fluorescent image er a
88. OGEN e fe Life Changing 8 3 Checking the Point Source Fitting Results 8 4 Exporting Results 3 Compare the 1 In the Results tab click Photon Density Maps The Photon Density Maps window appears P Pilon Density iapa 2 Selectthe image from ag BE E the Image sources drop down list simulated and measured photon densities To export all results 1 In the Results tab click Export results 2 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 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 In the dialog box that appears choose a folder for the results enter a file name txt and click Save Tool Palette x eSutece Tonoa O feat See Fang E Error Estimation Starting Chi 2 8 58e 09 Ending Chi 2 7 03e 08 Point Source Fitting Results Fitted value 0 1 43 crn 6 676 cm Parameters Mua Em Mus Em Mueff 1 710 cm Diff 0 049 cm Total source flux 2 22e 1 1 photon s x saava sh misoak source 0 181 Vetem K Photon Density Maps Density Maps Err A Save Results Name LMFIT_ 13 v gt FLIT 3D Reconstruction gt 3D Tools 97 a 8 Point Source Fitting 8 5 Managing Point Source Fitting Results
89. ROI in the image The ROI Properties box appears and displays the positions and dimensions of the selected ROI 2 Enteranew width or height value in the ROI Properties box 3 To lock the current ROI size choose the Lock Size option Note The ROI size cannot be changed until the Lock Size option is cleared 67 5 Working With Region of Interest ROI Tools Editing the ROI Line ROI Properties F pi ROI ROI 7 ROI Label Rol Bkg ADI Subj ROI Info Click Number CK20031215152405_001 v ROI _none_ v V Lock Position Height cm 1 0500 I _ _ _ Xefiem 33450 Yelem 73500 Angle deg 0 0000 M Lock Size Width cm 0 3450 Line Size f2 2 Line Color Ezam EA To edit the ROI line thickness 1 Enter a new value in the Line Size box Alternatively click the 4 arrows To change the color of the ROI line 1 Click the Browse button __ The Select Color box appears 2 To select a basic color for the ROI line click a basic color swatch and click OK 3 To specify a custom color drag the crosshairs in the custom color field adjust the brightness slider and click Add to Custom Colors 4 To select a custom color for the ROI line click a custom color swatch and click OK Pl Select color Basic colors P Basic colors Cross hairs in the custom color field Custom colors Brightness slider
90. T results that are associated with the organ atlas 2 Select File Import Organ Atlas on the menu bar The Import Organ Atlas box appears 3 Click Add Organ Files 4 Inthe 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 In the Select Skin Mesh drop down list select the skin organ file 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 Figure 10 26 Importing an organ atlas 140 CEG Edit view Tools Window Help JA K g Open Ctrl O hh BJ Browse Ctrl B HHE Sequence View 4 3D View 4 A Browse Biotrue Ctrl R i Save Ctrl Save As d gt DICOM Export Print Ctrl P Print Preview lo Qh Recent Files gt Exit Threshold Intensity Color T able C Reverse C Logarithmic Scale Source Yoxel Measurement Total Flux 0 00 phofoms sec Volume 800 mar3 Center of Mass 900 000 000 13 0 photons sec Perspective Source Intensity lt Pj Import Organ Atlas Select Skin Mesh PE Organ Files Generate Mesh Corefficients Organ Atlas Mame Add Organ Files Save Organ Atlas Close
91. Total source flux phot s DLIT result The sum of the bioluminescent source intensities Total fluorescence yield N mm The total sum of the fluorescent yield The quantity measured is FLIT result Fluorescence quantum efficiency for the excitation wavelength to emission wavelength photons Excitation wavelength photon absorption cross section Fluorophore number density Volume of voxel size 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 2 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 123 _ 10 3D Reconstruction of Sources N surt 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 60 for IVIS Imaging System 3D Series data or 70 for IVIS Spectrum or IVIS 200 or IVIS Spec
92. 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 152 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 D 7 To eliminate the excitation light as a variable from the measurement the data can be displayed in terms of efficiency Figure D 8 XENOGEN Living Image Software User s Manual Life Changing o Intensity Nn wo Ss o o N oo Pa kam Width cm Figure D 7 Illumination profiles for different FOVs on an IVIS Imaging System 100 Series measured from the center of the FOV To enable a more ARW20050826124002_001 quantitative Units Efficiency Display Overlay Mi Finto g comparison of fluorescent signals choose Efficiency EEA Min 4 43e 5 Max 5 07e 4 Efficiency Color Bar Min 6 86e 5 Max 4 69e 4 Figure D 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
93. When this option is chosen data you select in the Browse for Load as Group Folder box is added to the Living Image browser If this option is not chosen data selected in the Browse for Folder box overwrites the Living Image browser contents Enables you to select images acquired during different sessions and open them as a sequence To select adjacent click numbers in the browser press and hold the Shift key while you click the first and last file in the selection To select non adjacent click numbers 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 Note Load as Group is only available if single images not part of an image sequence are selected Browse Opens the Browse For Folder box Figure 4 1 Load Opens the selected image or image sequence Close Closes the Living Image browser Figure 4 2 shows how to open image data from the Living Image browser A sequence is a collection of images that are included 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 a sequence might include a spectral filter scan or images taken at different exposure time or different angle views on the IVIS Imaging System 3D Series Images acquired during different sessions can al
94. X 2 a aA a amp R Units Photons C Apply to all Sa SA EELE EEEE ECEECEE SESSE SESS S Units Photons Display Overlay gy 5 7 gt Image Adjust i ROI 4 0 416e 05 F Planar Spectral Imaging ai Min 5 25e3 p er Analyze Properties Results Sequence CX200372757504 9 SEQ Tissue Muscle Source Frey Select Filters Filter 560 1 0 s 6 520 ae ROI 3 2 633e 06 x10 500 520 540 560 ROI 5 5 486e 06 5 05 al Ed z p sec em 2sr z Color Bar Select All ROI ae sie Rls Al ts uae Te l gt Surface Topayranhy 2 Click Planar Spectral Imaging in the tool palette 3 In the Analyze tab select the emission filter wavelengths for the analysis e Click Select All to choose all wavelengths at which images were acquired e Alternatively select only particular wavelengths of interest Note It is recommended that you do not Include a wavelength in the analysis if the signal is less than or equal to the autoluminescent background If autoluminescent background is a concern you can create a background ROI and link it to the measurement ROI prior to planar spectral analysis For more details see Drawing an RO Using the Free Draw Method page 62 4 Select the ROIs for the analysis a Open the image with the measurement ROI s that include the source s to be analyzed b In the ROI drop down list select All or a particul
95. a larger attenuation of light due mainly to hemoglobin absorption A longer exposure time is recommended at these wavelengths Figure E 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 CK20031215150449_ 008 Units Photons Display Overlay x ino g Image Min 1 26e3 Max 9 76e5 p sec em 2 sr Color Bar Min 5 69e4 Max 4 48e5 Figure E 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 E 3 After the ROI is defined start the planar spectral analysis for more details see Performing Planar Spectral Image Analysis page 88 The software e Measures the total flux inside the ROI on each filtered image e Normalizes the data to the luciferase spectrum Plot of Intensity vs Lambda Figure E 4 e Fits the normalized data to the analyti
96. a quick guide to drawing measurement ROIs For more details on ROI drawing methods see ROI Drawing Method See Page Manual Automatic Free draw Measurement ROI Measures the signal intensity in an area of an image 60 61 62 ROI Types Average Background ROI Measures the average signal intensity in a user specified area of the image that is considered background Note Using this type of ROI is optional If the animal has significant autoluminescence or autofluorescence you can determine a background corrected signal in a measurement ROI by subtracting an average background ROI from a Subject ROI Identifies a subject animal in an image Note Using this type of ROI is optional 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 measurement ROI Available ROI Drawing Methods e Manual e Automatic e Free draw e Manual e Free draw e Manual e Automatic e Free draw Circle square grid or contour Available Shapes Circle or square Square 57 5 Working With Region of Interest ROI Tools 5 2 ROI Tools To display the ROI tools 1 Open an image 2 Click ROI Tools in the tool palette Note An image must be active to display the ROI tools If an image sequence Is active the ROI tools are absent from t
97. a tag to an image Organizing Images When multiple image windows are open you can organize them in a cascade or tile arrangement Figure 4 13 Figure 4 13 Image windows tiled left or cascade right 42 Living Image Software User s Manual XENOGEN ee Life changing Life Changing 4 6 Viewing Image Information Information about an image that is captured at acquisition includes all of the text information that is saved with every image for example camera parameters and user labels To view information about an image 1 Open the image or image sequence of interest For details on how to open data see Figure 4 2 2 Select View Image Information on the menu bar The Image Information window appears List of open image sequences Choose Individual Images from the list to show the open single images in the Images drop down list 1 To choose an image make a selection trom the Sequences drop down list and the Images drop down list 2 To view information of interest click a category in the upper box The information is displayed in the lower box For example if you select luminescent image the luminescent image acquisition parameters are displayed Figure 4 14 Viewing the image information Click Info to display the label set information and acquisition information for the image File Edit view Tools Window Help i gA BW a amp Units Counts v C Apply to all
98. ace 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 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 197 _ F 3D Reconstruction of Light Sources This page intentionally blank 198 Living Image Software User s Manual XENOGEN Life changing Life Changing Appendix G Menu Commands amp Tool Bar Figure G 1 Living Image toolbar Menu Bar Command File Open File gt Browse File gt Browse Biotrue File gt Save File gt Save As File gt Import Organ Atlas File gt Import gt DICOM File Import 3D Mesh File gt Import 3D Volume
99. acteria Bacterial luciferase 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 Xenogen Corporation 193 a F 3D Reconstruction of Light Sources You can view tissue optical property values up 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 Fat or muscle are good choices for general reconstructions NOTE Default tissue optical properties and source spectrum are specified in the Preferences box For more details see Appendix H page 207 F 2 Algorithm Parameters amp Options This section explains the user modifiable DLIT algorithm parameters and options Analyze Tab Tool Eee ss Taal Palette Tissue and a T L Planar Spectral Imaging O source are arer eeaeee specified in the V DUIT 3D Reconstruction 7 FLIT 3D Reconstru
100. age acquired at binning 4 and FOV 20 cm has the same spatial resolution as an image acquired at binning 8 and FOV 10 cm Due to the increase in binning the latter image has a four fold increase in sensitivity compared to the former A charge coupled device CCD is a photosensitive detector constructed in a two dimensional array of pixels After an image is acquired each pixel contains an electrical charge that is proportional to the amount of light that the pixel was exposed to The software measures the electrical charge of each CCD pixel and assigns a numerical value counts For more details on counts and other measurement units see Appendix A page 149 The resulting image data comprise a two dimensional array of numbers each pixel contains the counts associated with the amount of light detected XENOGEN ee ie Ba Living Image Software User s Manual p E 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 B 3 Binning 1 Binning 2 Binning 4 CCD pixel Signal 4 times larger Signal 16 times larger Spatial size doubled Spatial size quadrupled Figure B 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 fou
101. age selected from box A TLT20060510111201_006 wl TLT20060510111207_001 A TLT20060510111201_002 TLT20060510111201_003 TLT20060510111201_o004 TLT20060510111201_005 z TLT20060510111201_006 J o a Color Scale Limits for amp and B rom DOXx Full Result Color Scale Limits Full uo Min 0 O Auto Result A B k al New image 2 Select Tools Image Math for xx_SEQ on ka froo za generated by the menu bar the Result 7 with Photo from 4 s function The Image Math window appears i 3 In the Image Math window select an image of interest from box A and box B Note For more details on items in the Image Math window see Table 6 1 79 AULT _ Me00bOS 10011201 _ 005 New image overlay LEDE mode 4 Select a mathematical function from the Results drop down list 5 To include a scaling factor k in the function enter a value for k The Image Math window shows a thumbnail of image A image B and the new image To view the new image 1 Click Display Result for Measuring The mathematical result is displayed To save the new image 1 Click the Save button fg Alternatively select File Save on the menu bar 2 In the dialog box that appears select a directory and click Save A folder of data is saved to the selected location AnalyzedClickInfo txt Clicklnfo txt luminescent and photographic TIF images To export the new i
102. ain a histogram of the photograph select Photograph from the Display drop down list To display the line profile File Edit View Tools Window Help Ce Cay Gy A ka A amp k Units Counts C Apply to all E EEEE E EE EEEE EEEE Bed Units Counts Display Overlay ca gt Image Adjust ale 1 Open the image of interest i i TLT20050624122348_001 Series Male Nn 2 In the Image Information tools click the Hae ns T E Line Profile button AL Em Filter 560 Label Two traser beads Scruff nl Bin MJ8 FOV 12 6 f2 1s Comment Dorsal A line appears on the image and the Line 6 z1 E Unts Cm v Camera IIS 200 Beta IL SIB20EEV Profile window appears image Image Binning Min 48 Max 28314 3 To view the line profile at different Image X Y 8674 4 737 cm locations in the image put the pointer ape sh dias oS sii i Crop Distanc over the line When the pointer becomes pa 0S A 000000 B 000000 Pevcensecenry at drag the line over the image re ees 10000 at i 200000 Distance 0 00 The line profile is updated as the line 4 a sai moves over the image i Color Bar Min 397 Max 18712 The blue line determines 2 i s Aline Frolla Window E Je the pixel intensities that Line Orientation Horizontal Width 1 Position 30 L Bey are plotted in the line profile graph Move the Min 0 00 Max
103. all subject ROIs in the click 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 of the dialog box The Bkg ROI tab shows a drop down list shows all average background ROIs in the click number selected above that can be linked to a user specified measurement ROI or subject ROI selected from the drop down list at the top of the dialog box ID User entered information about a subject ROI Label Label name of the selected subject ROI Lock Position Choose this option to lock the position of the ROI selected in the image XC X 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 the ROI selected in the image for more details on setting the units see ROI Dimensions page 73 Height Height pixels or cm of the ROI selected in the image 65 5 Working With Region of Interest ROI Tools Items in the ROI Properties Box Description 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 g Done Click to close the ROI Properties box and apply any new settings including e Linkages b
104. 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 uep 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 C page 162 Itis 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 187 a E Planar Spectral Imaging This page intentionally blank 188 XENOGEN fe ezeas Life Changing Living Ilmage Soft
105. ant to save this collection of ROIs using the existing name click Overwrite Figure 5 12 Saving ROIs and loading ROIs onto an image o XENOGEN Living Ilmage Software Users Manual ae Life Changing Deleting ROIs You can delete ROIs from an image or permanently remove ROIs from the system To delete ROIs from an image Fie Edt view Tools Window Help gt i JAX S AH a amp hk Units Counts C Apply to all 1 In the ROI tools click the XJ button TR Units Counts Display Overy Info iy 2 Make a selection from the drop down list of delete commands m The specified ROIs are deleted Sie na Y Mane TIZI from the image B ype easuremen Note This does not delete ROIS esz Fe Meronen 200 y All Autos saved to the system global save SaveROls l ae Name ROI_1_KSA All Subjects 6000 ROI 1 ROI 1 7 845e 05 gt Planar Spectral Imaging ROI2 4000 ROI 2 2 903e 04 Color Bar Min 763 Max 9596 File Edit view Tools Window Help JAX i sga H ERE R i Units Counts io Apply to all HSS SSSA SSE Unk Couns iDisk Overy al E Courte x papy setes lt i Eve __ E Eonections 7 Fiteing E mage Information To permanently remove ROIs from o5 96 Y X Min AD the system C Apply to Sequence Type Measurement ROI vi 1 In the ROI Tools select the ROIs TS eat M Threshold E 25 gl that you want to del
106. ar ROI for the analysis If there is no measurement ROI open an image from the sequence and draw an ROI that includes the area for analysis For more details on drawing ROIs see Drawing Measurement ROIs page 59 Note You only need to draw an ROI on one image in the sequence The software copies the ROI to all other images of the sequence during the analysis The ROI should include as much of the light emission from a single source as possible without including too much background continued in Figure 7 4 Figure 7 3 Performing planar spectral image analysis continued in Figure 7 4 88 Living Ilmage Software Users Manual XENOGEN e fe Life Changing To perform planar spectral image analysis continued from Figure 7 3 Taal Palette El E Image Adjust cimchint hes E Conections Filtering E e Image Information gt ROI Tools ji f Planar Spectral Imaging i Analyze Properties Results Sequence A200 ZA tadaa Sig Tissue Afke Source Sre Select Filters Filter amp 560 s 620 Mean PEED E Surface Topography f ee 5 Choose the tissue properties a In the Properties tab make a selection trom 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 The software automatically sets the internal medium index of refraction based
107. arted Starting the Living Image Software 0 000 eee 5 Basic Living Image Software Tasks aooaa a 6 Living Image Help 444 4 aa a 7 For information on installing the software see the Installation Guide included on the Living Image CD ROM By default the software is installed to PC C Programs Xenogen Living Image 3 0 Macintosh Applications Xenogen LivingImage 3 0 2 1 Starting the Living Imagee Software All components of the IVIS Imaging System should be left on at all times because of 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 Living Image Alternatively click the Living Image software desktop icon FY Macintosh Users Click the Living Image software desktop icon or run the software from the application folder 2 When prompted select a user ID or enter a new User ID up to three letters and click Done The Living Image software user interface appears Figure 2 1 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 a
108. ates if column wise weighting was used Row Weighting Mode Indicates if row wise weighting was used Click to display the spectrum plot tab Plot Concentration Click to display the concentration plot tab 9 4 Spectral Unmixing Options The Options tab in the Spectral Unmixing tools shows the user modifiable parameters in the spectral unmixing algorithm Figure 9 8 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 E gt Image Adjust RB 5 Planar Spectral Imaging EA 7 Spectral Unmixing i Al ce Options Spectrum Results a Lock Unimod HP LP CI N Mia NAA E NAA Nea NAA f OE ort 0 Force Denise by PEA Medium Defautt Normalization Equal Height sl Unimod Tolerance 5 5 PCA Mode Correlation _ Explained Variance Boel A ales l Biplat ss Figure 9 8 Spectral unmixing options 106 Living Ilmage Software Users Manual Spectral Unmixing Option XENOGEN fe aa Life Changing Description Constraints Init Lock Unimod HP Sort Force Denoise by PCA Normalization Unimod Tolerance PCA Mode Explained arance The constraints for unmixing the components The method for generating the initial guess of the spectrum for the selected component Auto means this is automatically determ
109. ation V Render Organs Opacity 100 Se Organ Atlas Mouse Atlas Female Organs Adrenal O Bladder Blood Brain Cecum Colon Eyelnside EyeQutside eet Ba 1 Sequence View b 3D View Surface Topography JAKI photons sec Source Intensity H Rigid registration Performs linear transformation but keeps the shape of the atlas mesh A Full registration Performs linear transformation and volume deformation The organs are displayed in the anatomically relevant position on the mesh Note For an optimum fit when there is a large difference between the orientation or size of the atlas organs and mesh first use the transformation tool to manually register the mesh and atlas organs then click the or A tool to automatically fit the organs For more details on manual registration see Figure 10 24 6 If necessary adjust the opacity of the organs using the slider or enter a number in the box Note The organs in the mesh are easier to view if you do not select Skin in the Organs list 7 To clear all organs from the mesh click the Deselect All button es To remove a specific organ remove the check mark next to the organ name 8 To display a specific organ s choose the organ name To display all organs on the mesh click the Select All button Ca Figure 10 23 Displaying organs on the mesh 137 _ 10 3D Reconstruction of Sources To manually adjust th
110. ation Tools You can view information about the active image using the Image Information tools The tools enable you to make measurements in an image and view pixel data in different formats Image Information Description See x y coordinates and associated The x y pixel coordinates of the mouse pointer location in the Figure 4 20 page 50 intensity image and the intensity counts or photons at that location Histogram Histogram of pixel intensities in an image Figure 4 21 page 51 Line profile Plots a line graph of intensity data at each pixel along a user Figure 4 22 page 52 specified horizontal or vertical line in the image m To view the tools click Image Information in the tool palette Figure 4 19 gt Image Adjust gt Corrections Filtering 7 Image Information lo EE Units C Irae Binning amp Image Sf T2487 9818 cm Image Data 9 courts Crop Distance Taleg _ 00000 2002800 Distance 2 90 L ROI Toos gt Surface Topography Figure 4 19 Tool palette Image Information tools Image Information Tool Description See Click this button to display a histogram of pixel intensity Figure 4 21 page 51 Click this button to display a line profile Figure 4 22 page 52 Click this button to display the distance measurement cursor in the Figure 4 23 page 53 image window IN EE Click this button to draw and measure a rectangle on an image Figure 4 24 page 54 Click this button to di
111. ation of the source Z location of the source Starting ChiSqure Ending ChiSqure 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 help reduce the error 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 specified value Absorption coefficient at the emission wavelength Reduced scattering coefficient at the excitation wavelength Effective attenuation coefficient Mueff 3Mua Mua Mus Diffusion coefficient Diff Mua Mus 3 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 95 _ 8 Point Source Fittin
112. bout the photon density voxels and DLIT algorithm parameters Tool Palette E gt Planar Spectral Imaging E gt Surface Topography si L gt Surface Topography sid ve FLIT a Reconstruction en Analyze Parame Properties Results FLIT Results FLIT_1 Loaded ToolPalette LY DLIT 30 Reconstruction A a Analyze Params Properties Results DLIT Results DLIT_noSimplOLmsh Loaded Key Value us Er rai Total fluorescence yield N mm 2 1 7 2e 09 he Faas Fin nia 0 75 ia source flus prot R Sze 10 i Number of sources ad Final vsize 2 00 Reduced Chi2 7 58e 07 Number of sources 30 Starting WEIZe 6 00 Reduced Chiz 2 a0e U3 Nauri best 200 Starting vsize best 5 00 Ifat surf samples E000 kappa best 2 00 Threshold angle 70 00 Neurf best ang Unitor Surface Samolina TRUE Tatal sur saronles TANIA Photon Density Maps Save Results Name FLIT_1 O E ee 23D Toos gt Spectral Unmixing Save Results Name DUT noSimplDLmsh _ i Delete IE Chvervurite l E 3D Tools Figure 10 11 3D reconstruction results FLIT left and DLIT right For more details on the DLIT and FLIT algorithm parameters see Appendix F page 189 Sometimes adjusting the DLIT algorithm parameters improves the fit of the simulated photon density to the measured photon density data Item in the Results Tab Description Optimized fit parameters
113. 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 is stored with the luminescent image data and is later used to correct minor drift typically less than two counts pixel that may occur in the bias offset since measuring the dark charge background If a dark charge background is not available at the time of the luminescent image exposure the software checks to see if the selected image parameters warrant a dark charge measurement large binning and long exposure time If a dark charge image is not required the read bias will be used If a dark charge is recommended the software provides the option of using the read bias measurement instead Since the read bias is by far the largest component of background using a read bias measurement instead of a dark charge 159 _ C Luminescent Background Sources amp Corrections measurement is often acceptable If read bias is used instead of a dark charge background the read bias image is
114. cal expression in Equation 1 page 182 where S absolute total photon flux emitted by the bioluminescence source and d source depth Plot of Linear Fit Results Figure E 4 n XENOGEN Living Image Software User s Manual Ezz life Changing CK20031215150449_008 Units Photons Display Overlay Min 1 26e3 Max 9 76e5 1 0 p sec em 2st Color Bar Min 6 97e4 Max 6 44e5 Figure E 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 a File Edit Yiew Tools Window Help X 3 ce g a M a amp k i Units Photons s C Apply to all n nannan GHG EL Unisi Photons W Displey Overlay aem CCT o Corrections Filtering i gt imane information gt ROI Tools bce is 7 Planar Spectral Imaging pes ores Analyze Properties Results Spectral Results 6 0 ROIL Depth mm Total Flux phots ROL 1 250 0 105 14 3666 3 09e5 _ zo ROI 1 3 031e 06 am 40 z A x102 3 0 2 0 Se gt 1 0 7 Plot Linear Fit p sec om 2 st at Save Results After the analysis is completed same Spim 2 a Ge Reick click a button to display graphical paas ran Max 6 44e5
115. capability choose the appropriate emission filter for your application see Chapter 7 page 85 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 Photo Adjustment page 46 If this option is chosen the software automatically sets the exposure time to produce a good photographic image 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 QTH 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 filter
116. ce Type of Analysis IVIS Imaging System For Image Sequence Requirements See Lumina 100 200 Spectrum 3D Series Series Series Planar spectral imaging Optional J J J Chapter 7 page 85 Computes the total flux and average depth of a source below the surface Display multiple fluorescent or J J J J J Chapter 6 page 82 luminescent reporters Uses the Image Overlay function to display multiple luminescent or fluorescent images on one photographic image Subtract tissue autofluorescence J J J J J Chapter 6 page 79 using blue shifted background filters Uses the image math feature to subtract a background image from the primary image DLIT Analysis J J J Reconstructs the surface topography of the subject and the brightness and 3D location of luminescent sources FLIT Analysis J Reconstructs the surface topography of the subject and brightness and 3D location of fluorescent sources Chapter 10 page 109 Chapter 10 page 118 Spectral unmixing J Chapter 9 page 99 Removes tissue autofluorescence from a fluorescence image 22 XENOGEN Living Image Software User s Manual ee E To acquire an image sequence on an IVIS Imaging System 1 Click Sequence Setup in the control panel to operate in sequence acquisition mode The sequence editor appears E WIS Hii cals hen Cael nol Pa alx imaging Hode Capone as iinn FASiop Emerton Filim mie r Disia Phot
117. ce View 3D View kek Background Color Preferences Solid Color p Gradient Color too i Bottom These preference settings specify the background 3 color in the 3D View tab of the image window 13 photons sec Perspective Source Intensity lt Figure H 3 Preferences 3D View tab left and image window 3D View right Preference settings that affect the appearance of the 3D view in the image window Preferences 3D View Tab Description Solid Color Choose this option to apply a non gradient background color to the 3D view in the image window To select a different color or define a custom color click the zd button Gradient Color Choose this option to apply a gradient background color to the 3D view in the image window Top To select a different color or define a custom color for the top of the window click the button Bottom To select a different color or define a custom color for the bottom of the window click the __ button Reset to Defaults Click to restore the default settings 206 Living Ilmage Software Users Manual XENOGEN eT _Life Changing Life Changing Tissue Properties Preferences General User 3D View Tissue Properties DULIT Params Biotrue Please select default settings for the user and click Save e i v Preview Tissue Properties Muscle Source Spectrum Firefly e mM Hornalized Amplitude Index of Refraction 1 40 3 1 00 Display
118. ce of two or more images of the light emission from the surface of the subject acquired at different filter bandwidths Table F 1 Table F 1Filter center frequencies and bandwidths IVIS Imaging System Filter Center Frequency nm Flatow Uroltamcaliay 3 0 Series 570 35 600 35 660 80 200 Series 560 20 580 20 600 20 620 20 640 20 660 20 The IVIS Imaging System 3D and 200 Series and the VIS 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 F 3 Figure F 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 191 a F 3D Reconstruction of Light Sources Defining the Linear Relationship Between a Source and Photon Density Finding the Best Approximate Solution to the Linear System 192 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
119. chroic reflector The lamp output is delivered to the excitation filter wheel assembly located at the back of the VIS Imaging System Figure D 3 Light from the input fiber optic bundle passes through a collimating lens followed by a 25 mm diameter excitation filter The VIS 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 Collimating Fiber Optic Bundle Input Lenses Fiber Optic Bundle Exatation Filter Figure D 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 Living Image Software User s Manual XENOGEN ee ihe aie Life Changing D 2 Filter Spectra The fused silica fiber bundle splits into four separate bundles that deliver filtered light to four reflectors in the ceiling of the imaging chamber Figure D 1 The reflectors provide a diffuse and relatively uniform illumination of the sample stage Analyzing image data in terms of e
120. click Initialize IVIS System You will hear the motors move Sequence Setup Initialize IVIS system Figure 3 1 Control panel initializing the IVIS Imaging System The control panel is only available on the PC workstation that controls the imaging system NOTE 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 a 3 Initializing the System amp Acquiring Images 3 2 Checking the System Temperature 3 3 Imaging Basics 10 The temperature box in the IVIS System control panel indicates the temperature status of the charge coupled device CCD camera Figure 3 2 At startup the temperature box is red and 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 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 200 Series is 37 C but may be set to a temperature from 25 40 C IVIS Acquisition Control Panel hen f Rp Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter gv Luminescent fo 0 a sec Medium 1 x Blac Open
121. constructed mesh Restore Removes smoothing that was applied to a mesh 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 and Volume is recommended for IVIS 3D surfaces Smooth Initiates the smoothing specified Save Result Name The name for the mesh Delete Click to delete the mesh selected from the Name drop down list Load Click to load the mesh selected from the Name drop down list Save Click to save the mesh to the name entered in the Name drop down list 112 XENOGEN Living Image Software User s Manual me ead IVISe Imaging System 200 Series or Spectrum Reconstruct the surface topography mesh continued from Figure 10 2 Single View Surface Topography Analysis Mask purple PLT SS A 6 Draw a crop box that includes a one cm margin around the subject if possible 7 Click Next to display the mask The mask is a purple overlay on the subject image that defines the area of interest for the surface topography reconstruction The mask should match the underlying photograph of the subject as closely as possible without including any area outside the subject image 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
122. creen For more details on the VIS Imaging System please see the appropriate V S Imaging System Hardware 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 Numbered steps explain how to 1 To start the Living Image software click the carry out a procedure icon on the desktop td A dash precedes the The main window appears description 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 CAUTION A caution note warns you that your actions may have nonreversible consequences or may cause loss of data Important information 1 IMPORTANT ALERT Important information advises you of actions that are essential to the correct performance of the instrument or software Caution information oo _ 1 Welcome 1 3 Contacting Xenogen Technical Support If you need technical support please contact Xenogen at Telephone 1 888 810 8055 Toll Free in the United States 1 508 435 9761 E mail tech support caliperls com Fax 1 508 435 0950 Address Xenogen Corporation A Division of Caliper Life Sciences 68 Elm Street Hopkinton MA 01748 USA XENOGEN gt Life Changing Living Ilmage Software Users Manual 2 Getting St
123. ct Copy All from the shortcut menu that appears All of the results table is copied to the system clipboard To Export Results 1 Right click the results table and select Export Results from the shortcut menu that appears In the dialog box that appears choose a folder for the results enter a file name txt and click Save 91 a 7 Planar Spectral Image Analysis This page intentionally blank 92 XENOGEN ee _Life changing Living Image Software User s Manual Ufe Changing 8 Point Source Fitting Displaying the Point Source Fitting Tools aoaaa aa aa 93 Performing Point Source Fitting a aoaaa aa a 0088 96 Checking the Point Source Fitting Results 97 Exporting Results 4s Gee Reed A eR ELE we BE we we 97 Managing Point Source Fitting Results 98 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 o 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 8 1 Disp
124. ct at an angle 0 The angle is known by instrument calibrations of the distance between the structured light projector and the optical axis D and the distance between the stage and the structured light projector Figure F 2 SL projector ot CED VA ae ks Y A a Y K l 4 4 Ve Ah E Y z a iS ks j x lt lt Figure F 2 Structured light projector and subject D and l form two perpendicular sides of a triangle giving tan 9 D I Together Ax and h comprise a smaller version of this triangle The height A can be determined from h Ax tan Q by measuring the displacement Ax The software utilizes fast numerical methods to rapidly evaluate Ax over the entire image to determine the surface topography The surface topography determination is limited to the topside of the object facing the lens Living Ilmage Software Users Manual Converting Light Emission to a Photon Density Map XENOGEN nnn c nite Cumaiag Life Changing 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 bioluminescent light sources includes e A surface mesh that defines the surface of the subject e A sequen
125. ction Properties tab Analyze ess a Properties Results Anaia Params PE Pioperies Resuts ji Tasis Meas ae HEP Tissue feecle Source ALABEOE at Select Wave Filters Select Image Sources select the T iiai aaanice pare ExwLh Emil MinRadian acquisition in e520 640 720 28Ge 06 wavelengths ia B40 720 286er for the DLIT 620 z B40 720 2 Beale analysis E d 720 2 Bes If DLIT analysis iH H Egi r20 2 et 15 E40 720 2 aet 16 results are H B40 f20 266e 0 he 3D H Bat 720 9 4 93e 06 oe the oo 640 Tel 1 16e 07 ools are HSER available 230 Toos le 3D Tools Figure F 5 3D reconstruction tools Analyze tab DLIT left and FLIT right 194 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 than 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 Upp exhibits a large change The larger
126. d p sec om 2 st Color Bar Min 1 01e5 Max 1 79e6 4 If you selected or O Use the pointer to draw the ROI _ Use the pointer to click around the area of interest and draw line segments that Anfinn tha DAI Diaht nlinl varhan tha lant naint Figure 5 4 Drawing an ROI using the free draw method In this example the Contour shape G was selected for the free draw method 5 5 Measuring Background Corrected Signal If a subject has significant autoluminescence or autofluorescence you can obtain a background corrected ROI measurement by subtracting an average background 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 Adjusting Image Appearance page 45 and Magnifying or Panning in the Image Window page 41 Figure 5 5 shows how to measure background corrected signal 62 Living Image Software User s Manual XENOGEN ee _Life changing Life Changing To measure background corrected signal 1 Draw one o
127. d Surface Topography sid B Point Source Fitting Analysis Results E Units Counts v C Use Previously Saved Colors 7 Transillumination Location Params Properties Sequence CX20070222183912 SEQ Tissue Muscle Source A Select Single Image Source MinRadiance Image EswL EmWL 6 2 675 720 675 720 8 70e 06 2 82e 06 2 82e 06 Min 926 Mu 18151 Mim 2599 Mac 51284 F2 gt FLIT 3D Reconstruction Mae 2544 94 n File Edit View Tools Window Help S A eaa e omk Axi g Sequence View 2 Image Adjust _ Units Counts w C Use Previously Saved Colors Transillumination Location Surface Topography B Surface Reconstruction Structure Light C K 2007022218 3912001 Path Averaging Size Auto gt Restore Surface Smoothing Smoothing Level Low Heig Height v Smooth Loss Recovery Save Results see a a v aon Source a Params Properties Results Transillumination Fluorescence Y Spatial Filter 0 1 Analysis Model T ype ngle Limit deg 70 Min 926 Mue 18151 Parameters starting values mm 0 140 te MuaEm 1 em 0 04 MusEm 1 cm 11 80 MuaEx 1 cm 0 05 MusEx 1 em 14 28 lef Restore Defaults Mask No mask Statics Weighting ___LMFiting gt FLIT 3D Reconstruction a jef F yield N mm 210 0 id a a a Living Image Software Users Manual XEN
128. data 3 Repeat step 2 to display data for other voxels of interest The voxel data is updated 4 Toclear the voxel data click any where in the 3D view window Voxel data L Figure 10 15 Viewing voxel data A File Edit view Tools Window Help FAAR aage gt Planar pectral Imaging _ Surface Topography gt DLIT 3D Reconstruction 33 1 3D Tools RAQE BEBE PLE Mesh Volume Organs Animation 55 Render Mesh 207 174 Sagittall 2 0 Coronal 14 0 Sihadadeanescediqhdban dha AMllipeds andsandsaadh ecahandssndcandaananee C Render Photon Density Map Apply Simulated 300 Wavelenath 560 i oo J Threshold 554 391 4 07e6 Ey J Logarithmic Scale 30 0 Slice f Coronal sd oe i y Sagittal all oo os X J 1 Interisity Color Table Rainbow Transaxial Perspective O File Edit view Tools Window Help eGQad ae R AA Ea Sequence View l 3D View gt Planar Spectral Imaging gt Surface Topography gt DLIT 3D Reconstruction 39 1 3D Tools k 4 8 7 S s ELEL Mesh Volume Organs Animation E Render Volume Re IRA Min 7 29e6 Sagittall 2 0 Max 1 49e10 Cubes Threshold Intensity J B373 Color Table 55A 391 BlackRed H _ Reverse C Logarithmic Scale Coronal 14 0 Render voxels As Source Yoxel Measurement To
129. e PiACUCNumber of i Confirm that the signal of interest is above the noise level and below CCD saturation Check the image min and max at the top of the color bar A signal of interest greater than 600 counts and less than 60 000 counts is recommended If the signal level is unacceptable adjust the exposure time or T Appl To Sequence binning level Edit Image Labels box 10 In the Edit Image Labels box that appears enter the image information and click OK If you do not want to enter label information click Cancel Note For details on how to save or export the image data see Figure 3 17 page 29 Figure 3 7 Acquiring an image on the IVIS Lumina IVIS Spectrum IVIS Imaging System 100 Series or 200 Series continued from Figure 3 6 19 3 Initializing the System amp Acquiring Images To acquire an image on the IVIS Imaging System 3D Series 1 For luminescent imaging a Choose the Luminescent option b Confirm that the Excitation Filter is set to Block and the Emission Filter is set to Open For fluorescent imaging a Choose the Fluorescent option b Choose the Filter Lock option and select the excitation filter from the drop down list The software automatically selects the appropriate emission filter c Confirm that the Fluorescent Lamp Level is set to High Note For more information on fluorescent Imaging see Appendix D page 165 2 Set the binning Confirm the default binning l
130. e 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 10 page 109 and Appendix F page 189 E 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 in tissue optical properties Figure E 1 In this portion of th
131. e Dala se se EERE OE AER E RGR H AS OS ORE 149 Quantifying Image Data 6424 8i nen e Redd dade a eva ws 151 Fiat Fielding e s sore p e a a ee a a ee 153 Cosmic Ray Corrections aooo e a ER Ow GS Bes 154 A 1 Image Data Scientific Image Data Scientific image data is a two dimensional array of numbers Each element of the array pixel is associated with a number that is proportional to the light intensity on the element A charge coupled device CCD camera used for scientific imaging 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 Graphic Image Data A graphic image is a two dimensional array of pixels with a color assigned to each pixel There are several schemes for digitally storing the images For example a common scheme assigns a red green blue RGB color code to each pixel The RGB code 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
132. e 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 pages 14 156 Note lo 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 Set the focus Do either of the following 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 Select Manual focus from the Focus drop down list For more details on manual focusing see Figure Focus use subject height Temperature ita Locked Initialize vIS system 6 Set the photographic image settings a Choose the Photographic option b Enter an exposure time or choose the Auto option c Confirm the binning and f stop defaults or specify new settings for the photographic image 7 mage Setup 7 If necessary click in the control panel to operate in single image mode Note In single image mode the Sequence Setup button appears in the control panel Click this button to set up sequence acquisition For more details on acquiring a sequence see Figure 3 10 page 23 8 Click Acquire During image acquisition the Acquire button becomes a Stop button To cancel the acquisition click Stop continued in Figure 3 7 Figure 3 6 Acquiring an image on the IV
133. e Image Information box that shows the label set and image acquisition information for the active data View Image Information View ROI Properties View gt ROI Measurements 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 View Image Layout Windo View Acquisition Control Panel Displays the control panel Acquisition Background Replace Opens a dialog box that enables you to select an instrument luminescent background This background measurement is subtracted from luminescent images Acquisition Background View Available Dark Charge Opens a dialog box that enables you to view the dark charge measurements for the system Acquisition gt Background Clear Available Dark Charge Acquisition Background Measure 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 Acquisition Fluorescent Background Add or Replace Fluorescent Background Opens a dialog box that enables you to select an instrument fluorescent background measurement for the active image data If the Sub Fluor Bkg option is chosen in the control panel the background measurement is subtracted from the image data Acquisition Fluorescent Starts a measurement o
134. e 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 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 VIS Imaging System 100 Series The IVIS Imaging System 200 Series has a larger lens with a smaller flat field correction 153 a A Image Data Display amp Measurement You may notice an increase in noise near the edges and corners of the FOV when flat field correction is applied this is normal A 4 Cosmic Ray Corrections 154 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
135. e as the equivalent luminescent exposure C 2 Background Light On the Sample An underlying assumption for in vivo imaging is that all of the light detected during a luminescent image exposure is emitted by the sample This is not accurate if there is an external light source illuminating the sample Any reflected light will be detected and is indistinguishable from emission from the sample The best way to deal with external light is to physically eliminate it There are two potential sources of external light a light leak through a crack or other mechanical imperfection in the imaging chamber or a source of external illumination IVIS Imaging Systems are designed to be extremely light tight and are thoroughly checked for light leaks before and after installation Light leaks are unlikely unless mechanical damage has occurred To ensure that there are no light leaks in the imaging chamber conduct an imaging test using the Xenogen High Reflectance Hemisphere Figure C 1 A more subtle source of external illumination is the possible presence of light emitting materials inside the imaging chamber In addition to obvious sources such as the light emitting diodes LEDs of electronic equipment some materials contain phosphorescent compounds 160 XENOGEN fe Bizas Living Image Software User s Manual tite Changing Do not place equipment that contains LEDs in the imaging chamber Phosphorescence is a physical process similar
136. e generated by image math in an image window 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 6 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 6 2 Table 6 2 Emission excitation and background filters used to acquire data that can be corrected for tissue autofluorescence Emission Filter Excitation Filter Background Filter Background Image Primary Image 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 79 _ 6 Performing Image Math Operations 80 The objective of using a background filter is to excite the tissue autofluorescence without exciting the fluorophore To reduce autofluorescence signal in the primary image data use the image math tool to subtract the background filter image from the primary excitation filter image 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 background signal primary signal The backgro
137. e imaging components 165 167 K kappa 123 kappa limits 124 196 Living Ilmage Software Users Manual L label information 44 lens aperture 155 line profile 52 Living Image browser 31 luciferase spectrum 183 luminescence reconstruct 3D sources 109 117 luminescent image 10 M manual conventions 3 manual focus 28 measurement ROI 57 auto ROI method 61 free draw 62 manually draw 60 measurement ROIs 59 62 measurements 53 menu commands 199 mesh 109 118 drawing style 133 lighting style 133 miscellaneous material autofluorescence 173 175 multiple reporters per photograph 82 83 N N surface limits 124 196 NNLS optimization 197 weighted fit 197 NNLS optimization 124 NNLS weighted fit 124 normalization 167 O opening an image 34 opening image data 32 34 optical density 168 optical properties for planar spectral imaging 183 overlay 150 overlay image 10 overlaying images 82 83 p PCA biplot 104 PCA explained variance 105 XENOGEN ee E Life Changing photographic image 10 photon density 125 photon density map measured 124 simulated 124 photon radiance 152 photons 152 pixel 149 pixel data 50 planar spectral analysis optimizing precision 187 planar spectral image analysis 88 89 planar spectral imaging 181 187 diffusion model 182 luciferase spectrum 183 optical properties 183 planar spectral imaging tools 86 87 point source fitting 93 98 preferences 203 210 user IDs 210 pseudoc
138. e 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 acquiring a sequence see Figure 3 10 page 23 Click Acquire During image acquisition the Acquire button becomes a Stop button To cancel the acquisition click Stop continued In Figure 3 9 Figure 3 8 Acquiring an image on the IVIS Imaging System 3D Series Procedure continues in Figure 3 9 20 XENOGEN ee te Living Image Software User s Manual Ute Changing To acquire an image on the IVIS Imaging System3D Series cont from Figure 3 8 After image acquisition Click Info in the image window to display the label set information is completed the image l window displays the RSE Edit view Tools Window Help IEK overlay image The tool AAH a Ag E Unte Counts 0 Apply toal palette and Edit Image Aa he A Er ETT A eLA i Labels box appear mmi Unita Counts Counts Display Overlay ely wfe a lafnau itai a Glick TLT20050624122348_003 Seres Male Nn E Corrections i Basa j S Fri Jun 24 2005 Experiment DOB 02 28 05 Em Filter 600 Label Two traser beads Scrutt Tool palette Image Information Bir Me FOW 12 6 F 1s Comment Dorsal Camera VIS 200 Beta ll SIBZ0EEV ROL Tools gt Planar Spectral Imaging C tuiface Topography zI DLIT Image Min 43 Max 27604 20000 15
139. e 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 182 In such cases high levels of binning may be appropriate up to 10 or 16 depending on the 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 157 a B Detection Sensitivity B 3 Smoothing 158 The IVIS 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 B 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 B 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 acqu
140. e 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 Autofluorescence FLIT Choose this option to take the autofluorescence signal into account Autofluorescence and fluorophore emission contribute to the photon density signal at the surface The autofluorescence signal is modelled in order to isolate the signal due to the fluorophore only where an average homogenous tissue autofluorescence yield is determined experimentally XENOGEN eT _Life Changing Living Image Software User s Manual caine Autofluorescence contribution to the photon density at the surface is forward modelled Simulated photon density data due to autofluorescence at the surf
141. e scale or location t vew Tak ww nep FAAN RER of organs 1 Follow step1 to step 4 in Figure 10 23 2 Click the Transform tool button E The transform tool appears 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 5 Press the Tab key to put the transform tool in scale mode A red cube ff appears at each corner of the transform tool 6 To increase or decrease scale the size of the organ drag a red cube at a 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 gt DLT 3D Re Reconstruction 2 aren ao amp Volume Organs Animation M Render Mesh sonsescsseesacneseeacnsessncnecscncccsatgumpasccecnsccsacescesaeneen Saensevencocessassvecsocncnasscansvensselocossasssansseocwanacee C Render Photon Density Map Ay i Threshold Intensity a Color Table Reverse _ Logarithmic Scale Slice Coronal bka 43 Sagittal 2t Transaxial t 1 4 Uke Tals kato sevtch bawie ieenstomnation took i RE _ 7 Press the Tab key again to put the transform tool in rotate mode A red green and blue circle appear around the mesh Figure 10 24 How to manually co register organs on the mesh Use the transtorm tool to move scale or rotate the organ In this example skin is selected from the
142. e selected organs and remove all organ diagrams from the mesh Update scene Click to display the selected organs on the mesh Viewing Voxel Data The voxels are automatically displayed when the 3D reconstruction is complete In the 3D tools the Volume tab displays the voxel intensity and other data and provides tools for voxel display Tool Palette E Planar Spectral Imaging Er TEE A QE 2 es Hale m X Mesh Volume Organs Animation l F Render Volume E Mins 4 89e6 Render vowels As Max B54e9 Cubes j w Threshold Intensity J 1 71e8 al Color Table Ecke e Reverse Logarithmic Scale l Source Voxel Measurement Total Flu O00 phate tee Volume O80 am Center of Hass A02 00d gon Figure 10 14 3D tools Volume tab 130 Living Image Software User s Manual XENOGEN Ceti Life Changing Figure 10 15 shows how to view the data total flux combined volume and the center of mass for user specified voxels For descriptions of the tools in the Volume tab see page 129 To better view the voxel data 1 In the Mesh tab reduce the mesh opacity use the slider or enter an opacity value 2 Clear the Render Photon Density Map option To display voxel measurements 1 Click the Measure Voxels button fg 2 On the mesh click the voxel of interest Alternatively draw a box around a group of voxels The Voxel tab displays the selected voxel
143. e 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 181 _ E Planar Spectral Imaging Optical Properties of Mouse Tissue Ex vivo muscle tissue integrating sphere measurement Firefly Luciferase Spectra PC3M Luc cells 0 9 0 8 0 7 0 6 0 5 0 4 mm 0 3 0 2 0 1 0 450 550 Wavelength nm 650 30 ssue Transmission Window er vitre vivo Intensity a u 400 500 600 roo 800 Wavelength nm Figure E 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 using PC3M cells Diffusion Model of Light Propagation Through Tissue 182 Light propagating through tissue under
144. e 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 ophotons 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 Source Voxel Click the es button then click a voxel in the 3D reconstruction to display measurements for the Measurement voxel Total Flux The total photons sec measured for the voxels selected using the voxel tool Volume Volume of the selected voxels 129 a 10 3D Reconstruction of Sources 3D Tools 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 Total N mm measured for the voxels selected using the voxel highlight tool Fluorescence Yield Organs Tab Organs Choose this option to enable the selection and display of organs on the mesh Requires the optional digital mouse atlas Organ database Choose the male or female organ database from the drop down list Organs A list of the organs in the selected organ database Select the organ s that you want to display on the mesh Reset Click to display the selected organs in their default positions aA Click to select all organs in the database and display them on the mesh m Click to clear th
145. e window to view the current folder path setting Figure H 1 Window size position Applies the active image window size and position settings to subsequently opened image data Recently Used Datasets Save List Displays recently opened files on the Recent Files menu List Size Enter a number or click the up down arrows to specify the number of files to display 204 XENOGEN Living Image Software User s Manual Life Changing User Preferences j Preferences 3D View Tissue Properties DLIT Params Biotrue User Setting s Existing User ID KSA New User ID Delete User ID Preferences Defaults Label Name Set Xenogen Universal l v Edit User Label Choices Default Units Counts a Figure H 2 Preferences User tab Preferences User Tab Description User settings Existing User ID Drop down list of users New User ID Opens the Add New User box A new user is added to the Existing User ID drop down list Delete User ID Deletes the user selected from the Existing User ID drop down list Preferences Defaults Label Name Set Drop down list of factory installed label name sets Edit User label Choices Opens a dialog box that enables you to edit a label set Default Units Specifies the units photons or counts for image display 205 a H User Preferences 3D View A Preferences A CK20050624151158_SEQ DOR Sequen
146. ect the structured light well DLIT analysis cannot be performed without the reconstructed animal surface that is derived from the structured light images If the study requires a furred mouse strain then the fur must be removed from the mouse body before imaging by shaving or applying a depilatory 109 a 10 3D Reconstruction of Sources Acquiring an Image Sequence For DLIT Analysis On the IVIS Imaging System 200 Series amp IVIS Spectrum Acquiring an Image Sequence For DLIT Analysis On the IVIS Imaging System 3D Series 110 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 The IVIS Imaging System 3D Series requires about 45 seconds for the instrument components filter wheel stage and mirror to rotate into position and acquire a photographic and structured light image at a particular view For all the views this adds about six minutes to the total acquisition time As a result the luminescent exposure time per view and emission filter is limited with respect to the luciferin kinetic p
147. ection from the Shading style drop down list in the 3D tools Surface face Figure 10 18 Mesh shading styles 134 c Smooth surface face Lj r gt E e m An S X D ee ee ee FEI Render Mesh Opaciy J C Render Photon Density Map Apply Wavelength _ A Threshold Intensity 407e6 ie 17 ri Color Table i 7 2 Reverse Logarithmic Scale h 5 Reflect smooth surface face G Reflect surface face XENOGEN Living Image Software Users Manual T p d Changing the View Figure 10 19 shows how to view a 3D image from different perspectives Perspective Figure 10 20 shows examples of the other available views ad oe toh a amp vide a EDLIT 30 Reconstruction pare l EAL To change the view e Select to change the view Render Mesh i pact _ aa e Alternatively click the mesh o Render Photon Densiy Map then press the V key to cycle sx f through the different views a E resho w of the mesh Figure 10 20 maa iF Select to display the Color Table 3 _ le perspective view Benbow EN iwl Reverse _ Logarithmic Scale A n Note Only the perspective as eee view can be rotated or moved in the 3D view window Figure 10 19 Changing the view of the mesh This view of the me
148. eeeeeeeeeesbee tebe Hote He eee Eee eueeeseeebetesseeHessmpensHses des Ttek a Ve ceresosdetvccons icinicsaddssenesebessueHogpubecewetetebotpudsetedsteneHeseHeRoboepbencbestgedsCosobods wececongncecnsdoRoWoMoNoto gp eoatuenencdosentecsgecdshotoqecsecdensCeteRodeboaal No Measurement Types Click Attributes or ROI Dimensions selected Click Attributes RO Dimensions ec All In the ROI Measurements table click pone stone m noe Configure f Refresh Export The Configure Measurements box appears E Beko It shows the types of measurements and data in the selected configuration as well as Click to reorder the available additional items that can be added to the table items in ascending or descending alphabetical order Do either of the following A ConfiquieMenwsrenents e Select a configuration that you want to x 5 o jf Update modify from the User Lists Selected Items OR Analysis Comment bagi Analysis User ID Stdev Counts e Select Customized Unsaved from the User Angle Min Counts Li S ts Animal Model bay Counts Animal Number Animal Strain Area cm2 To add an item to the table select the item of B aAA interest in the Available Item list and click Add Avg Dark Charge Counts Avg Efficiency To remove an item from the table select the Avg Fluorescent Bko Counts item that you want to remove in the Selected Je ie tetas Items list and click Remove Cell Line Cell Line
149. el and below CCD saturation It is recommended that the signal of interest is greater then 600 counts and less than 60 000 counts If the signal level is unacceptable adjust the exposure time or binning level 11 In the Edit Image Labels box that appears enter the image information and click OK It you do not want to enter label information click Cancel Note For details on how to Save or export the image data see Figure 3 17 page 29 IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter SIF Luminescent 1 0 4 sec gt Medium zj f2 z 605 x 720 z IM Fuorescent J7 Transilumination _Setup Lamp Level Hish z xl nv Photograph 52 Medium 8 117 Reuse 22 Structure JV Overlay J Lights JV Alignment Grid Field of View C System Status Service fis4 cm ide Acque Sequence Subject height 1 50 cm Image Setup Focus use subject height v Temperature I Locked Initialize IVIS System 640 High Yes 1 50 6410 7638 605 660 High No lc 150 6410 7638 605 680 High No lc 150 6410 7638 605 700 High No c 1 50 6410 7638 a 720 High No c 1 50 6410 7638 i 0 File Edt view Tools Window Help SAAR Age MTT Oa x Sequenice View 2lImageAdiust B Units Photons C Use Previously Saved Colors Planar Spectral Imaging
150. elect this option to apply a log scale to the y axis Click this button to choose a grid line pattern to display in the line profile window ig Click this button to export the line profile data to a csv or txt file Ea Click this button to copy the line profile graph to the system clipboard Click this button to open the Print dialog box Making Image Figure 4 23 shows how to measure distance on an image Measurements Figure 4 24 shows how to display a measurement box on an image To measure distance C File Edit wiew Tools Window Help 14 X S A A el A amp EEEE EEEE ii jaiii 5 Units Counts Display Overlay Info ag 1 Open the image of interest and in the Image Information tools click the Distance Measurement Cursors button a A measurement cursor is displayed on the AaB une Nine 48 image The position and length of the jabe msi cursor are displayed in the tool palette Binning 8 Image x Y 72 248 0737 cnr 2 To change the cursor position or size drag REDON A cir the A or B end of the cursor to a new rahe E A 1563 B i 9456 30 location on the image va ae cA y i 890 000 Distance 6 0 cm The measurement information in the todl palette is updated Max 18712 Position and length of the measurement cursor ae Pixel x y coordinates of Measurement cursor bestest location A on the cursor ae Pixel x y coordinates of a location B on the cursor Len
151. elected from the Name drop down list Click to save the mesh to the name entered in the Name drop down list 121 a 10 3D Reconstruction of Sources 10 3 Reconstructing 3D Fluorescent Sources After the mesh is generated the 3D reconstruction of the light sources can proceed Figure 10 10 shows the steps to reconstruct 3D sources To reconstruct 3D fluorescent sources Tool Palette l Ed Tool Palette gt PlanarSpectrallmaging sd Planar Spectral Imaging p a E V FLIT 3D Reconstruction Anapze Params Properties Results J Analyee Parame Properties Results Angle Lirit ideg Fo IN Surface 200 Tissue Properties Bladder Internal medium index of refraction iE Starting Voxel Size E Auto Fluorescence T Uniform Surtace Sampling a NALS Weighted Fit Restore Defaults _ _ 7 gt 3D Tools gt Spectral Unmixing 5 In the Properties tab make a selection from the Tissue Properties and 2 In the Params tab confirm Source Spectrum drop the defaults or enter new down lists values For more details on the parameters and options see Appendix F page 189 1 In the tool palette click FLIT 3D Reconstruction Muscle is usually the best choice of tissue properties for general in vivo reconstructions Note Ihe internal medium index of refraction is automatically entered when you select a 3 In the Analyze tab e For images acquired on the
152. elections Select the type of ROI or image data to Include in the table Measurements Types Figure 5 14 Viewing the ROI measurements table Item in the ROI Description Measurements Table Measurement Types Make a selection from the drop down list to specify the type of ROI measurements to include in the table None Exclude ROI measurements from the table Counts 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 for a pixel inside the ROI Max counts highest number of counts for a pixel inside the ROI For more details on count units see page 151 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 72 Living Image Software User s Manual Item in the ROI XENOGEN eT Life changing Life Changing Description Measurements Table Photons Efficiency Total Flux the radiance photons sec in each pixel summed or integrated over the ROI area cm x 4r Average Radiance the sum of the radiance from each pixel inside the ROI number of pixels or super pixels ohotons sec cm sr Stdev Radiance standard deviation
153. em the Select User ID box appears Select a user ID or enter a new ID and click OK The Preferences box appears Figure H 1 NOTE Any changes made in the Preferences box are implemented at the start of the next session 203 a H User Preferences General Preferences Preferences Biotrue 3D View Tissue Properties DUT Params RR General User Preserve Settings Color Folder Path F Window size position Startup Defaults C Show Activity Window Dock Tool Panel Left Right Window size i 2 Reset to Defaults Width 55 Height 65 Recently Used Datasets Save List List Size 5 C Show Advanced Options Tool palette docked at the left side of the main window Activity window j hidden by default Some preference settings in the General tab specify how the main application window is organized O Fie Edit Yiew Tools window Help AAH Ra bu Units Counts C Apply to all 2 ae El Units Counts v Display Overlay gt l gt Corrections 7 Filtering a T Fri Jun 24 2005 Em tersB00 Binc MJ8 FO Y 12 6 f4 1s _ ROI Tools Camera I VIS 200 Beta ll SIB20EEV gt Planar Spectral Imaging Ss Surface cn Topography DLIT Series Male Nn nu Experiment DOB 02 28 05 Label Two traser beads Scruff Comment Dorsal Image Min 43 Max 27604 2000
154. en 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 C 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 cm2 sr and 1000 photons s cm2 sr respectively Since these values are well above the lower limit of detection of the IVIS Imaging System 100 photons s cm 2 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 5 page 57 Note that the background light emission is not uniform over the entire animal In Figure C 2 images of control animals mice show a somewhat higher background component originating from the abdominal and thoracic regions Therefore care must be taken when selecting a representative background area 4 O00 4 000 3500 550 S000 3000 2500 2300
155. ent also known as analog digitizer units ADU or relative luminescence units RLU is proportional to the number of photons detected in a pixel Counts are uncalibrated units that represent the raw amplitude of the signal detected by the CCD camera A signal measured in counts is related to the photons incident on the CCD camera The signal varies depending on the camera settings for example integration time binning f stop or field of view setting All IVIS Imaging Systems include a CCD digitizer that is a 16 bit device which means that the signal count range is from zero to 65 535 Sometimes the displayed signal count may appear outside of this range due to corrections applied to the image data for example background corrections gILizoosonzaiasso7_ooa Uniti Couet S Dipl Dvel Sito 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 A 2 Image window and ROI Measurements table counts mode 151 A Image Data Display amp Measurement Photons Efficiency 152 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
156. ential setup and edit the exposure times and filters in the sequence Eeor For more details see Working In the Sequence Editor page 27 i Display ae 2 fF aean o ea a T T M 2 Opn Mo 150 FO 30 oo Ss a ERE mg o Medium 150 EN i Open 1 50 b 3s Delay foo 4 min Apply to All a Remove Aup T al Inert a Figure 10 1 Sequence editor showing sequence acquisition parameters on the IVIS Imaging System 3D Series for DLIT analysis NOTE For details on how to specify a sequential setup and acquire an image sequence see Chapter 3 page 17 After the image sequence is acquired reconstructing 3D light sources is a two Step process Table 10 2 Steps to reconstruct 3D sources from a bioluminescent image sequence Step Description For Details See IVIS 200 or IVIS Spectrum IVIS 3D 1 Perform structured light Figure 10 2 page 112 Figure 10 2 page 112 analysis to reconstruct and A the subject surface al topography mesh Figure 10 3 page 113 Figure 10 4 page 114 2 Set the DLIT algorithm Figure 10 5 page 115 Figure 10 5 page 115 parameters and d d reconstruct the position an geometry and strength Figure 10 6 page 116 Figure 10 6 page 116 of the bioluminescent Sources 111 a 10 3D Reconstruction of Sources To reconstruct the surface topography mesh 1 Load the image sequence that you want to analyze 2 In the tool palette click Surface Topography
157. equence C Me bk few deh Wieder Heb E sH AURRASY To edit an image sequence TER gt 3 1 Open the image sequence that you want to edit For details on how to open image data see page 31 2 If you want to add an image s to the sequence brows for the image s in the Living Image browser Note Only individual images not an image sequence can be added to the sequence you are editing a 3 In the image window click the Edit button f l a i The Edit Sequence window appears E Edit Sequence 4 To remove an Image from a sequence h li k b h S Sequence Clicks Browser Images select the click number in the sequence TL 20060624122348 _001 Aina yep Clicks windowpane and click Retire TLT20080624122348_002 WUH200606307 42125 002 l TLT20050624122348_003 JJH20050630142125_003 The Image IS removed from the sequence TLT20050624122348 004 palits 3 i z Single images in the and the file number is added to the TLT20050624122348_005 amit ta J TLT20050624122348_006 Living Image Browser Retired Clicks windowpane r that can be added to the Note A retired image does not appear in EEE sequence you are editing the Living Image browser and cannot be ve sequence added to another sequence Retired Images 5 To add an image to the sequence select the click number in the Browser Clicks lad Images that have windowpane and click Copy l been removed from i the active sequence The image
158. equence For FLIT Analysis On the IVIS Spectrum 118 The steps to perform 3D reconstruction of fluorescent sources include e Acquire a fluorescent image sequence in transillumination mode on the IVIS Spectrum e Generate the surface topography mesh of the subject e Specify the user modifiable FLIT algorithm parameters for example transillumination source positions and tissue properties e Reconstruct the position geometry and strength of the fluorescent SOUTrCeS Table 10 4 shows the recommended image sequence Acquire the images in transillumination mode using the same excitation and emission filters from at least four source locations Table 10 4 Example image sequence for FLIT analysis Image Type Source Location Photographic Structured light Fluorescent First location J J J Subsequent locations Use previous photo Use previous photo J For details on how to specify a sequential setup and acquire an image sequence see Chapter 3 page 17 XENOGEN ee _Lfe changing Living Image Software User s Manual Ufe Changing Reconstructing the After the image sequence is acquired reconstructing 3D light sources is a two Surface Topography step process Mesh Table 10 5 Steps to reconstruct 3D sources from a fluorescent image sequence Step Description For Details See 1 Perform structured light analysis to reconstruct the Figure 10 8 page 119 subject surface topography mesh
159. erature is locked 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 3 1 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 camera controller a component of some IVIS 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 IYIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter To initialize the system EAP Luminescent foo 2f see z Medun AN A foen I 1 Start the Living Image Aa PES software For more Ai SS EEE details see Chapter 2 OF Oveiy I Loli P Alignment Gid page 9 Field of View fe Service 13 4 cm Subject height fi 50 cm Focus use stibject haighk System Status System need initialization Click on Initialize button to proceed Teoh I rocked 2 In the control panel
160. erest Hk 2 Select Edit Image Labels on the menu bar The Edit Image Labels box appears Max 28314 3 In the Edit Image Labels box edit the information of interest You can also select a new label set to apply to the image or Edit Image Labels UserlD TLT x LabelSet menogen Default Check any S fields Series Male Mndnu v Z Experiment DOB 02 28 05 v Label Two traser beads Scuff Comment Dorsal w Analysis Comment test C Apoly To Sequence Figure 4 15 Editing image label information Max 18709 XENOGEN Living Image Software User s Manual OO a TE 4 7 Adjusting Image Appearance You can adjust the appearance of an image using the Image Adjust tools Figure 4 16 Not all tools are available for all image display modes File Edit view Tools Window Help 16 X ws QA W A Ee Rs Unite Counts 7 Apply to al E EE EE EEEE EEE EEEE zi Units Counts Display Overlay v g Image Adjust Image data Q2ana ROH Min and Max Photo Adjustment i l aty i mage Brightness 100 Min 48 Gamma J 15 al Max 28314 Opacity Lo a Image Color Scale 15000 adjust tools Min 397 Color bar Max F teres Color Scale Limits 10000 Auto Full Manual Individual Color T able 5000 oo a Reverse O Logarithmic Scale Cant nese
161. ete from the KaSS Name drop down ist Name ROI_1_ks x 6000 2 Click Delete E Planar Spectral imeging RO 2 2 903e 04 2000 Counts Color Bar Min 763 Max 9596 Figure 5 13 How to remove ROls from an image and delete ROIs from the system 71 5 Working With Region of Interest ROI Tools 5 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 151 To view the ROI Measurements table click fe Edt view Tools Window Hep ues p i B A A H A amp R Units Counts o Apply to all the Y button Alternatively select View gt Saena C ea e ROI Measurements on the menu bar eek j Meee 11711 C Apply to Sequence Type Measurement ROI 8000 E Threshold J 25 8 Save ROls Name ROI_1_KSA x 6000 l Column headers include ROI information aes AOI 22 Seer si ail a engl ROI measurements and dimensions and information about the image recorded at acquisition ROI Measurements Click Number ROL Image Layer Total Counts Avg Counts Stdev Counts Min Counts Max Counts TLT 200506241 45507_004 Overlay 4 825e 06 1 029e 04 1 128e 04 1 339e 03 5 899e 04 gt Customized S
162. etween a measurement ROI and subject ROI for more details see Drawing an ROI Using the Free Draw Method page 62 e ROI size dimensions or position e Subject ROI ID information Changing the ROI Position e Drag the ROI to a new location e Edit the settings in the ROI Properties box Figure 5 8 There are two ways to move an ROI on an image You cannot move ROls created using the auto ROI feature IA TLT20050624122348_002 Units Counts Display Overlay Info iy 20000 Counts Color Bar Min 352 Max 38915 To move an ROI using the pointer 1 Place the pointer over the ROI so that the pointer becomes a ef arrow 2 Click and hold the mouse button while you move the ROI 3 Release the mouse button when the ROI is at the location of interest ROI Properties mp ROI RO 2 ROI Label ROI 2 Bkg ROI SubjRO Info Click Number TLT20050616134836_005 ROI _none_ X F Lock Position xel pix f 45 8934 B Ye pix 161 2964 Angle deg 0 0000 Lock Size Width pix 21 3902 3 Height pix 23 5394 4 Line Size Line Color Position size line color and line point size of the ROI selected in the image Figure 5 8 Moving an ROI on an image 66 To move an ROI using the ROI Properties box 1 Double click the ROI in the image The ROI Properties box appears and displays the positions and dimensions of the selected ROI To c
163. evel or select a new level for the luminescent or fluorescent image For more details on binning see page 13 and page 156 3 Set the exposure time Confirm the default exposure time or enter a new setting for the luminescent or fluorescent image For more details on Image exposure see page 13 and page 156 4 Set the focus Do either of the following 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 eSelect Manual focus from the Focus drop down list For more details on manual focusing see Figure 3 16 page 28 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 Imaging Hode D F Stop Emettion Filter JF Lunrerncert fio bec Hedum JIE Open OF olograph f 4 Madum 16 Ee Stuc E Ovai O Lights Ven rade z i Sptlem Status Angelncement 0 4 ide Aege Gubet bagehi Slem Sequence Setup Wee subject height Tamp sus TE Locked rotate 21S center Set the photographic image settings a Choose the Photographic option b Enter an exposure time or choose the Auto option c Confirm the binning and f stop defaults or specify new settings for the photographic image If necessary click Image Setup in the control panel to operat
164. f interest for more details on opening an image see page 31 2 In the ROI tools select Measurement ROI from the Type drop down list If the image is a member of a Sequence and you want to apply the ROI to the other images in the sequence choose the Apply to Sequence option 3 To automatically draw all ROIs detected by the software click an ROI shape button Circle OJ Square Gj or Contour and select Auto All from the drop down list The ROIs appear on the image To automatically draw one ROI at a user specified location a Click an ROI shape button and select Auto 1 from the drop down list The create tool appears on the image b Use the ring 2 to move the create tool to the location of interest c Click Create on the ring tool The ROI appears on the image To measure the signal in the ROI s click the Measurement button jy The ROI label displays the intensity signal and the ROI Measurements table appears Figure 5 2 For more details on the ROI Measurements table see page 72 Note For information on how to save ROIs see page 70 File Edit View Tools Window Help FAAA la amp R Units Photons 7 Apply to al EEEEEEEE EEE EES EEFE a Units Photons Display Overlay E mage Information ERO Tao ooa o o 9 Y X C Apply to Sequence Image Min 5 25e3 Max 2 03e6 Type Measurement ROI x F a 4 Thre
165. f the instrument fluorescent background Background Measure Fluorescent Background Opens a dialog box that enables you to select a fluorescent background measurement Acquisition Fluorescent Background Add or Replace Fluorescent Background Opens a dialog box that enables you to remove the fluorescent background measurements from the system Acquisition Fluorescent Background Clear Available Fluorescent Background 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 Acquisition Fluorescent Background View Available Fluorescent Background Tools Image Math for Opens the Image Math window for the active data Tools Image Overlay for Opens the Image Overlay window for the active data Window Close Window Close All Closes the active image window Closes all image windows 200 XENOGEN Life changing Life Changing Living Image Software User s Manual Menu Bar Command Toolbar Description Button Window gt Cascade Organizes the open image windows in a cascade arrangement Figure 4 13 page 42 Window Tile Organizes the open image windows in a tiled arrangement Figure 4 13 page 42 Window 1 xx Window
166. ff Image TLT20050624122348 001 Fri Jun 24 2005 12 24 53 Em Filter 560 Bin MJ8 FOV 12 6 f2 1s Comment Dorsal Camera I IS 200 Beta Il SIB20EEY Image Min 48 E Max 23314 15000 10000 5000 Counts Color Bar Min 397 Max 18712 Note By default the Auto min max range of the image data determines the histogram range and bins the software sets the min and max values to optimize image display and suppress background noise Click Full to use the actual image min max data to generate the histogram range and bins Low and high intensity bin A Histogram Window Min Bin 103 S Max Bin 38915 TLT 200806241 22345 003 Overlay Figure 4 21 Image histogram S Bins 512 x r Number of intensity bins x axis To export the histogram data 1 Click the Export button H 2 In the dialog box that appears select a directory and enter a file name 3 Select the file type csv or txt and click Save To copy the graph to the system clipboard 1 Click the Copy button Ez The histogram plots number of pixels y axis per intensity bin x axis 51 _ 4 Working With Images Viewing a Line Profile The line profile plots pixel intensity data that you select by placing a horizontal or vertical line on the image The line profile is automatically updated when you change the line position In the Overlay display mode the line profile plots the luminescent data To obt
167. fficiency 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 152 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 No of Emission Filter Como P VEUT Wheel Positions Fluorescence Filters 200 Series 24 two levels each with 22 60 mm diameter 12 positions Lumina 8 3D 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 D 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 bandpass region and block absorb or reflect all wavelengths outside the bandpass region This spectral band is like a window characterized by its central wavelength and its width at 50 peak transmission or full width half maximum Figure D 5 shows filter transmis
168. file in the selection 15000 e o select non adjacent Images in the 10000 browser PC users Press and hold the Ctrl key while you click the images of interest in the browser 5000 Macintosh users Press and hold the Cmd key apple key while you click the images of interest in the browser Color Bar Min 26 Max 18709 TLT20050624145907_0014 3 Click Load as Group The image thumbnails are displayed together in an image window Note For details on how to save or export the image data see Chapter 3 page 29 Figure 4 8 Creating an image sequence from images acquired during different sessions 38 XENOGEN Living Image Software User s Manual Or a TE 4 5 Working With a Single Image Choose the image display units Select a display mode from the drop down list A VLT20050624122346_003 A TLT20050624122348_003 Units Counts Display Overlay Overlay R Photograph Luminescent Bias Saturation Map Fino e g Units Counts _ Display Overlay 0 a Click TLT20050624122348 003 Series Male Nn nu Fri Jun 24 2005 Experiment DOB 02 28 05 Em Filter 600 Label Two traser beads Scruff Bin MJ8 FOV 12 6 f4 1s Comment Dorsal Camera IVIS 200 Beta Il SIB20EEV Min Max 27604 To display or Image 20000 10000 j Min 43 hide the image Max 27604 EF o 3 30000 information d click Info Counts Counts Color Bar Mi
169. fined by the read noise another quantity that must be determined for quantitative image analysis Saturation Map Displays image regions that saturated the CCD digitizer gt 65535 counts 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 3D Series 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 F page 189 39 _ 4 Working With Images Item inthe Image Description Window Export Opens the Export Active View As Image box so that the active image data can be exported bmp jpg ong tiff or postscript format Info Click to display or hide information about the image in the image window Figure 4 14 QCk20050624 51158_005 Overlay QCkrn0s0624151158_ 005 Une Camii ipla Sano Ma Ira G ACEZD0MIEZAI M150 S60 3D View 4 CRA0050624151159_ 005 EER Lipe Cara Hiph Phsgrash l E ia atj HE Photograph A CK20050624151158_001 Unir Cost
170. fresh 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 Displays the Save Measurements box so that the data can be saved to a txt or csv file Close Closes the ROI Measurements table 5 Working With Region of Interest ROI Tools Configuring the ROI You can customize the data and information column headers in the ROI Measurements Table Measurements table Several predefined categories are available in the Measurement Types Click Attributes and ROI Dimensions drop down lists To configure the ROI Measurements table To reorder the columns drag a column header 1 To create a custom table configuration 1 left or right in the table Make a selection from the Measurement Types Click Attributes or ROI Dimensions drop down lists The table is updated with the new data AROI Measurements DOR Click Number ROI Image Layer Subject Subject Label Subject ID Bkg AOI CK20031215150449 001 lt BKG1 Overlay Subject1 Subject 1 NuMouseELI21 none TESTUEETEELESESTAT TELS STEEL T LES EerET OTe irre Sete e Cheese eeeeeeecee Cree rseriry sere eee ieee rier errs Seeeeeer eraser errr errr reer serie rere reer ere re eee ee ee re ert i sete res creer S Henan enced es eee ewe e sede ease cede eee ee suet e meee tenstesstetpedssteustensHetete teenagers neseeeseseeeHede pe
171. g 8 2 Performing Point Source Fitting Point source fitting is performed separately on each image in a sequence 1 Open the image sequence that you want to analyze 2 In the Analysis tab select an image _ in the sequence 3 In Surface Topography tools generate or load a mesh Note It is recommended that you use the smoothing tool to generate a good quality mesh 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 5 If you want to fix a parameter Starting value click the unlocked icon Ef so that it becomes a closed lock B 6 If you want to construct the source only in a region of interest make a section 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 96 a Edit view Tools Window Help 1 x AH t RARAN Fh Sequence View nnn ial Sequence View Le Image Adjust gt Surface Topography si
172. ge analysis include e Acquire two or more images at different wavelengths e Measure the total integrated intensity on each image e Fit the measured values to the exponential function of Equation 1 The results of the fit are the total flux of the bioluminescence source S and the source depth d Living Ilmage Software Users Manual XENOGEN fe Bizas Life Changing E 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 ug is a function of the absorption and reduced scattering coefficients Hef SHa U s M 2 Calculation of the function K in Equation requires all three coefficients u4 H 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 assumes that the tissues are optically homogeneous and the Living Image software provides several factory set
173. ging 9 2 Spectral Unmixing Results Window Concentration Plot The results are displayed in the four tabs of the Spectral Unmixing Results window The Concentration Plot 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 Concentration Plot Spectrum Plot PCA Biplot PCA Explained Variance Bey a Double click an unmixed result to display it ina separate image window Figure 9 2 Spectral unmixing results concentration plot Item in the Concentration Plot Click to show the controls to adjust the fluorescence opacity Image Adjust 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 101 a 9 Spectral Unmixing Spectrum Plot 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 3 File Edit View Tools Window Help ME RRE AB EAE a ARR Concentration Plot SpectumPFlot PCA Biplot PCA Explained Variance IF ell E Image Adiust JO show Legend aa I gt Planar Spectral Imaging E A list of the spectra in the fh Sanechal inan A Unmixed 1 results Add re
174. gnal Levels and f stop Settings D 4 Image Data Display Fluorescent Efficiency 170 E CCD Array gt Imaging Lens Tissue Autofluorescence Photon Scattering Mean free path 0 5 mm Bioluminescent or Fluorescent Source Figure D 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 1s 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 155 Fluorescent image data can be displayed in units of counts or photons absolute calibrated or in terms of efficiency calibrated normalized For more details see Quantifying Image Data page 151 If the image is displayed in photons you can compare images with different exposure times f stop setting or binning level
175. goes 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 IA SK X CXp Howd 1 where S is the absolute total photon flux emitted by the bioluminescent source and d is the source depth The term pg is the effective attenuation coefficient It is determined by the tissue coefficient of absorption u and reduced scattering u that quantify the two main phenomena light undergoes in tissue The function K A 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 4 and the function K are dependent on the wavelength Equation shows that if the total integrated intensity ROI measurement is measured at several wavelengths it is proportional to an exponential function of the product of the depth and the optical property Uep Therefore the steps to planar spectral ima
176. gth 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 Figure 4 23 Measuring distance with the measurement cursor a 4 Working With Images To display a crop box on the image C Fie Edit view Tools Window Help I X 1 Open the image of interest and in the gescccce eccoci Image Information tools click the Image Crop button i773 ff cae Se ees BEERS EEE EEE Units Counts Display Overlay v zino g brunete Ma ES z The pointer changes to a teens eos Nee a laa A AAEN Unit Cm Max 28314 2 Draw a rectangle on the area of e Binning 2 interest n 15000 Image x Y 9548 72 077 cm ee Image Data 77 counts 3 To change the size or position of the ce ae ino crop box drag a box corner or side A meray E7 877572 AA 0907 Distance 178 4 To delete the crop box from the EAS TE image click the F71 button EROI Toos Poun _ Surface Topography raged m Crop box position and dimensions Max 18712 Tome x y coordinates at the upper left corner of the box Crop box T x y coordinates of lower PENSAT right corner of the box Box width and height Distance Length ofthe diagonal from the upper left to lower right Figure 4 24 Measuring distance with the crop box 4 10 Image Layout Window The Image Layout window provides a convenient way to annotate a
177. hange ROI position drag the ROI in the image Alternatively enter new Xc x coordinate and Yc y coordinate values in the ROI Properties box To rotate the ROI clockwise enter the degrees in the Angle deg box and click outside the box To lock the current ROI position choose the Lock Position option Note he ROI position cannot be changed until the Lock Living Ilmage Software Users Manual XENOGEN ee _Life changing Life Changing Editing ROI Dimensions There are two ways to resize a circle or square ROI e Drag a handle on the ROI e Edit the settings in the ROI Properties box NOTE You cannot change the size of an ROI that was created using the auto ROI or free draw tool QI LT20USUB24122348 ODF DER j ROI Properties BES Uris Certs oe Displae Cheri To resize an ROI using the pointer 1 Select the ROI and place the pointer over a handle mi on the ROI 2 When the pointer becomes a X arrow drag the handle Figure 5 9 Changing the ROI size ROI ROI 7 ROI Label ROI BkgRO SubjRO Info Click Number CK20031215152405_001 X Rol _none_ bd Lock Position Kel cm g 3450 Yelcm 7 3500 Angle deg o o000 gt pahi dale fa W Lock Size width cm 10 9450 Height cm oson Line Size 2 2 Line Color Done To resize an ROI using the ROI Properties box 1 Double click the
178. he GJ button and select 1 Position the subject ROI so that it includes the measurement ROI s and the associated average background ROI Method 2 Right click the measurement ROI and select Set BkG ROI to Bkg X from the shortcut menu that appears Figure 5 5 Measuring background corrected signal Three methods are shown for associating the average background ROI with a measurement RO s Method 3 In the ROI Properties For aaam Eaa L AUI Properbes R BG a ie mG Eana Seay mi aa Y F Ua a BRG hen flat Fs ii te CR SS ee OO ic CD Eniro sequence Right click a background ROI and select Properties on the shortcut menu ROI Properties box that appears click the Bkg ROI tab and put a check mark next to Use as BKG for future ROIs in Choose the image name or the Entire sequence option 63 5 Working With Region of Interest ROI Tools 5 6 Managing ROls In the ROI Properties box you can e View information about the selected ROI Figure 5 6 page 64 e Change the position of the ROI on the image Figure 5 8 page 66 e Edit the ROI label and line characteristics Figure 5 11 page 69 Viewing ROI Properties To view ROI properties do one of the following e Double 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 Proper
179. he pixel intensities in an Histogram image Figure 4 21 The software sorts the intensities into groups bins and plots the number of pixels per bin NOTE In the Overlay display mode the histogram plots the luminescent data To obtain a histogram of the photograph select Photograph from the Display drop down list 50 Living Image Software User s Manual XENOGEN Life Changing Life Changing To display the image histogram 1 Open the image of interest 2 Inthe Image Information tools click the Image Histogram button Laat The histogram is displayed 3 To display the histogram using the full intensity range of the image click Full in the Histogram window 4 To edit the minimum or maximum bin intensity enter a new value in the Min Bin or Max Bin box or click the ua arrows 5 To edit the number of bins enter a new value in the Bins box or click the wa arrows nme The histogram is updated O File Edit view Tools Window Help E x a Qi W la amp k Units Counts pais Apply to all E Corrections Filtering wa OE 4 ca PY E Units Cm Image Binning 2 Image X Y 6274 473 cm Image Data 28374 counts Crop Distance Romo E woooey 00 000 Distance 000 gt ROI Tools gt Surface T opography _ Units Counts v Display Overlay x E Series Male Nn nu Experiment DOB 02 28 05 Label Two traser beads Scru
180. he spectrum Figure B 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 Ilhumimated E front lkmimated Image Intenafer Quantum Efficiency 200 400 600 B00 000 Wavelength nm Figure B 1 Quantum efficiencies Bialkali photocathode A back illuminated CCD and front illuminated CCD E 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 2 For maximum sensitivity select f 1 the largest aperture setting on the IVIS Imaging System Figure B 2 This provides the greatest light collection efficiency but results in the minimum depth of field for the image The depth of field refers to the depth over which the image appears to be in focus and is determined by the f stop and the field of view FOV At f 1 the depth of field ranges from 0 2 cm at FOV 3 9 cm IVIS Imaging System 200 Series only to 2 cm at FOV 25 cm You can use the manual B Detection Sensitivity Image Exposure Time Field of View FOV B 2 Binning 156 focus option on the Control panel to easily assess the depth of
181. he tool palette ROI tools Quick guide to drawing measurement ROls 1 In the ROI tools select Measurement ROI from the Type drop down list 2 Click the Contour button and select Auto All from the drop down list 3 If it is necessary to adjust the ROI boundaries use the slider or the arrows to change 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 4 Click the Measure button W The ROI measurements are displayed on the image and the ROI Measurement table appears Note After ROIs have been created right click an ROI to view a shortcut menu of ROI commands Ctrlk click for Macintosh users The shortcut menu provides easy access to many functions for managing ROIs and viewing ROI properties The ROI Measurements table displays data for all ROIs created 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 RO Measurements Table page 72 File Edit view Tools Window Help 0 X A A W A amp R Units Counts o Apply to all ETD ope Coote Dipin veda a i gt Image Adjust gt E mage Min 40 O 0 9 Y x 10000 C Apply to Sequence 2000 Type Measurement AOI s Threshold a Sa
182. his option to display the source spectrum Save as default settings for DLIT Choose this option if the settings are for the Properties tab in the Surface Topography DLIT tools Spectral Imaging Choose this option if the settings are for Properties tab in the Planar Spectral Imaging tools 207 a H User Preferences DLIT Parameters fl Preferences General User 3D View System VIS 200 Angle Limit dea 70 za Lower Upper Kappa Limits 0 5 4 Surface Topography Settings Path Averaging Size Auto C Smooth Mesh Surface N Surface Limits 200 200 Voxel Size Limits 6 J8 Voxel Size Increment 1 7 Uniform Surface Sampling f NNLS Optimization C NNLS Weighted Fit These preference settings specify the defaults for the Params tab in the Surface Topography DLIT tools Tool Palette _ Biotrue aah ete pia J gt Planar Spectral maging pi Surface Topography DLIT i Analyze Params Properties Results Angle Limit degk 70 lower upper Kappa Limits 0 5 a H Surface Limits 00 i Vowel Size Limita lB fe Reset To Default Voxel Size Increment Hniform Surface Sampling M NALS Optimization CI NNLS Weighted Fit Surface Topography Path Averaging Size Auto E C Smooth Mesh Surface Restore Defaults Figure H 5 Preferences DLIT Params tab left and Surface topography DLIT tools Params tab right P
183. image sequence specified in the Sequential Setup table Sequence Setup Click to display the Sequence Editor so that you can specify and manage sequence acquisition parameters or open sequence acquisition parameters xsq For more details on acquiring an image sequence see page 23 24 Image Setup Click to close the Sequence Editor Initialize IVIS Click to initialize the IVIS Imaging System For more details on initializing the system see page 9 system 14 XENOGEN Pe eo Life Changing Living Image Software User s Manual Table 3 2 Additional IVIS System Controls for the IVIS Imaging System 200 Series or IVIS Spectrum Item in the Control Panel Description IVIS Imaging System 200 Series Alignment grid Choose this option to activate a laser generated alignment grid on the stage when the imaging chamber door is opened The alignment grid is set to the size of the selected FOV The grid automatically turns off after two minutes If subject alignment is not completed in two minutes place a check mark next to Enable Alignment Grid to turn on the grid Note The horizontal cross hair of the alignment grid is offset appropriately to take into account the height entered in the Subject height box Focus Scan Mid Image Choose this option in the Focus drop down list to set the focal plane at the maximum dorso ventral height of the subject at the middle of the animal This focusing method uses the laser to scan horizo
184. in the Results Tab Description Overwrite If you reanalyze saved results click to save the new results and overwrite the previous results Viewing Photon Density 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 is 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 125 a 10 3D Reconstruction of Sources 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 Tool Palette C Sutace Topography O Z DLIT 3D Reconstruction Analyze Params Properties Results DLIT Results DLIT_noSimplDLmsh Loaded Note The voxels are also automatically displayed when the 3D reconstruction IS f V e 5 82e 10 pis Total source flux phot s Final vsize z Number of sources 30 Reduced Chi2 2 38e 03 Starting vsize be
185. ined by the software Alternatively you can used a loaded spectrum as the initial guess 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 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 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 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 Choose this option to automatically sort the unmixed spectra in ascending order of their center wavelength Choose this option to force the first component to non zero ever
186. inescent sources IVIS iy Imaging System 200 or 3D Series only For more details on 3D reconstruction see Chapter 10 page 109 Animation 51 21 8 13 8 Wavelength 580 Threshold Intensity Color T able Rainbow Reverse Slice Coronal Sagittal Transaxial GLI 2 E F C Logarithmic Scale Jess photons sec Source Intensity L Image window 3D View tab __ Perspective Figure 4 6 Image sequence acquired on the IVIS Imaging System 3D Series top and DLIT 3D reconstruction of bioluminescent sources bottom 36 XENOGEN ome ee Life Changing Living Image Software User s Manual Item in the Image Window Description Sequence View tab Units Choose counts or photons for the image data from the drop down list For more details on counts and photons see Appendix A page 151 Use Previously Saved Colors Choose this option to display an image using the color table that was last applied to the image data Click this button to open all images in a sequence Click this button to close all open images in the active sequence Click this button to open the Edit Sequence window that enables you to add or remove images from the active sequence For more details on editing a sequence see Figure 4 7 page 37 Click this button to export the active image in DICOM or other graphic file format for example omp jQ Editing an Image S
187. ing the night like dark charge background is After you acquire a fluorescent image inspect the signal to determine if a fluorescent background should be subtracted Figure D 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 NOTE 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 177 a D Fluorescent Imaging 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 D 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 D 7 Subtracting Tissue Autofluorescence Using Background Filters 178 High levels of tissue autofluorescence can limit the sensitivity of detection of exogenous fluorophores particularly in
188. inning 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 measured 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 1s relatively flat 10 over the range from 500 700 nm which includes the spectral variation found in bacterial or firefly luciferase Therefore calibration is accurate over this range The fluorescent signal detected from a sample depends on the amount of fluorophore present in the sample and the intensity of the incident excitation light The excitation light incident on the sample stage is not uniform over the field of view FOV At FOV 10 there is a slightly dished illumination profile due to the close proximity of the stage to the illumination reflectors while the XENOGEN Living Image Software User s Manual Life Changing A 3 Flat Fielding profiles for the other stage locations are peaked near their center The illumination intensity profile varies by up to 430 across the entire FOV Figure A 4 100 80 p 6
189. 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 F 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 F 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 as the difference between where the line should fall on 189 a F 3D Reconstruction of Light Sources 190 the stage in the absence of the subject and where it appears in the image due to occlusion by the subject Ax F 4 Figure F 1 Parallel laser lines projected onto a subject Given knowledge of the angle 9 the height of the subject h can be determined by analyzing the displacement Ax of the laser lines as they pass over the object The parallel lines are projected onto the surface of the subje
190. ion in the control panel the IVIS System acquires only one photographic image for the entire sequence If this option is not chosen the system acquires a photographic image for each image in the sequence 6 To specify a time 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 fl b In the dialog box that appears select a directory for the Tile enter a file name and click Save Note You can add or delete an image to the sequence or edit a parameter value For more details see page 27 and 28 continued In Figure 3 11 Figure 3 10 Acquiring an image sequence Procedure continues in Figure 3 11 23 a 3 Initializing the System amp Acquiring Images To acquire an image sequence on an IVIS Imaging System continued from Figure 3 10 GIVES Acquisition Control Par imaging Mode Exposwe Tee Pinning F Stop 8 Toacquire the images click Acquire Sequence in the control panel e aa aE a e a During image acquisition F Ovela Uh the Acquire Sequence ee ee or button becomes a Stop rT a Bc T the smsa 2 button To cance a EAT acquisition click Stop A File Edit Yiew Tools window Help ec QA Lo amp When all of the images are acquired and displayed in the image window confirm that the signal of interest is above the noise level and below CCD
191. ired Conceptually soft binning is the same as hardware binning groups of pixels are summed and a smaller lower resolution image is produced However in soft binning the summing is performed digitally on the stored image data not on the electronic charge before readout as in hardware binning Although soft binning does not improve the signal to noise ratio for read noise it may enhance the signal visibility because it reduces the statistical scatter of nearby pixel contents Usually hardware binning is preferred but if it is not possible to take another image applying soft binning to the data may provide a worthwhile solution 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 B 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 Center pixel value the mean value of the nine pixels in the 3x3 neighborhood Figure B 4 3x3 pixel neighborhood Living Image Software User s Manual XENOGEN a Camaag Life Changing Appendix C Luminescent Background Sources amp Corrections Electronic Background i ek id ei OER ER ee HERD ERE 159 Background Light On the Sample
192. irm that the Excitation Filter is set to Block and the Emission Filter is set to Open SEM Structure W Overlay For fluorescent imaging E WIS Acquisition Control Panel Imaging Hode g Luminescent jon 3 Sec Medium 2 Block r Open al Fluorescent Transillumination DUM Photcoraph po TAE Exposure Time Binning FfStop Excitation Filter Emission Filter Lamp Levet High Medium E Lights i Alignment Grid System Status a Choose the Fluorescent option Field of View C itis Service 3 4 cm dle b IVIS Spectrum only Choose the ea res Se Transillumination option if you want to use Subject height 1 50 2 em Sequence Setup bottom illumination For top illumination leave this option unchecked c Choose the Filter Lock option and select the excitation filter from the drop down list The software automatically selects the appropriate emission filter d Confirm that the Fluorescent Lamp Level is set to High Note For more information on fluorescent imaging see Appendix D page 165 Set the binning Confirm the default binning level or select a new level for the luminescent or fluorescent image For more details on binning see page 13 and page 156 Set the exposure time Confirm the default exposure time or enter a new setting for the luminescent or fluorescent image For more details on Image exposure see page 13 and page 156 Set th
193. is displayed on the associated grayscale photographic image Figure A 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 emission 1s low Living Ilmage Software Users Manual XENOGEN ea Camaag Life Changing A 2 Quantifying Image Data The Living Image software can quantify and display scientific image data using three types of measurements Figure A 1 e Counts e Photons e Efficiency for fluorescent images only Data Display Choose This to Display Recommended For Counts An uncalibrated measurement of the photons incident Image acquisition to ensure that the camera on the CCD camera settings are property adjusted Photons A calibrated measurement of the photon emission ROI measurements on bioluminescent images from the subject Efficiency A fluorescence emission image normalized to the ROI measurements on fluorescent images incident excitation intensity radiance of the subject illumination intensity Counts Select Counts Photons or Efficiency for the image data 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 A 2 The counts measurem
194. ition the mouse pointer over the vertical or horizontal border When the 4h or appears click and drag the border Figure 10 22 Making measurements and displaying point coordinates in the coronal sagittal and transaxial plane 136 Living Image Software Users Manual XENOGEN ee ite Changing 10 6 Displaying Organs on the 3D Reconstruction Life Changing The Xenogen digital mouse atlas enables you to display 3D mouse organs on the 3D reconstruction Figure 10 23 shows how to display organs on the mesh The software automatically aligns the organs on the mesh However you can also manually adjust the scale or location of organs on the mesh Figure 10 24 and Figure 10 25 You can also import a custom organ atlas created from Open Inventor files iv To display organs on the mesh In the 3D Tools click the Organs tab _ 2 Confirm that the mesh is in the perspective view click the button or press the R key 3 Choose the Render Organs option 4 Select an organ atlas All of the organs in the selected atlas appear on the mesh 5 To co register the digital organs and the mesh a Click the Fit organs to the mouse button n b On the drop down list that appears click a button to choose an option O Fie Edt view Tools Window Help S A A ed La amp Planar Spectral Imaging gt DLIT 3D Reconstruction Z 3D Tools R4 e 8 6 PLE Mesh Volume Organs Anim
195. k to display the manual transform tool g El N 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 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 Figure 10 21 olala Click to show or hide a bounding box around the subject 128 XENOGEN Life nai Life Changing Living Ilmage Software Users Manual 3D Tools Description EE Click to show or hide a grid under the subject Mesh Tab Mesh Choose this option to display the mesh in the 3D view window It may be helpful to turn off the mesh to better view the photon source 3D reconstruction Opacity Controls the mesh opacity A lower number makes the mesh 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 reconstruction of the Maps bioluminescent source has not been generated this option is not available Apply Select the simulated computed by the DLIT algorithm or measured photon density for the photon density map Wavelength Drop down list of acquisition wavelengths for the active image data Select an acquisition wavelength for Threshold Choose this option to apply a photon
196. ke a selection from the Preset drop down list To select a saved user defined setup a Click Load b In the dialog box that appears select the directory and animation setup xml of interest To edit the key frame sequence 1 To add a key frame a Adjust the 3D view to show the properties of interest b Click the Fh button 2 To reorder a key frame in the sequence select the key frame and click the or arrow 3 To update a key frame a Select the key frame of interest b Adjust the 3D view c Click the 4j button 4 To delete a key frame a Select the key frame that you want to remove b Click the 8 button and select Delete Current To save the animation setup 1 Click Save 2 In the dialog box that appears select a directory and enter a Tile name xml Figure 10 31 Editing and saving an animation setup 146 Ss sak Total Duration secs 5 ae EBENE Frezet Animations Presets Spin CCW orn Anis Frame Factor 1 Animation Setup Time Scale 4 J Key Frame 1 kep Frame 2 Kev Frame 3 Kev Frame 4 Kev Frame 4 Kev Frame 6 w F Se Be le es Frames Per a 0 i S Load Save Living Image Software User s Manual XENOGEN Life Changing Life Changing 1 1 Biotrue Collaborative Data Management System The Biotrue Collaborative Data Management System CDMS is an optional web based application that provides secure online data s
197. l of interest must exceed in order to be detected 173 _ D Fluorescent Imaging Figure D 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 D 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 It is recommended that you take control measurements to characterize all materials used in the IVIS Imaging System 180 160 140 120 100 80 60 40 Autofluorescence Relative to Black Lexan 20 Figure D 12 Comparison of autofluorescence of various laboratory materials to that of black Lexan 174 XENOGEN Life Changing Living Image Software User s Manual tite Changing Despite the presence of various background sources the signal from most fluorophores exceeds background emissions Figure D 13 shows the fluorescent signal from a 96 well microplate
198. lane through the subject 1 Click the 7 button Measurement cursors and cursor length mm are displayed in the Coronal Sagittal and Transaxial windowpanes 2 To measure a distance drag each end of the cursor to the position of interest The distance measurement is updated To display x y coordinates 1 In the Coronal Sagittal or Transaxial windowpane click 34 4 the position of interest The x y coordinates mm of Coronal sagittal and transaxial windowpanes show the slice through the mesh taken by the associated plane ENA ma ag elk a Senet ereere che caer ecee seer ebCCcResSeeGeRe 5 oO Sequence View t 3D View Planar LT O EN pE a g gt Surface Topography gt Surface Topography B Ternat 5 T Help gt DLIT 3D Reconstruction a A 3D Tools LAQ 8 6 22 7 mm Measurement cursor Bow Mesh Volume Organs Animation 554 Render Mesh 207 TA 10 Opacity J M g Sagittal 1 8 C Render Photon Density Map Apply Simulated Z Ai Stara xg lO Wavelenath 560 3 0 0 eshold 55 4 39 1 Intensity 4 0 6 05 Color T able a eron Transaxial 8 1 __ Logarithmic Seale 30 0 hee ai 1 e m Lo 2 4 1 8 a0 X 8 1 il photons sec Perspective 39 455 Source Intensity the position are displayed If you drag the cursor the coordinates are updated 0 0 2 Til To change the windowpane size pos
199. laying 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 gt Preferences klGoce on the menu bar Startup Defaults Preserve Settings 2 In the dialog box that C Show Activity Window bike appears put a check mark peer ee es Z Folder Path next to Show Advanced ie Window size postion Window size Recently Used Datasets Options Width 55 amp Restore Defauts 3 Save List Height 65_ List Size 5 3 Click OK Show Advanced Options The point source fitting tools appear in the tool palette Note If the image sequence does not include a structure light image the point source fiting tools do not appear in the tool palette Figure 8 1 Preferences dialog box 93 a 8 Point Source Fitting Analyze tab Tool Palette gt Surface Topography 7Point Source Fitina O Analysis Params Properties Results Sequence CX200702227 89972_SEQ Tissue Muscle Source MA Select Single Image Source l Image EswL EmwWL MinRadiance O 1 675 720 2 44e 07 OD 01mm D y 20 6 Ho 2 675 720 8 70e 06 HO 3 675 720 2 82e 06 HO 4 675 720 2 82e 06 j j gt FLIT 3D Reconstruction _ gt 3D Tools Analysis tab shows the active image sequence Click the sign to display the position of the bottom illumination so
200. le txt the system clipboard Figure 5 16 Copying or exporting the ROI Measurements table 15 76 5 Working With Region of Interest ROI Tools This page intentionally blank XENOGEN Living Image Software User s Manual a 6 Performing Image Math Operations Using Image Math to Create a New Image 78 Subtracting Tissue Autofluorescence aooaa a 79 Overlaying Multiple Images n nooo 2 0028 ee eens 82 The Living Image software provides tools that enable you to mathematically combine two images to create a new image Living Image Tool Use This Tool To See Page Image Math Mathematically combine add multiply subtract or 78 divide two user specified images Image Math Remove autofluorescence from a fluorescent 79 Image Image Overlay Coregister multiple fluorescent or luminescent 83 images on the same photographic image to view multiple reporters in a single image To perform image math open an image sequence see Figure 4 3 page 34 or a group of images see Load as Group page 32 T7 6 Performing Image Math Operations 6 1 Using Image Math to Create a New Image To create a new image using image math 1 Load the image sequence of interest Image Math window iimage Math Window Sequence TLT20060510111201_SEQ A TLT20060510111201_001 TLT20060510111201_002 TLT20060510111201_003 TLT20060510111201_ 004 TLT20060510111201_005 gt Im
201. le name 3 Click Save To export the image data to DICOM files dem 1 Select File Export DICOM on the menu bar 2 Inthe dialog box that appears select a destination or create a new directory for the data and click OK A directory containing the dcm Tiles and a SequencelInfo txt is created at the specitied location Figure 3 17 Saving or exporting image data 29 a 3 Initializing the System amp Acquiring Images This page intentionally blank 30 XENOGEN Living Image Software User s Manual Life Changing 4 Working With Images 4 1 Opening Image Data Browsing Image Data 1 Click the Browse button Gy rowse Alternatively select File from the menu bar The Browse For Folder box appears 2 In the Browse for Folder box select the data of interest and click OK Note When you start the Living Image software and open the browser the software automatically returns to the last folder visited 3 The selected data are displayed in the Living Image browser along with the user ID and label information and camera configuration information The sign indicates an image SEQUENCE 4 Click the sign to display the data in a sequence To view data properties right click the item of interest and select Properties from the shortcut menu Note For details on how to open an Image or image sequence see Figure Opening Image Data 2 a a a 31 The Tool Palette
202. lter 7 Bremen F 4 se Se J or Open Fl NS Protogeph fT 3 Modum lt 6 Zi fass F Ovetay T Uphte Field of View f nei Shpa heaghe 7 50 2 em _ image Setup Focus use subject height Temperature IT incked ntishre VIS system Delay 010 min Apply tc Al 3 Remove A Update Chime oaa To edit a parameter value in the sequence editor 1 Double click the table cell that you want to edit 2 Enter a new value in the cell or make a selection from the drop down list 3 Press Enter or click outside the cell Note Press Enter on the keyboard to add a row to the end of the table using the information in the control panel Figure 3 15 Editing a parameter value in the Sequential Setup table 3 6 Manual Focusing The IVIS Imaging System automatically focuses the image based on subject height see Focus page 14 If you do not want to use the automatic focus feature you can manually set the focus Figure 3 16 To manually set the focus 1 In the control panel choose Manual Focus in the Focus drop down list The Manual Focus window appears j TVIS Acquisition Control Panel x Imaging Mode Exposure Time Binning F Stop Excitation Alter Pee Filter gr Luminescent 1 0 oo sec Medium jf Pick Jf B Fuorescent l EEF Phtooash ME In the Manual Focus Window jor 2 Select the size of the step increment that the stage moves choose
203. lts 3 7 mm just below the BLT Results DLT noSimplDLmsh Loaded surface Key Value Total source flux phot s 5 82e 10 Final size 2 00 Number of sources 30 Reduced Chiz 2 soe 03 Starting vsize best 6 00 Results tab displays the results data and the DLIT S2Ppa best oie Neurf best agd parameter values Total surf samples Save Results Name OLIT_noSimpelDLmeh gt 3D Tools Perspective Loo Fie Edit view Tools Window Help LR Ra ke gt Surface Topography DLIT 3D Reconstruction Tobest view the light EiT ae sources voxels AQ E 1 In the 3D tools a e H move the slider or enter an opacity Mesh Volume Organs Animation value to adjust the MI Render Mesh mesh opacity pacity S eel oe r Fender Photon Density Map 2 Clear the Render Photon Density Map 70 option to display the era mesh without the Treni photon density map Intensity _ 26 5 Color Table ro Reverse Logarithmic Scale Slice Coronal 20 Sanittal A 5 0 Transasial r 4 T Figure 10 7 Viewing DLIT 3D reconstruction results Results tab top and 3D tools bottom Perspective Image window 3D view voxels 14 GA Source Intensity es photons sec Source Intensity 117 a 10 3D Reconstruction of Sources 10 2 3D Reconstruction of Fluorescent Sources Acquiring an Image S
204. mage to a graphic file 1 Click the Export button mm Figure 6 1 Creating a composite image Image math enables you to create a new image by mathematically combining two images 78 XENOGEN Pe eo Life Changing Living Image Software User s Manual Table 6 1 Items in the 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 he 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 k k Image Math window A user specified scaling factor applied in the results function k Fluorescent Background Subtraction window The software computes k the ratio of the autofluorescent signal 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 Display Result for Measuring Opens the imag
205. move a check E n Unenixed 2 Anal Opt Spect Result maliz mark to show hide the L mi Ri pectrum esults f Loo Ha ed Armplitude spectrum in the spectrum plot ae et Show Type Mame Label Color E SoL Unmied gt Unmixed 1 Pa Urnmsed Unimiked 2 Select a spectrum row to i Spectrum Plot show in the preview pane ls actus Preview Ed 0 50 al Normalized Ariplitude 30 720 760 a0 a4q Wavelength mi Fail 740 F l 780 200 230 g4 Wavelength mm Figure 9 3 Spectral unmixing tools and spectrum plot Item in the Spectral Description Unmixing Tool Palette Type The type of spectrum 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 EXT A spectrum loaded from the external data provided by the user This mode is currently disabled 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 a
206. multiplied by 1 4 from the primary filter image The 6 week old female Nu nu mouse was injected subcutaneously with 1x 106 HeLa luc PKH2 cells in the left flank 1 2 Em Filter Ex Filter Bkg Filter Py 1 0 i PA Excitation i bei wassers Emission 2 0 8 i J ae Avitofluorescence af AG as we amp a HE 0 4 Na 1 O G2 EN S s 400 450 500 550 600 650 700 750 Wavelength nm Figure D 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 179 a D Fluorescent Imaging This page intentionally blank 180 XENOGEN gt Life Changing Living Ilmage Software Users Manual Appendix E Planar Spectral Imaging Planar Spectral Imaging Theory 002 8825 e 181 Optical Properties 1 o o e a a 183 Luciferase Spectrum oaao a 4 a 183 An Example of Planar Spectral Imaging 183 Optimizing the Precision of Planar Spectral Analysis 187 The unique spectral signatures of the luciferase emission spectrum and the optical properties of tissue enable the Living Image software to determine th
207. n 103 Max 38915 Color Bar Min 103 Max 38915 Note For details on how to browse data and open images see Figure 4 1 page 31 and Figure 4 2 page 33 Figure 4 9 Image window overlay display mode Item inthe Image Description Window Units Choose counts or photons from the drop down list for the image data For more details on counts and photons see Appendix A page 151 Display To choose an image display mode in the image window make a selection from the Display drop down list Figure 4 9 See Figure 4 10 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 or fluorescent 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 camera background acquired with the camera shutter closed See Appendix C page 159 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 de
208. nalysis features Macintosh users have access to only the analysis features of the Living Image software a 2 Getting Started Men bar eG 40 Ae Toolbar VY If the workstation controls the IVIS Imaging System the control panel appears For more details on the control panel see Chapter 3 page 9 Figure 2 1 Living Image software user interface at startup For more details on the menu bar and toolbar see Appendix G page 199 2 2 Basic Living Image Software Tasks Here are links to some common tasks in the Living Image software Table 2 1 Basic Living Image software functions Task See Page Acquire an image IVIS Lumina or Spectrum 18 19 IVIS 50 100 or 200 Imaging System Series 18 19 IVIS 3D Imaging System Series 20 21 Acquire an image sequence 23 24 Acquire an image sequence for bioluminescent 3D tomographic analysis IVIS 200 Imaging System Series 110 IVIS 3D Imaging System Series 110 Acquire an image sequence for fluorescent 3D tomographic analysis IVIS 118 Spectrum only Save or export image data 29 Browse and open image data 31 Adjust the image display 45 Correct or filter image data 46 Make image measurements 48 Draw a region of interest ROI on an image 57 Mathematically combine images 78 Overlay multiple images 82 Remove tissue autofluorescence from a fluorescent image 79 Using spectral unmixing IVIS Spectrum
209. nd Sources amp Corrections 159 C 1 Electronic Background 2 2 a 159 C 2 Background Light On the Sample 2 2 00000 160 C 3 Background Light From the Sample 0 2 04 162 Appendix D Fluorescent Imaging 0 4 165 D 1 Description and Theory of Operation 0 0002 ee ee eee 165 D2 Wile Specs s s se ew seen ee SA eee eRe eee sb hpa betes 167 D 3 Working with Fluorescent Samples 2 20000 552 0G 169 D 4 Image Data Display a oa oa aaa ee 170 D 6 Subtracting Instrument Fluorescent Background 177 D 7 Subtracting Tissue Autofluorescence Using Background Filters 178 Appendix E Planar Spectral Imaging 181 E 1 Planar Spectral Imaging Theory 2 000000 000 181 E 2 Optical Properties o a a a a ee 183 E3 L ciferase Spectrum zs 65844466448 SSE Ee OERGA EE REE SS 183 E 4 An Example of Planar Spectral Imaging 183 Appendix F 3D Reconstruction of Light Sources 189 F 1 Determining Surface Topography 000 ee eee ee eee 189 F 2 Algorithm Parameters amp Options ooo a ee 194 Appendix G Menu Commands amp Tool Bar 199 Appendix H User Preferences 0 02 2008 203 Hl Viewine User Preferences es aoe dw Gee ee eee SEES HEBER EB HES 203 Living Ilmage Software Users Manual 1 Welcome
210. nd plot the spectrum at this pixel in the spectrum preview 102 XENOGEN Living Image Software User s Manual eis Changing Modifying the Spectrum Plot You can modify the appearance of the spectra in the spectrum plot and add or remove spectra from the plot To add a spectrum plot C Fie Edt view Tools Window Help EA 1 In the spectral unmixing eal amp AS e tools click the Spectrum tab 25 25 E Concentration Plot Spectrum Plot PCA Biplot PCA Explained Variance l peimad Sual E k e Show Legend E 2 Right click the spectrum L gt Planar Spectral Imaging Eam table and select Add on the V Spectral Unmixing shortcut menu Analyze Options Spectrum Results pg Nonmalized Amplitude ame Spectrum Show Type Name Label Color SOL Unmteed Unmixed 1 mM SOL Unmived Unmixed 2 3 In the Spectrum dialog box that appears Spectrum Pj Tope O Name AFBBOEm i Spectrum Preview Loo Sidi Amplitude Label AFB80Em Line Color pes A n n i i 0 0 Cancel 720 760 00 240 Dae Wavelength mri a Select the type of spectrum SOL LIB ROI EXT b Make a selection from the Name drop down list 720 740 760 T80 00 820 a4 Wavelength nr c To select a color for the spectrum graph click the button d Click OK The spectrum graph appears in the s
211. nd save an image to a graphic file e To open the Image Layout dimage Layout Window aE window select View gt OD SSBC ZA K SF Laynrtsyle Fiee syle w Image Layout Window on the menu bar elo paste the active image into the Image Layout window click the button Note If you do not clear the layout before you close the Image Layout window the same window contents are displayed the next time the window is opened Annotation ar 42 A Figure 4 25 Image Layout window 54 Living Image Software User s Manual XENOGEN eT Life Changing Item in the Image Description Layout Window Clears the Image Layout window 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 Oval drawing tool AM Sy a a Arrow and line drawing tool a EN 4 Bring forward h WY Send backward Send to back 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 Layout Style PETTEE dw A drop down list of formatting options for the Image Layout window For example the 2x2 layout style provides
212. nd the emission filter DsRed Figure D 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 improvement reduces the minimum number of cells necessary for detection from 1 5x 105 to 6 7x 103 XENOGEN Living Image Software User s Manual a Te a Primary excitation filter b Background filter c Corrected image DSRed DSRed Bkg BOD y eH 3 F 1 7 x10 a 3 wo a A 200 11x10 3 5 4 5 100 Signat Background 6 5 Scale Factor 1 4 Sigal AMS error 150 Min detectabie cells 1 58 10 Min detectable cells 6 7 10 Figure D 17 Example of the autofluorescent subtraction technique using a background excitation filter a primary excitation filter DsRed b blue shitted background excitation filter DsRed Bkg and c corrected data The corrected image was obtained by subtracting the scaled background filter image
213. ne the source location In Figure C 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 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 C 3 Background Light From the Sample 162 Another source of background is the natural light emitted from a sample that is not due to emission from the source of interest in the sample This type of background may be due to a material associated with the experimental setup For example the cell culture medium may phosphoresce Materials should be screened
214. new session the Select Add Select Add User ID EIE me i Known UserID Hews f Living Image v3 0 Software Open Look in E CK20031215152406 SEQ 7 emee My Recent K20031215152405_002 Documents CK20031215152405_003 L E ck20031215152405_004 gt i C3 ck20031215152405_005 Desktop E ck20031215152405_006 aceon CK20031215152405_008 SavedDLITResults txt 2 SayedResults txt EJ Sequenceinfo txt 09CK20031215152405_001 K20031215152405_001_orig My Documents My Computer x Text files txt X My Network File name Places Files of type User ID box appears Figure 4 3 Opening image data without the Living Image browser 4 2 The Tool Palette Cancel 3 Enter your user ID or make a selection from the drop down list and click OK The image s and tool palette are displayed Figure 4 2 The tool palette contains information about the active image s and organizes the image analysis tools The tools available in the tool palette depend on whether an image or an image sequence is active Tool Palette gt Image Adjust gt Corrections Filtering Image Information _ Tool Palette VimageAdust Click to expand a tool 34 gt ROI Tools _ Planar Spectral Imaging Surface Topography Color Scale Limits Auto Ful Individual Color Table ra Reverse Manual
215. nt source fitting Items in the Point Source Description Fitting Tabs Analysis Tab Image ExWL EmMWL MinRadiance X Y Params Tab Model Type Angle Limit deg Spatial Filter Parameter starting values x y OF Z F yield Power MuaEm MusEm 94 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 196 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 Living Image Software User s Manual Items in the Point Source Fitting Tabs XENOGEN eT Life changing Life Changing Description MuaEx MusEx Restore Defaults Mask Statistics Weighting LM Fitting Properties Tab Tissue Properties Internal medium index of refraction Results Tab MuaEm MusEx Mueff Diff X location of the source Y loc
216. ntally 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 recommended Structure 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 t stop and exposure time are automatically set to defaults for the structured light image f 8 and 0 2 sec respectively The spatial resolution of the computed surface depends on the line spacing of the structured light lines The line spacing and binning are automatically set to the optimal values determined by the FOV stage position and are not user modifiable Transillumination Setup Choose this option to display the transillumination setup window that enables you to IVIS Spectrum only select the locations for image acquisition using bottom illumination that originate
217. o the active image P 58 Living Ilmage Software Users Manual XENOGEN fe aa Life Changing Item in the ROI Tools Description 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 Click and select Auto All to automatically draw ROIs in the image using the auto ROI parameters Click and select Auto 1 to automatically draw one ROI around a user specified area For more details on using the auto ROI features see Figure 5 3 page 61 S oe Click to display the ROI Measurements table Click to display a drop down list of options to delete an ROI s in the active image For more details see Deleting ROIS page 71 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 Average Bkg or Subject ROI 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 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 fr
218. of a new session The settings are applied when this user ID is selected Preferences CRE General User 3D View Tissue Properties DUT Params Biotrue To add a new user ID User Setting s Existing User ID KSA x New User D 1 x In the Preferences box click New User ID Preferences Defaults Label Name Set Xenogen Universal all Edit User Label Choices Default Units Counts Add New User Known UserID KSA New UserID Cancel 2 Inthe dialog box that appears enter a new user ID and click OK The user ID is added to the system To delete a user ID 1 Select the ID that you want to delete from the Known User ID drop down list 2 Click Delete User ID Figure H 7 Adding or removing a user ID from the system Living Ilmage Software Users Manual Index Numerics 3D fluorescent sources 118 121 3D image displaying planes 136 rotate or move 132 3D luminescent sources 109 117 3D perspective 135 3D reconstruction bioluminescent sources 115 125 displaying organs 137 139 fluorescent sources 118 121 parameters amp options 194 197 3D reconstruction results 123 125 A acquire image sequence 23 24 acquire single image 18 20 IVIS 3D 20 21 adjusting image appearance 45 angle limit 195 autofluorescence 62 172 miscellaneous material 173 175 See tissue autofluorescence subtract using background filters 17
219. of numbers but unlike a scientific image the numbers are only color codes and are not related to light intensity A graphic image can be exported to a graphic display application Pseudocolor Images An image can be generated from scientific image data by assigning a color to each numerical value and plotting the array so that each pixel is filled with the color that corresponds to its numerical value A color table defines the relationship between the numerical data and the displayed color For example a grayscale color table assigns black to the smallest number in the array white to the largest number and shades of gray to the values in between Figure A 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 A 1 149 a A Image Data Display amp Measurement A 1LT20050624145507_004 Units Counts v Display Photograph aae A 1LT20050624145507_004 zino i g Units Counts m Display Overlay Info iy Image Image Min 96 Min 49 Max 2307 Max 58989 2000 50000 40000 30000 20000 10000 Counts Counts Color Bar Color Bar Min 96 Min 405
220. 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 151 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 151 Click Attributes None All Possible Values All Populated Values Xenogen Defaults 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 click attributes from the table Includes all of the click attributes label name settings and camera settings in the table Includes only the click attributes with values in the table Includes all Xenogen Default label name settings in the table ROI Dimensions Make a selection from the drop down list to specify the ROI dimensions to include in the table None Excludes the ROI area x y coordinates and dimensions from the table Pixels Includes ROI area x y coordinates and dimensions in pixels in the table cm Includes ROI area x y coordinates and dimensions in cm in the table Copy Selected Copies the selected row s in the table to the system clipboard All Copies all rows in the table to the system clipboard Re
221. ographic Setting Limneroeni fo a f e Feim f 560 s a a a Block DP Peco 4 fiom je gees i Medan Block SBE No i5 o oo FEY Stuchae 1 Medaum 1 Bleck BED Yen 150 4 oo FF De T ugh i 1 Medan 1 Block ED No 1 50 4 oo I i 1 Medan 1 Black SEU Ye 150 w n Gyabem Sima vew OO O a 1MM i 6 1 Meda 1 Biozk Sa No 150 a on Angie Increment E5 3 de Saas Serene EA i I Medium 1 Bleck BAD Yen 150 1 o0 IL a pa 1 Medam 1 Block 560 Ma 150 1 a Subic height 150 em Image Setup a ee t Medam 1 Block BED es aa Pocus use subyect haghi Tenperatue B ose lrviiakze TAS syriam Deap 3 min iu x mar tind ar Fiver Chas 5 ____________________ Sequence Editor __________ 2 If necessary click Femve and select All to clear the table 3 In the control panel specify the settings for the first bioluminescent or fluorescent image in the sequence and the photographic image e For IVIS Imaging System100 Series 200 Series or Lumina See Figure 3 6 page 18 for more details on image acquisition settings e For IVIS System 3D Series See Figure 3 8 page 20 for more details on image acquisition settings 4 In the sequence editor click Ey Add The acquisition parameters are added to the table Each row in the table specifies the acquisition parameters for one image in the sequence 5 Repeat step 3 for each image in the sequence Note If you choose the Use Previous Photograph opt
222. olor image 149 R radiance photon 152 radiance units 152 read bias 159 reconstruct 3D fluorescent sources 118 121 reconstruct 3D luminescent sources 109 117 reduced Chi2 123 region of interest See ROI ROI 57 background corrected signal 62 63 delete 71 edit dimensions 67 edit position 67 free draw 62 measurement ROI auto ROI method 61 free draw 62 manually draw 60 measurement ROIs 59 62 Measurements table 58 move or edit label 69 ROI line 68 save 70 213 _ Index tools 58 59 ROI Measurements table 72 73 configure 74 copy or export 75 ROI properties 64 66 ROI types average background 57 62 measurement 57 subject 57 S save image data 29 ROI 70 scientific image data 149 single image acquisition 18 20 IVIS 3D 20 21 smoothing 47 158 source spectrum 124 spectral imaging See planar spectral imaging spectral unmixing 99 options 106 parameters 105 PCA biplot 104 PCA explained variance 105 starting the system See system initialization steradian 152 subject ROI 57 system FOV 16 initialization 9 manual focusing 28 optics autofluorescence 177 temperature 10 T tag an image 42 technical support 4 temperature 10 threshold angle 124 tile images 42 tissue autofluorescence 178 179 eliminate by spectral unmixing 99 subtracting with background filters 79 81 tissue properties 124 214 tool palette 34 35 correcting filtering 46 47 image information 48 49 ROI tools 5
223. om the system Note This permanently removes the ROI from the system Applies the ROI set selected from the Name drop down list to the active image Saves the ROI set in the active image Note This is a global save the ROI is saved to the system and the ROI set can be loaded onto any image If you use the File gt 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 70 5 3 Drawing Measurement ROIs To obtain the intensity signal in an ROI draw a measurement ROI on the image and click the Measurement button There are three ways to draw measurement ROIs e Manual Figure 5 2 page 60 e Automatic Figure 5 3 page 61 e Free draw Figure 5 4 page 62 59 5 Working With Region of Interest ROI Tools Manually Drawing Measurement ROIs 1 Open the image of interest Note An image must be active to display the ROI tools If an image sequence is active the ROI tools are absent from the tool palette 2 In the ROI tools select Measurement ROI from the Type drop down list 3 If the image is a member of a Sequence and you want to apply the ROI to the other images In the sequence choose the Apply to Sequence option 4 To specify the ROI shape click the Circle 0 Square O or Grid button J On the drop down list that appears select the number of
224. on 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 The pointer becomes a fe 2 To move the Image in the image window click and hold the pointer while you move the mouse Note This feature helps you view different areas of a magnified image If the image has not been magnified you cannot pan the image Figure 4 11 How to magnify an image or pan the image window 41 a 4 Working With Images Tagging an Image You can apply a tag to a user specified location in an image Figure 4 12 The tag displays the x y pixel coordinates of the location and the pixel intensity z counts or photons A TLTS00S 0824145507 _002 Alias QQ TLTS0S0624145507_002 Uris Cowes Ooms To tag a location To move the tag 1 Right click the location of interest in the image 1 Position the pointer over the label 2 Select Insert Tag on the short cut menu 2 When the hand tool appears use a click and drag operation to move the tag then click the To remove a tag mouse to set the tag location 1 Position the pointer over the tag A line between the pixel and the tag identifies the location associated with the tag 2 Right click the image and select Remove Tag on the shortcut menu 3 To remove all tags right click the image and select Remove All Tags on the shortcut menu Figure 4 12 Applying
225. on the selection in the Tissue Properties list Figure 7 4 Performing planar spectral image analysis continued from Figure 7 3 Tool Palette E E Image Adjust nimi ikea E Corrections f Filtering _ me gt RO Tools Planar Spectral Tirana Analyze Froperties Results Tissue Properties Muscle l Internal medium indes of refraction Source Spectrum Firefly Plot Tissue Properties Wavelength ror i gt Surface Topography E 6 In the Properties tab make a selection from the Source Spectrum drop down list 7 Click Analyze in the Analyze tab Tool eet E Conections Filtering gt Image Information _ gt ROI Tools E Planar Spectral Imaging Analyze Properties Properties Results Spectral Results Unsaved ROI Depth ram ik Flux phota gt Plot Intensity p a Plot Linear Fit Save Results Name Splm_3 Delete urface T opography The Results tab displays the computed average depth mm and total flux ohoton sec of the bioluminescent point source in the specified ROI s 89 a 7 Planar Spectral Image Analysis 7 4 Viewing amp Exporting Graphical Results Tool Palette ai To view a graph of the results z Image Adiust J 1 In the Results tab select an ROI 2 Click Plot Intensity or Plot Linear Fit gt ROI Tools Y Planar Spectral Imaging Analyze Properties Results A Plot of Intensity Vs Lambda
226. 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 begins with large voxels specified by the voxel size limit the length of a sid
227. only 99 By subtracting a fluorescence background image 79 Planar spectral image analysis 88 Reconstruct 3D bioluminescent sources 109 n XENOGEN Living Ilmage Software Users Manual ae Life Changing Table 2 1 Basic Living Image software functions Task See Page Display organs on a 3D reconstruction 137 Upload or download data from the Biotrue Collaborative Data 147 Management System an optional feature 2 3 Living Image Help There are several ways to obtain help on the software features e To view a tooltip about a button function put the mouse cursor over the button e To view a brief description about an item in the user interface click the Wf toolbar button then click the item e Press F1 or select Help User Guide on the menu bar to display the Living Image 3 0 Software User s Manual pdf a 2 Getting Started This page intentionally blank XENOGEN Living Image Software Users Manual Life Changing 3 Initializing the System amp Acquiring Images Initializing the IVIS Imaging System 0 0 00 eae 9 Checking the System Temperature aoao 5004 10 Imaging Basics occ kd hd a a a 10 About the Control Panel na aoao a a a eee ees 12 Acquiring ImageS naaa a 17 Manual Focusing lt x oe we ee OS eS a 28 Saving or Exporting Image Data 500205000 29 The IVIS System is ready for image acquisition after the system is initialized and the demand operating temp
228. ons available in the image window and tool palette depend on whether an image or an image sequence is displayed When you open an image sequence the image window displays thumbnails of the images in the collection Figure 4 6 A single color table is applied to the images For details on how to open an image sequence using the Living Image browser see Figure 4 2 page 33 peep Re Se ayy To open all images ina ri a AUNTIE Py EE sequence click the Display All cas FRRGS GEE Use Peviousb Spree Colon m gg Dutton To hide all open 240 H Be lane click the Hide button Prete celine Gamma 7 is g ies La haku Cok Toa re To open any image in a separate Max W image window double click the robr Sram Levels thumbnail 3 Aio 0 Fa aria Choose Individual to apply a separate color scale to each image thumbnail ina sequence Finirai je Fiere L Tool palette Image window __ Fie Edt view Tools Window Help Inirdvicisal Cake Tialda q Chen PEAS 1S8_030 LI Logrihai Erai Sequence View tab JAK SAAM Ea nRa R Boo Mesh Volume Render Mesh Opacity F V Render Photon Density Me Apply 5i imulated gt Planar EA Imaging gt Surface Topography 5 a EAEN e Organs 5 A Sequence View 4 3D View If DLIT analysis results are loaded click the 3D View tab to a y display the 3D reconstruction of the lum
229. onstruction 2 Enter your user ID or make a selection trom the drop down list and click OK The image s and tool palette are displayed Min 2140 Mus 36446 3 To open an image in a sequence double click the thumbnail To open all images in a sequence Je Beer click the Display All button Ta To close all images click the Hide button 5 Tool palette The image window displays thumbnails of the images in a sequence using a single color table 586 Mim 1122 Mas 10348 96 Mae 19546 For more details on working with a ei File Edit view Tools Window Help JA X sequence in the image window see ga QB BB NP Unie Cane O eh oa page 36 E Units Counts Display Overlay y Eno 3 For more information on the tool H IA palette see page 34 Plana Spectralimaging sd Min 46 Max 4405 gt Surface Topography 3000 2000 1000 Max 3432 L Single image in the image window Figure 4 2 Opening an image sequence or an image The tools available in the tool palette depend on the image data 33 a 4 Working With Images Opening Image Data Without the Living Image Browser 1 Click the Open button amp on the toolbar Alternatively select File Open on the menu bar 2 The Open box appears Double click the data of interest Alternatively select the data and click Open At the start of a
230. ore details meest on opening an image sequence see page 31 2 Click Planar Spectral Imaging in the tool rae palette The planar spectral imaging tools are displayed in three tabs Figure 7 2 Figure 7 1 Planar spectral imaging tools Tissue amp source selected in the Properties tab Active image sequence Tool Palette maea O O Planar Spectral Imaging Analyze Properties Results Sequence CA 200372 75750499 SEQ Tissue Muscle Source fref Select Filters Fiter 560 500 600 M620 540 cca _ Select All Display ROls _none_ v Analyze gt Surface Topography gt gt DLIT 3D Reconstruction i Analyze tab a File Edit View Tools Window Help SAA a ag Ke 11 Bere ttre enero H Sequence View Planar Spectral Imaging 7 Plata Saectallmagng a n Analyze Properties Results Sequence CX20037275750449_ SEQ Tissue Muscle Source Freh Select Filters e500 i 520 500 520 540 ROls _none_ x Analyze gt Surface Topography 4 gt DLIT 3D Reconstruction Tool Palette Ea gt tmageAdust Hl l 5 Planar Spectral Imaging Analyze Properties Results Internal medium index of refraction 1 40 Source Spectrum Firefly Plat Source Spectrum Normahzed Amplitude 1 00 600 800 Wavelength nra gt Surface Topography gt DLIT 3D Reconstruction Propertie
231. p tool OR The length of the distance measurement cursor Figure 4 24 page 54 Figure 4 23 page 53 Figure 4 24 page 54 Figure 4 23 page 53 49 _ 4 Working With Images Viewing X Y Coordinates amp Intensity Data To view x y coordinates information Fie Edt view Tools Window Help 20K amp associated intensity S AA H A R i Units Counts 40O Apply toal 1 In the Image Informations tools mati Units Counts al Display Overlay x tino C choose Cm or Pixels from the Units CO Image TLT20050624122348_001 Series Male Nn nu drop down list torn ing Fri Jun 24 2005 12 24 53 Experiment DOB 02 28 05 Erm Filter 560 Label Two traser beads Scruff re Bin MJ8 FOV 12 6 f2 1s Comment Dorsal 2 Put the mouse pointer over the laa OE CaP Hefin a Camera viS 200 Beta Il sI620EEV location of interest in the image Image P f Binning 2 Min 48 The x y coordinates and intensity Mak 28314 data are displayed in the tool Mase Y 6274 4731 cm palette mage Data 283374 counts The information is updated when SST Y at 909000 B 000000 you change the pointer position TAL i E Eee N 200 000 Distance 000 gt ROI Tools fi gt Surface Topography Color Bar Min 397 Max 18712 Figure 4 20 Position the mouse arrow in the image window to display x y coordinates and intensity data Viewing an Image The image histogram plots a frequency distribution of t
232. pears 2 In the dialog box that appears choose a folder for the results enter a file name and click Save Figure 10 27 Managing results 141 _ 10 3D Reconstruction of Sources 10 8 Creating an Animation Key frame box shows the key frames in the current animation setup Click a key frame to display the associated 3D view and time stamp position in the time scale 0 100 at which the frame occurs in the animation Figure 10 28 Animation tab 142 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 mesh 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 For More Details See View a preset animation generated from a predefined Figure 10 29 animation setup page 144 Create a custom animation generated from your custom Figure 10 30 animation setup page 145 Edit an animation setup Figure 10 31 page 146 Save an animation to a movie Figure 10 31 page 146 File Edit View Tools Window Help 0 X aa a E a E eja s E Mesh Volume Organs Animation Preset Animations Presets Spin CCW on Y Anis Frame Factor 1 Animation Setup Time Scale B Key Frame 1 Key Frame
233. pectrum plot tab To edit a spectrum plot 1 In the Spectrum tab of the tools right click the spectrum row that you want to edit 2 Int pectrum dialog box that appears enter a new label or click the button to select a new color for the spectrum graph 3 Click OK To remove a spectrum plot 1 In the spectrum tools tab right click the spectrum row that you want to delete and select Delete on the shortcut menu The spectrum graph is removed from the plot Figure 9 4 Adding editing or removing a spectrum plot 103 _ 9 Spectral Unmixing PCA Biplot 104 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 Concentration Plot Spectrum Plot PCA Biplot PCA Explained Variance P ero s l OSOS O OMl RAs Component 1 Y Ass Component E ae a L0 08 06 04 2 00 034 04 06 08 10 Figure 9 5 PCA biplot Living Image Software User s Manual XENOGEN Life Changing PCA Explained Variance The PCA Explained Variance histogram shows the part of variance y axis that can be explained by a number of principal components x axis g Pat BT Figure 9 6 PCA explained variance 9 3 Spectral Unmixing Parameters ET The Results tab in the Spectral Unmixing tool palette shows
234. ple 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 CH meonta OOOO 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 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 Figure 3 16 Temperature The temperature box color indicates the temperature and status of the system System not initialized System initialized but the CCD temperature Is out of range 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 10 Acquire Click to acquire an image using the settings and options selected in the control panel or to acquire an
235. r more measurement ROls on the subject For more details see page 61 2 Draw an average background ROI on the subject a Select Average Bkg ROI from the Type drop down list b Click the Square O or Circle Q button and select 1 The ROI is added to the image For more details on adjusting the ROI position or dimensions see page 66 and page 67 Note The average background ROI and measurement RO 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 three methods shown below to associate the average background ROI with one or more measurement ROIs 4 To obtain the background corrected signal click the Measure button J The ROI label displays the background corrected intensity and the ROI Measurements table appears more details on the ROI Measurements table see page 64 Three ways to associate an avg bkg ROI amp measurement ROls Units Photons Display Overlay a Image Subject 2 Min 5 25e3 Max 2 03e6 1 5 10 x106 0 5 p sec eom 2 st Color Bar Min 3 00e5 Max 1 79e6 Method 1 Draw a subject ROI that includes the measurement ROI and the average background ROI To draw the subject ROI using the auto ROI feature select Subject ROI from the Type drop down list click the button and select Auto All To manually draw the subject ROI select Subject ROI from the Type drop down list click t
236. r pixels that comprise a 2x2 group on the CCD are summed prior to read out and the total number of counts for the group is recorded Figure B 3 This produces a smaller image that contains one fourth the pixels compared to an image at binning 1 However due to summing the average signal in each pixel is four times higher than at binning 1 At binning 4 16 pixels are summed prior to read out Binning significantly affects the sensitivity of the 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 sinc
237. references DLIT Params Tab System Select the IVIS Imaging System 200 Series or 3D Series from the drop down list Angle Limit deg Kappa Limits Description The angle between the object surface normal and the optical axis For more details see page 195 Kappa x is a parameter that is searched during a reconstruction to determine the N Surface Limits best fit to the image data Small values of kappa tend to favor deeper sources while large values favor more shallow sources For more details see page 196 The maximum number of surface intensity points to use in the reconstruction at a Voxel Size Limits 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 Voxels are the small cubes of space inside a subject each of which contains a light Voxel Size Increment 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
238. rlap 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 Living Image Software User s Manual XENOGEN eT Life Changing Fluorescent Filters and Imaging Wavelengths Eight excitation and four emission filters come standard with a fluorescence equipped IVIS Imaging System Table D 1 Custom filter sets are also available Fluorescent imaging on the VIS 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 D 1 Standard filter sets and fluorescent dyes and proteins used with IVIS Imaging Systems Emission Passband Dyes amp Passband Passband nm nm AEU Excitation GFP 445 490 DsRed 500 550 Cy5 5 615 665 ICG 710 760 GFP Background 410 440 DsRed Background 460 490 Cyb 5 Background 580 610 ICG Background 665 695 515
239. rofile Table 10 1 shows the recommended image sequence for DLIT analysis Analyzing more images usually produces more accurate results Ata 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 10 1 Recommended image sequence for DLIT analysis on the IVIS Imaging System 200 Series amp IVIS Spectrum 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 For details on how to specify a sequential setup and acquire an image sequence see Chapter 3 page 17 The IVIS Imaging System 3D Series has a translational stage and rotating mirror which enables views from multiple angle perspectives around the mouse For DLIT analysis the image sequence acquired on the VIS Imaging System 3D Series must include a e Photograph every 30 e Photograph and structured light image every 45 e Luminescent image acquired through two different filters every 90 XENOGEN Living Image Software User s Manual Life changing Reconstructing the Surface Topography Mesh shows the recommended image sequence NOTE If an appropriate sequential setup xsq already exists you can import the sequ
240. rror bars represent noise in the image Figure 7 5 Viewing graphical results of planar spectral image analysis and exporting the data 90 Living Ilmage Software Users Manual XENOGEN Life Changing Life Changing 7 5 Managing Planar Spectral Imaging Results 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 Analyze E gt Corrections f Filtering ea Image Information gt Rol Tools lf Planar Spectral Imaging Properties Results mE Rei Unsaved Total Flu phot rL Ter ote EEES ETE R AA a A O E A EEE TTA Boat La m gt Plot Linear Fit Plot Intensity Save Results Name Spim_3 J Delete Load E Swtace Topography Figure 7 6 Saving and managing planar spectral imaging results Tool Palette ir gt Image Adjust BE To copy selected results I Right click the results row of interest and select Copy Selected from the shortcut menu that appears he selected results are copied to the system clipboard To copy all results 1 In the Results tab right click the results table and sele
241. s Display Stuctee v irio g CR cO0S0624151159_00 Bias Figure 4 10 Display modes for a single image The software coregisters the luminescent and photographic image to generate the overlay image 40 Ck2O050624 51158_ 00 re Lipi Cour TS oaan al ein Luminescent AOR P00508 2415171 SH _ O01 Umir Coots ipiy Seterence Reference XENOGEN ee _Life Changing Living Image Software User s Manual tite Changing Magnifying or Panning in the Image Window File Edit view Tools Window Help JIA Xx gt H ret teed La amp K i Units Counts io Apply to all Ie Imape Ae S Units Counts 1 Display Overlay v mo 4 i gt Imoge Information ____ gt Planar Spectral Imaging Max 10944 8000 6000 Insert Tag Remove Tag 4000 Remove All Tags Copy All ROIs Paste ROI Hide ROI Tags 2000 Delete All ROIs 4rea Zoom Zoom In Counts TONAR Color Bar Min 547 Max 8622 To incrementally zoom in or out on an image eClick the E or S button Alternatively right click the image and select Zoom In or Zoom Out on the shortcut menu To magnify a selected area in an image 1 Click the button Alternatively right click the image and select Area Zoom on the shortcut menu The pointer changes to a 2 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 magnificati
242. s beneath the stage Table 3 3 Additional IVIS System controls for the IVIS Imaging System 3D Series Item in the Control Panel IVIS Imaging System 3D Series Angle The starting position of the CCD camera relative to the imaging stage The first image in a sequence is acquired at this angle Inc The number of degrees between each successive position of the CCD camera during the acquisition of an image sequence Structure Choose this option to take a structured light image an image of parallel lines scanned across the subject when you click Acquire For more details see Structure in Table 3 2 15 a 3 Initializing the System amp Acquiring Images Table 3 4 Additional IVIS System controls for the IVIS Spectrum Item in the Control Panel IVIS Imaging System 3D Series Transillumination Setup Choose this option to for bottom illumination excitation light Click to display the Transillumination setup window Figure 3 12 Structure Choose this option to take a structured light image an image of parallel lines scanned across the subject when you click Acquire For more details see Structure in Table 3 2 16 Table 3 5 Typical field of view FOV settings FOV Setting IVIS Imaging System Lumina 100 Series 200 Series FOV cm A 4 10 3 9 B 7 15 6 5 G 10 20 13 D 12 25 19 5 E 26 There are no FOV settings for the IVIS Imaging System 3D Series because the instrument
243. s for Planar Spectral Image Analysis 85 Planar Spectral Imaging Tools a 86 Performing Planar Spectral Image Analysis 88 Viewing amp Exporting Graphical Results 90 Managing Planar Spectral Imaging Results 91 The Living Image software enables you to apply planar spectral image analysis to an 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 E page 181 7 1 Image Sequence Requirements for Planar Spectral Image Analysis Table 7 1 shows the recommended image sequence for planar spectral image analysis Analyzing more images usually produces more accurate results 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 nm Table 7 1 Recommended image sequence for planar spectral image analysis on the IVIS Imaging System 200 Series Image Type Emission Filter Photographic Select the Reuse Photographs option in the control panel Luminescent J J J For details on how to specify a sequential setup and acquire an image sequence see Chapter 3 page 17 85 a 7 Planar Spectral Image Analysis 7 2 Planar Spectral Imaging Tools To display the planar spectral imaging tools i 1 Open an image sequence For m
244. s 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 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 a 3 Initializing the System amp Acquiring Images Table 3 1 General controls for all IVIS Systems Item in the Description Control Panel Field of View Sets the size of the stage area to be imaged by adjusting the position of the stage and lens The FOV FOV is the width of the square area cm to be imaged A smaller FOV gives a higher sensitivity measurement so it is best to set the FOV no larger than necessary to accommodate the subject or area of interest The FOV also affects the depth of field range in which the subject is in focus A smaller FOV results in a narrower depth of field Select the FOV by choosing a setting from the drop down list For more details on the calibrated FOV positions A E see Table 3 5 page 16 Service Click to move the stage to a position for cleaning Load Click to move the stage from the cleaning position back to the home position 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 exam
245. s tab Figure 7 2 Planar spectral imaging tools and results 86 Units Photons C Use Previously Saved Colors fri 4 3 Gs ked gt eS IMin 3 6603 Mur 392e4 3 Maur shtet j Min 8 54e3 Mac 1375 Min 5 18e3 Missie E Tool Palette gt gt Image Adjust CS e o A gt Corrections Filtering E ie gt Plot Lineal Fi Save Results Name Splm_3 D elete _ Surface Topography Results tab Living Image Software User s Manual Item in the Planar Spectral Imaging Tab XENOGEN eT Life Changing Description Analyze Tab Sequence Select Filters Select All Display ROIs Analyze Drop down list of open image sequences Click the acquisition wavelengths for the images in the selected sequence To select non sequential 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 Click to select all wavelengths Click to display the images from the sequence acquired at the selected wavelength s A drop down list of the ROIs in the active image Click to perform the spectral analysis Properties Tab Tissue Properties Internal medium index of refraction Source Spectrum Display Tissue Properties Source Spectrum Results Tab Spectral Results ROI Depth Total Flux Plot Linear Fit Plot Intensity Save Results Name Delete Load Save
246. s tab eee an Te u continued in Figure 10 6 Figure 10 5 Reconstructing 3D bioluminescent sources 115 a 10 3D Reconstruction of Sources To reconstruct 3D bioluminescent sources Tool Palette i Tool Palette F continued from Figure 10 6 L Planar Spectral Imaging a teign Z Sutace Topography S 4 In the Analyze tab DLIT 3 Reconstruction Z DLIT 3D Reconstruction Anal Anal Pat P t Fesults o VIS Imaging System 200 Series or IVIS nalyze Params Properties Results nalyze anams roper Properties sults ji e Sequence fi T202 22a SEG Sequence CAMs IDE SER Spectrum Select the acquisition wavelengths 560 660 nm Tissue dusch Source Ereti Tissue dunne Source Srat Select Wave Filters Select Wave Filters 7 IVIS Imaging system 3D Series select the i Minimum Radiance Filter Views Minimum Radiance acquisition filters 550 585 nm 585 620 ery San Ea nm and 620 700 nm All view angles are itt HO 4 65e5 selected by default for each acquisition ey B01 2 Be oe Seis filter i 7 e400 90 7 7Se 03 Siena 135 7 7Ge 03 o l 180 7 75e 03 5 If necessary edit the minimum radiance 2 7 756403 associated with an acquisition wavelength 4 E5e 05 or angle For more details on the minimum radiance see Appendix F page 189 Reconstruct Reconstruct Note It is recommended that you only aD Tods OOOO 3D Tools analyze images that have signal well above the noise Anal
247. saturation It is recommended that the signal of interest is greater then 600 counts and less than 60 000 counts If the signal level is unacceptable adjust the exposure time or binning level gt Surface Topography DLIT 10 In the Edit Image Labels box that appears enter the image information and click OK If you do not want to xenogen Universal UserlD KSA z LabelSet 1 Bloch 1 Blot y 5 1 Bick 5 5 1 Bibo 60 Ye i 3 i Medaan 1 Block SSON 150 3 og 1 Medium 1 Bloch SED Yes 150 1 o0 Medam 1 Bick EBD No 150 15 oo Medam 1 Bleck FED Yes 150 180 oO Delay f00 Ejmin _Anehioas Xe Remove GA Update O irose Qaa Soe ee iuit Ea T Sequence view T 3D View 2ImageAdust ____ B _ Planar Spectral Imaging I X Units Counts C Use Previously Saved Colors 2 C Mins 66 Nur 9769 Min 86 Mic 16843 Image window Mins 69 Mar 9603 Edit Image Labels xi Check any 5 fields enter label information click Cancel ie p V Group Ee a Note For details on how to save or Ao oO export the image data see Figure 3 17 Fema TE ommen uae page a I Time Point Animal Number hs Animal Strain i 3a Animal Model Sex aa T View es T Cell Line Po I Reporter On I Treatment as M LucinectionTime S YS M WACUCNumber T Apply To Sequence Edit Image Labels box Figure 3 11 Acquiring an image sequence continued from
248. see lable 6 1 page 79 4 Inthe Image Math window select the primary image in box A Select the background image in box B 5 Click Compute k tam Role and select the ROI created in step 2 from the drop down list Mav 247e10 The background corrected signal is displayed iq image Math Window 6 To view the mathematical result overlay mode in a separate image window click Display Result For Measuring Note If necessary use the Color Scale Min and Max sliders in the Image Adjust tools to adjust the image display Sequence TLT20061220101746_SEQ A TLT20051221160239 TLT20051221160320 Primary image selected from box A B TLT20051221160239 TLT20051221160320 Color Scale Limits for amp and B A To save the new image Background image selected from box B 1 Click the Save button f Alternatively select File gt Fu O Auto Save on the menu bar Ful D Aut Result Color Scale Limits 2 In the dialog box that appears select a directory and e 1 Mn 0 click Save Result A B k v Sati mS k 1 00 ATLT_M20051221160239 A folder of data is saved to the selected location Unte F Decir Oroi e l A AnalyzedClicklnfo txt Clicklnfo txt luminescent E CT z and photographic TIF images To export the new image to a graphic file 1 Click the Export button mg 2 Inthe dialog box that appears select a directory en
249. sh called the perspective view is the default 4 4 F Top Bottom ome Back i Left Figure 10 20 Alternative views of the mesh 135 a 10 3D Reconstruction of Sources Displaying Planes Through the 3D Image To display the planes 1 Click the button The Coronal Sagittal or Transaxial windowpane FAAN a R amp W EEEE PRH A C Sequence View I 2 View Fr Srettingng a Eotonal 10 5 shows the intersection of the plane with the image slice 2 To change the location of a plane move the coronal sagittal or transaxial slider left or right Alternatively click the Opacity a s s Mesh Volume Render Mesh C Render Photon Density Map Sagittal plane Transaxial plane k 2G Organs Animation 55 4 ova a Saaittal 1 8 Eg Figure 10 21 and Figure 10 22 show how to display planes through the 3D image and make measurements a H Help Apply Simulated j 30 0 s x10 slider then press the lt or wavelengh 560 ll lee keyboard arrow keys Threshold 554 391 Intensity 4 feb The Coronal Sagittal or Re Transaxial windowpane Is Ranbon a Transavial 1 a utom at ca ly u odated Reverse Logarithmic Scale Slice Coronal ir 105 li Coronal plane Sagittal 1 8 aa Transaxial gt i il SARF os bo Figure 10 21 3D view window and 3D tools To measure distance in a p
250. shold Save ROIs Name ROI_2_KSA v 1 0 Delete Load gt Planar Spectral Imaging gt Surface Topography p sec om 2 st Color Bar Min 1 01e5 Max 1 79e6 PAS CAIEIFA LETETI E E LN TEE Units Photons Display Overlay Info iy Image Min 5 25e3 Max 2 03e6 1 5 10 196 Ri 0 5 p sec em 2 sr Color Bar Min 1 01e5 Max 1 79e6 Figure 5 3 Drawing measurement ROls using the auto ROI method The software can identify all ROIs on the image that meet the auto ROI parameter thresholds top or one ROI at a user specified location bottom 61 5 Working With Region of Interest ROI Tools 5 4 Drawing an ROI Using the Free Draw Method To draw an ROI using the free draw Ei Fie Edt view Toos window Help aE method A A wl a amp R i Units Photons 7 Apply to all Units Photons Display Overlay v Info iy 1 Open the image of interest for more details on Opening an image see page 31 a 2 Select the type of ROI that you want to draw O O Y x y 1 Man 20368 C Apply to Sequence from the Type drop down list Type Measurement ROI x Threshold J z s 3 Click an ROI shape button Circle OJ eset Smee Square J or Contour and select Free rer 10 sof Draw from the drop down list Note The ROI shapes that are available depend on the type of ROI selecte
251. sion 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 167 D Fluorescent Imaging 168 100 O Excitation Pi Filter O gt lransmission o ro _ 0 001 Wavelength nm Figure D 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 Emission Optical Density Wavelength Figure D 5 Typical attenuation curves for excitation and emission filters In Figure D 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 VIS Imaging Systems transmission in the bandpass region is about 0 7 OD 0 15 and blocking outside of the bandpass region is typically in the OD 7 to OD 9 range The band gap is defined as the gap between the 50 transmission points of the excitation and emission filters and is usually 25 50 nm There is a slope in the transition region from bandpass to blocking Figure D 5 A steep slope is required to avoid ove
252. sion spectra for several different luciferases A digital Xenogen Mouse Atlas male and female nude mouse and the ability to automatically coregister the atlas with your DLIT 3D data Import a custom organ atlas Ability to import Open Inventor files of segmented CT or MRI images and coregistering with a DLIT 3D image using a non rigid registration algorithm Create and export movies of tomographic data from multiple perspectives Reconfigured menu bar that matches the standard layout of Windows Macintosh applications Living Image software remembers the path of recently accessed folders and provides auto save capability during imaging Integrated Instrument control image acquisition and analysis tools written in C for enhanced stability and performance Extensive help tools and PDF manual with quick links for rapidly finding information See Page 147 85 109 109 i5 137 140 145 IVIS Imaging System z J J J J A J J A J J A J pf ft J J J J A J J OO D S Living Ilmage Software Users Manual XENOGEN fe Bizas Life Changing 1 2 About This Manual Conventions Used In the Manual This user manual explains how to acquire images on an IVIS Imaging System and analyze image data 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 s
253. so 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 Creating an Image Sequence from Individual Images page 38 XENOGEN eife Living Image Software User s Manual tite Changing 5 Living Image Browser 1O open amined Or imeds CickNunber UserID Series _ Exeeimert Label Comment Date and Tine sequence ee CK 20031215152 3405 SEG _CK B16F10 1 1VIS200 nu nu ventral mou 5x10 5 cells Day 13 o E B16F10 IV Day 13 12 15 2003 15 25 15 1 In the Living Image browser double i ventral 42 15 2003 15 26 31 j ie A 12 15 2003 15 28 46 gt click the file or folder name p 2 OER ean Alternatively select the file or folder i i 5s 13 15 2003 15 3215 of interest and click Load j 4 fabio gee ens F i ventral 12 15 2003 15 34 44 At the start of a new session the i inu nu ventral 12 15 2003 15 36 09 Select Add User ID box appears For details on how to open the Livin ieee browser see eae 4 1 S iaasa m Z M atta tie renee oana Load Close f Select Add User ID FR 7 Fie Edt view Tools Window Help EJA X Known User ID _none_ New User ID pe ak F Sequence View Units Counts C Use Previously Saved Colors A ig e Planar Spectral Imaging mim ai gt Surface Topography gt DLIT 3D Rec
254. splay hide a scale on the x and y axis of the Figure 4 20 page 50 image window Click this button to display hide a grid the image window Figure 4 20 page 50 BCE E 48 Living Image Software User s Manual Image Information Tool Description Choose the units cm or pixels for distance measurements in the XENOGEN ee ife Changing Life Changing ti l image window Image Binning The binning applied to the image Note If soft binning is applied to the image data and the binning level is changed from 8 to 16 the new binning is indicated as 8x2 Image X Y The x y pixel coordinates of the mouse pointer location in the Figure 4 20 page 50 image Image Data The intensity counts or photons at the pixel location of the mouse Figure 4 20 page 50 Crop Distance pointer Hees The x y pixel coordinates at the upper left corner of the crop tool Figure 4 24 page 54 LA OR The x y pixel coordinates at the A end of the distance Figure 4 23 page 53 measurement cursor a The x y pixel coordinates at the lower right corner of the crop tool Figure 4 24 page 54 OR The x y pixel coordinates at the B end of the distance measurement cursor For details see Figure 4 23 page 53 Distance The width and height of the image crop tool OR Ax Ay from the A to B end of the distance measurement tool The length of the diagonal from corner A to corner B in the image cro
255. st 8 00 Kappa best 2 00 Nsurf best 800 Tatal surf saronles igli completed For more details on viewing the voxels see page 130 Save Results Name DLIT_noSimplDLmsh Overwrite 2 To display the measured and simulated E GOJIGENMES photon density profiles Wavelength EE Select one or all wavelengths for display Pj Photon ensily Maps Wavelingth DDRII na of View 7 LIJ LL 11 0 lepih Sea Move the wheelto the left or right to rotate the meshonthe vertical axis Y Angle of View ra Poe e E E aI C Dogatithmic Scale a Select a wavelength ae b Drag the crosshairs to the location of interest The horizontal and vertical photon density profiles are updated R i Note In a good reconstruction the photons mm 3 measured and simulated photon density curves are close together ea 3j Photon density photons mm 3 20 A 10 D 10 Position rem Position 40 Horizontal Profile a Simulated ao m WWleasured i Vertical Profile Simulated io m Wleasured 3 5 Photon density photons reun 3 20 D 20 40 60 Position mm Pasition FF Figure 10 12 How to view photon density maps Left Photon density maps for wavelengths Right Photon density map at 620 nm 126 Close XENOGEN Living Image Software User s Manual a 10 5 Working With 3D Images The 3D tools appear in the tool palette when a surface topography mesh or
256. 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 tB2 lt 1000 where T is the exposure time seconds and B is the binning factor Under these conditions dark charge contributes less than 0 1 counts pixel and may be ignored Dark Charge 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 VIS Systems has been cycled or if the camera temperature has changed a new dark charge measurement should be taken The dark charge is measured with the camera shutter closed and is usually performed automatically overnight by the Living Image software The software acquires a series of zero time exposures to determine the bias offset and read noise followed by three dark exposures The dark charge measurement usually takes more than three times as long to complet
257. t Photograph fre 4 Medium vl f8 jr Hane Lights V Alignment Gnd Field of View C n 8 de I Service i23 om ide rane A Subject height 1 50 H cm Image Setup Focus use subject height x Temperature I Locked Initialize VIS System Delay 0 0 aj min Apply to All X Remove v A Update Insert Add Figure 3 14 Control panel left and sequence editor right 4 Item in the Sequence Editor Function Displays a dialog box that enables you to select and open a sequential setup xsq Displays a dialog box that enables you to save a sequential setup xsq Display Photographic Settings Choose this option to include the photograph exposure time binning and F Stop in the sequence editor Delay TE T Specifies a time delay between each image acquisition Apply to Al Applies the selected cell value to all cells in the same column Deletes the selected row from the Sequential Setup table Remove ee Updates the selected row in the Sequential Setup table with the acquisition parameters GUpdete in the control panel Inserts a row above the currently selected row using the information from the control lrisert panel Adds a new row below the currently selected row using information from the control asd panel 27 a 3 Initializing the System amp Acquiring Images 2 WIS Aoquisiten Control Panel imaging Mirbe Exposure Time Finning Step ciation Filter Emission Fi
258. t 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 Living Image Software Users Manual XENOGEN ee _Life changing Life Changing Source amp Tissue Properties DLIT analysis of spectrally filtered images requires knowledge of the spectral dependence of bioluminescent light emission Table F 2 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 E E Planar Spectral Imaging E Surface Topography k 7 DLIT 3D Reconstruction Analyze Params Properties Results Selecta tissue or organ from the igsuePiopeties Muscle _ drop down list The associated 12 internal medium index of refraction is automatically Source Spectrum Firefly i entered Plot Source Spectrum Select a bioluminescent source spectrum Internal medium index of refraction 1 40 Normalized Amplitude Choose the Source Spectrum from the Plot drop down list to display the selected spectrum 1 00 600 800 Wavelength mri pume id Figure F 4 DLIT 3D reconstruction tools Properties tab Table F 2 Source spectra Source Spectrum Description B
259. t view of the subject If necessary resize the crop box to include the subject and a small margin around the subject Repeat this step to confirm the proper position of the crop box in all views Ei Fie Edit view Tools Window Help Surface Generation Options Photo Back Projection Surface Smoothing Parameters Smoothing Level Low Save Results Name SURFACE_3 EJ Analyze Params Properties Results Sequence CK20050624151158_SEQ issue Loss Recovery Height Perspective Mask ourple 8 Click Next to display the mask The mask is a purple overlay on the subject image that defines the area of interest for the surface topography reconstruction The mask should match the underlying photograph of the subject as closely as possible without including any area outside the Subject image 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 10 Click Next to display the next view of the subject It necessary adjust the threshold value 11 Repeat step 10 until the mask is properly adjusted in each view of the image cae 12 Click Finish The mesh and 3D tools appear For more details on the 3D tools see page 127 Note It may take several minutes to generate the mesh If you selected both the Surface and DLIT options
260. tal Flux 2 84eT 0 phofons sec 30 0 Volume 787788 mm 3 n Center of Mass 7 0875 22 2236 97379 r E 1 Perspective fea Sequence View 4 3D View xi 18 photons sec Source Intensity A X gt a H Help f y x101 photons sec Source Intensity 131 a 10 3D Reconstruction of Sources Rotating Moving or Zooming On a 3D Image Select a tool from the drop down list File Edit view Tools Window Help S QA ed a amp SSESESESESESESE SESE siaiac ce cececesesesesesesesesesessecececes XIE gt Planar Spectral Imaging pra Suface Topography JO DLIT 3D Reconstruction TET Ey J4 OR 4M L ES R E olume Organs Animation r Mesh Q n s C Render Photon Density Map lass Apply Simulated 30 0 Sequence View 4 3D View Wavelength so 8 og Threshold Intensity Color Table 407e 6 S Rainbow Reverse Slice en Logarnthmic Sca e Coronal Sagittal 81 20 7 17 1 Transaxial To rotate the image 1 Choose the l or d 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 Select the arrow in the 3D tools and drag the image Alternatively press the Shift key while you drag the image Figure 10 16 Working with images in the 3D Vie
261. ter a file name and select the file type from the Save as type drop down list 3 Click Save Figure 6 2 Subtracting tissue autofluorescence 81 a 6 Performing Image Math Operations 6 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 luminescent or fluorescent images on one photographic image The tool provides a convenient way to view multiple reporters in one image To do this e Acquire a fluorescent or luminescent image sequence using the appropriate filters for each reporter Alternatively create a sequence from images acquired during different sessions For more details see Chapter 4 page 38 e Load the sequence of interest in the Image Overlay window Figure 6 3 e Select a photographic image e Select the fluorescent or luminescent images that you want to coregister and specify a different color table for each image Figure 6 4 To coregister multiple images IA ARW20050826124002_SEQ i Sequence View t 3D View 1 Open the image sequence of interest Units Efficiency C Use Previously Saved Colors a A Note To view all images in the sequence click the Display All button to open each image overlay mode in a separate image window 2 Open one of the images and optimize the image display using the color scale Min and Max sliders in the Image Adjust tools 3
262. the difference in Ups the higher the quality of information that the wavelength data adds to the analysis The recommended XENOGEN Life Changing Living Image Software User s Manual Tp E 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 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 cm2 sr and adjusting the color bar Min to be above the level of noise in the image Parameters Tab Tool Palette gt Planar Spectral maging gt Surface T opography i lt FLIT 3D Reconstruction Analyze Params Properties Results T Angle Lirit deg 70 NM Surface 200 Tool Palette 2 Planar Spectral Imaging sis Planar Spectral Imaging ee V DLIT 3D Reconstruction Results Analyze Params Properties Anale Limit deg E0 lower Upper F Kappa Limits 05 E iM Surface Limits 800 Starting Voxel Size 6 Auto Fluorescence Mosel Size Linnits E Uniform Surface Sampling oxel Size Increment C NALS Weighted Fit
263. 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 175 D Fluorescent Imaging 176 Regular Rodent Food GFP DsRed Cy5 5 ICG Alfalfa free Rodent Food GFP DsRed Cy5 5 ICG Figure D 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 D 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 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 0x1
264. the optimized fit parameters used by the software to derive the spectral unmixing results Figure 9 7 Tool Palette gt Image Adjust Spectral Unmising Results tem a Number of Iterations 16 Humber of Components 2 Number of Wavelenghts Number of Samples 2F00 Lack of Fika PC4 3 50637 Lack of Fit EXP 3 79593 Divergence counter Maximum Iterations 200 Denoise PCA Medium Pees Save Results Name SPUM_ f Erel te Figure 9 7 Spectral unmixing tools Results tab 105 a 9 Spectral Unmixing Item in the Results Tab Description Number of Iterations The number of iterations that the algorithm used Number of Components The number of components unmixed Number of Wavelengths The number of wavelength pairs used in the analysis Number of Samples The number of pixel samples used in the analysis Lack of Fit PCA The fitting residue compared to the data filtered by principal component analysis Lack of Fit EXP The fitting residue compared to the experimental data Divergence Counter The number of divergences that occurred Maximum Iterations The maximum number of iterations allowed Denoise PCA Indicates how much of the data was filtered by principal component analysis Normalization The normalization method used in the analysis Non negativity Method The non negativity method used in the analysis Weighting Mode The weighting method applied to the data Column Weighting Mode Indic
265. the software generates the mesh and performs 3D reconstruction of the bioluminescent Sources Figure 10 4 Generating the surface topography IVIS Imaging System 3D Series continued from Figure 10 2 114 Living Image Software User s Manual XENOGEN ee _Life changing Life Changing Reconstructing 3D Bioluminescent Sources After the mesh is generated the 3D reconstruction of the light sources can proceed Figure 10 6 shows the steps to reconstruct 3D sources and Figure 10 7 shows example results For more information on the DLIT algorithm and user modifiable parameters see Appendix F page 194 To reconstruct 3D bioluminescent sources 1 In the tool palette click DLIT 3D gt Planar Spectral Imaging Reconstruction Surface Topography DLIT 30 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 retraction is automatically entered when you select a tissue a Analyze Params Properes Results Tissue Properties Muscle Intemal medium index of refractions i A Source Spectrum Firefly 7 3 If you want to view the tissue properties Ua Hefi Us Of source Spectrum for the tissue and light source selected above make a selection from the Plot drop down list in the Propertie
266. 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 6 2 page 79 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 6 page 79 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 D 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 D 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 Bkg the primary excitation filter DsRed a
267. ties box appears Note The items in the ROI Properties box depend on the type of ROI selected To view properties for another ROI either e Click another ROI in the image The ROI Properties box is automatically updated OR e Select an ROI from the ROI drop down list at the top of the ROI properties box Figure 5 6 ROI properties 64 TLT20050624122348_002 Units Counts Display Overlay a ROI 1 Resize Rotate Copy ROI BKG 1 Copy All ROIs Paste ROI pe a Duplicate ROI 4 Syeee Set Bkg ROI to BKG 1 Set Bkg ROI to none Set Subject ROI to Subject 1 Set Subject ROT to none Hide ROI Tag Delete ROI Delete All ROIs Unlock Position Unlock Size Eee Image Min 39 Max 47071 30000 20000 10000 Counts Color Bar Min 352 Max 38915 Living Ilmage Software Users Manual XENOGEN fe aa Life Changing ROI selected in the image ROI Properties ROI BKG 2 ROl Label BKG2 Bkg ROI Use as BKG for future ROIs in CK20031215152405_001 c Entire sequence C Lock Position ef em 8 0279 Yelem 6 4606 Angle deg 0 0000 C Lock Size Width cm 1 1657 Line Size Line Color Bkg ROI tab average background ROI selected in the image Subj RO Info La bel name of ROI Properties DR ROI Properties DE the selected Rol ROI2 Drop down list of ROI ROI E ROI
268. ting 0 0 0 000000 2 eee ee 93 8 1 Displaying the Point Source Fitting Tools 0000 93 8 3 Checking the Point Source Fitting Results 0 000 97 8 4 Exporting Results aoaaa 97 9 Spectral Unmixing 20 0 2 99 9 1 Performing Spectral Unmixing resci 2 eee ee ee ee 99 9 2 Spectral Unmixing Results Window 0 0000 eee eee 101 9 3 Spectral Unmixing Parameters 2 2 ee 105 9 4 Spectral Unmixing Options 2 106 EE Contents 10 3D Reconstruction of Sources 0 109 10 1 3D Reconstruction of Bioluminescent Sources 2088 109 10 2 3D Reconstruction of Fluorescent Sources 2 000040 118 10 4 DLIT amp FLIT Results 22 4 25 5446448464444 8b 66 24444 SG 123 11 Biotrue Collaborative Data Management System 147 11 1 Uploading or Loading Image Data 00 4 147 Appendix A Image Data Display amp Measurement 149 Ad Inace Data Ban gen haba pees BAe eee ee eRe eee eRe 149 Was Flat Fieldiig se seses emagena wk See ERR eee Ree tees eee 153 A 4 Cosmic Ray Corrections 1 2 ee 154 Appendix B Detection Sensitivity 0 4 155 B 1 CCD Detection Efficiency 2 0 0 0 00 ee ee 155 De i006 oa nha eo eee ee ee ERE RE ESSERE REGS EERESD ES 156 Bo SMOOUMNG Gon ee neer ke errs eeeereceakeeseGerts ated 158 Appendix C Luminescent Backgrou
269. tion 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 143 _ 10 3D Reconstruction of Sources Viewing a Preset Animation File Edit View Tools Window Help To view a preset animation FAAR Lao amp 1 Open the DLIT results of interest 2DLIT 3D Reconstruction _ Te TEIE sE Mesh Volume Organs Animation Preset Animations Presets Spin COW on Axis
270. torage and management capabilities You can upload data from the Living Image software to the Biotrue CDMS or load data from the Biotrue CDMS to the Living Image software For more details on managing the Biotrue CDMS see the Biotrue CDMS User Manual 11 1 Uploading or Loading Image Data To open the Biotrue browser C File Edit view Tools Dar Help SAARA 1 Click the Browse Biotrue button 3 Alternatively select File Browse Biotrue on the menu bar ne T Sequence View To upload data to the Biotrue CDMS 1 Confirm that the data of interest is the active window 2 In the Biotrue Browser select the directory for the data and click Upload The image data is added to the directory 3 Click Refresh to update the Biotrue browser and view new data on the Biotrue CDMS File EGR View Tou Window Heb EESE eae ASW To load data from the Biotrue CDMS 1 In the Biotrue Browser select the data Piare 1 ea directory of interest and click Load Biotrue Browser Use Label Set Kerogan Delna Petre Make a selection from the drop down list to view label set information Figure 11 1 Uploading or downloading data from the Biotrue CDMS amy 147 a 11 Biotrue Collaborative Data Management System This page intentionally blank 148 XENOGEN ome 8 eae Life Changing Living Ilmage Software Users Manual Appendix A Image Data Display amp Measurement imag
271. trum data For more details see Angle Limit page 195 Kappa limits 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 page 196 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 Tit Starting Voxel Size FLIT The length of the side of the voxel cube in mm units that is evaluated by the algorithm to determine the optimum solution 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 F
272. und 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 Figure 6 2 explains how to use the image math tool to subtract tissue autofluorescence after you acquire an image sequence that includes a primary and background image For more details on acquiring an image sequence see Chapter 3 page 17 For more details on tissue autofluorescence see Appendix D page 178 XENOGEN ee TAAA Living Image Software User s Manual Ute Changing To subtract tissue autofluorescence TLT20061220101746 SEQ 1 Load the image sequence that includes the primary iniii 027 r and background fluorescent images Units Photons ii C Use Previously Saved Colors TLT20051221160239 TLT20051221160320 2 Open either the primary or background image and a Optimize the image display using the color scale Min and Max sliders in the Image Adjust tools b Draw a measurement ROI on an area of the animal that represents background signal area where no fluorophore signal is present Note You only need to draw the ROI on one of the images The software copies the ROI to the other image 3 Select Tools Image Math for xx SEQ on the menu bar The Image Math window appears Note For more details on items in the Image Math window
273. urce read from the click info x y or x y z depending on the image model Params tab Tool Palette gt Surface Topography Point Source Fitting a Analysis Params Properties Results Model Type Epillumination Fluorescence Angle Limit deg 70 Spatial Filter 0 1 Parameters starting values 0 140 Power photon s Tel 0 MuaEm 1 cm 0 54 MusEm 1 cm 8 81 Restore Defaults Mask No mask RA Statistics Weighting LM Fitting li gt FLIT 3D Reconstruction gt 3D Tools Starting parameter values Properties tab Tool Palette gt Image Adjust l gt Surface Topography Point Source Fitting Analysis Params Properties Results Tissue Properties Muscle Internal medium index of refraction 1 4 Plot Tissue Properties ern 1 Wavelength nra E FLIT 3D Recursion Select other starting values for the optical properties here Total source flux TERI Lai tab Results tab Tool Palette Analysis Params Properties Results Error Estimation Starting Chi 2 8 58e 09 Ending Chi 2 7 03e 08 Point Source Fitting Results Fitted value 0 143 crm 6 676 cm 1 710 cm 0 049 cm 2 22e 11 photons x location of the source 0 181 crm LAOI 3 Parameters Photon Density Maps Export results Save Results gt FLIT 3D Reconstruction _ gt 3D Tools Point source fitting results Figure 8 2 Tool palette poi
274. ve ADIs Name ROI_2 KSA sac D elete Load 5 ave 3000 Color Bar Min 586 Max 10348 File Edit view Tools Window Help S X ge QA A Se R Unite Counts O Apply to all EEEE EHHH EEE ixi Units Counts Display Overlay x Finto Le 7 Image C Apply to Sequence 2000 Type Measurement ROI w pem ROI 1 7 845e 05 6000 Threshold J 25 Save ROls 3 Name ROI_2_KSA x ROI 2 2 903e 04 en Delete Load z e a fi Color Bar Min 1103 Max 9596 ROI Measurements DOR Click Number Image Layer Total Counts Avg Counts Stdev Counts Min Counts Max Counts TLT 200506241 45507 001 PF ca Overlay 1 136e 05 _ 1 495e 03_ 1 022e 03 2601e 02 4 406e 03 TLT20050624145507_ 003 a ROLI Overlay 2 48e 06 5 739e 03 7 690e 03 4 633e4 4 280e 04 TLT20050624145507 003 ROL2 Overlay 5 305e 05 2101e 03 1 413e 03 5 95le 02 16 048e 03 TLT 20050624145507 004 ceed ONT Overlay 2 434e 06 3 202e 04 1 112e 04 1 697e 04_ 5 8992404 TLT 20050624145507 006 ccna MONT Overlay 2 670e 06 2 4376 03 3 140e 03 3 424e 02 2 245e 04 Customized Selections Copy Measurements Types Click Attributes ROI Dimensions Slated Al Counts _ i EZ _none_ pa j vi _none_ vi Figure 5 1 Displaying the ROI tools and quick guide to drawing ROIs Item in the ROI Tools Description Click to select the number of circle ROIs to add to the active image f Click to select the number of square ROIs to add t
275. w window 132 30 0 10 5 z amp 1 8 o0 Mie Perspective EF X x010 0 5 photons sec Source Intensity Axis shows image orientation To zoom in or out on the image L Select the gt arrow in the 3D tools o zoom in on the image magnify right click Ctrl click for Macintosh users and drag the amp toward the bottom of the window TO zoom out On the Image right click and drag the EL toward the top of the window XENOGEN Living Image Software Users Manual etude Selecting a Drawing or You can choose a different drawing and lighting style to change the appearance Lighting Style for the Mesh of the mesh Figure 10 17 shows how to choose a mesh drawing style Figure 10 18 shows how to choose a mesh shading style gt Planar Spectral Imaging gt Surface Topography gt DLIT 3D Reconstruction To choose a drawing style make ry a selection from the Drawing iler REX style drop down list in the 3D ame VAE tools a Opaciey FJ Render Photon Density Map Apply Wave S60 Threshold Intensity jir 3 Color Table os ae iw Reverse _ Logarithmic Scale Wire frame amp surface face mesh Point cloud mesh FAN Wire frame mesh FY Surface face mesh Figure 10 17 Mesh drawing styles 133 _ 10 3D Reconstruction of Sources To choose a shading style make a sel
276. ware Users Manual Appendix F 3D Reconstruction of Light Sources Determining Surface Topography 2 5 2805 189 Algorithm Parameters amp Options 004 194 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 density photons mm3 just beneath the surface of each element of the mesh e Divides the interior of the subject
277. ywhere 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 Determines how to normalize the unmixed spectra Equal Height normalizes by the maximum of the spectra Equal Length normalizes by the sum of the spectra The threshold for the unimodality constraint It is the percentage of overshoot allowed for the second spectral peak 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 107 a 9 Spectral Unmixing This page intentionally blank 108 Living Ilmage Software Users Manual XENOGEN oe ee Life Changing 1 0 3D Reconstruction of Sources 3D Reconstruction of Bioluminescent Sources aooaa oaa a 109 3D Reconstruction of Fluorescent Sources a a o oaa a 118 DLIT amp FLIT Results n aonana 123 Working With 3D ImageS naoa a eew i ew dew a eae 127 Displaying Organs on the 3D Reconstruction 137 Managing Results s 4 v2 e eee ek wee eee eee we Ee REG HS 141 Creating an Animation 0 0 ee ee ee 142 The Living Image software includes algorithms for 3D reconstruction of bioluminescent or fluorescent sources tomographic analysis
278. yze tab Analyze tab IVIS Imaging System 200 Series IVIS Imaging System 3D Series or IVIS Spectrum 6 To edit the minimum radiance double click the entry and enter a new value 7 In the Params tab confirm the parameter Tool Palette defaults or enter new values gt Planar Spectral Imaging For more details on the parameters see PA a Appendix F page 194 8 In the Analyze tab click Reconstruct m Limit deg E0 Angle limit default The reconstruction requires about 1 5 aaa a oe Fens Sm minutes depending on the parameter KeppaLimiss 05 3D Series data or settings and the processor speed NSuface Limits B00 jaan 70 for IVIS Figure 10 7 shows example 3D pedretes e M a ie reconstruction results orel Size Increment 1 Spectrum data E Uniform Surface Sampling DLIT algorithm C NNLS Simplex Optimization user modifiable z NNLS Weighted Fit param eters gt 3D Tools Params tab Figure 10 6 Reconstructing 3D luminescent sources 116 Living Image Software Users Manual XENOGEN aaa e a Life Changing The 3D View in the a File Edit view Tools Window Help image window 5 displays the mesh the ae By A ki Ea a amp 3D reconstruction of T ence la 3D View the bioluminescent light sources inside the subject voxels gt Planar Spectral Imaging E Surface Topography and the photon 7 DLIT 3D Reconstruction density map photons Analze Params Properties Resu

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