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IVIS Spectrum Manual - University of Manitoba

1. Pon iA I SEE EAR AA i 4 a PATA E TLT20050624145507 sEQ baba IG Edit Sequence EJES ra C O Sequin Mew Spectra Sequence Clicks Browser Images Units Counts ba Use Saved Colors Options v Info R a TLT20050624145507 001 JJH20050630142125_001 TLT20050624145507 002 JJH20050630142125 002 TLT20050624145507_003 JJH20050630142125 003 TLT20050624145507_004 4Copy JJH20050630142125 004 TLT20050624145507 005 TLT20050624145507 006 Retired Images Images in the active Mins 2140 lt lt Reactivate Mayo 36446 Images that have osue been removed cove Dow from the active Min 2949 i gt Mins 1122 Max 51182 95 Max 10348 76 Max 19545 4 Choose the image s to add or remove retire from the sequence in the Edit Sequence box that appears Figure 4 31 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 88 To add an image to the sequence select an image from the Browser Images and click Copy To remove an image from the sequence choose an image from Sequence Clicks and click Retire 5 To restore a retired image to the sequence select the retired image and click Reactivate 6 To reorder the sequence select an image and click Move Up or Move Down M NOTE The Move Up and Move Down buttons are only available wh
2. Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools Table 13 3 3D Multi Modality tools for rendering slices Item Description Slice Color Table Choose this option to apply the color table selected from the Color Table drop down list Volume Color Table Choose this option to apply the volume color table of the volume color opacity map that was selected in the Volume tab Color Table Color table options Choose the Reverse option to apply the inverse color table Jb Reverse Opacity Move the slider to adjust the color opacity Color Scale Min Sets the intensity level associated with the lowest color scale value Max Sets the intensity level associated with the maximum color scale value NOTE Black areas that appear around the optical sources in the overlay with the 3D volumetric data slicees are due to the black color level at the low end of the color palette To correct this go to Sources tab in the 3D Optical Tools and move the low end colorbar slider up from the black level Cl Viewing Slices Figure 13 10 Viewing slices Volume Slice Viewer A Volume Slice Viewer A EA Orientation Coronal v Slice Spacing 1 50 mm Total Slices 21 Q A Tool Palette 1 gt ROI Tools Jol L gt Planar Spectral Imaging h o Spectral Unmixing and DyCE Jal Render T
3. 6 2 Drawing a 3D ROI 1 Load DLIT or FLIT results 2 Click the 3D ROI button in the ROI tools Figure 6 3 A red bounding box appears in the 3D View M If you do not see the red bounding box in the 3D View do either of the following m Select the Maximum Intensity Projection MIP option in the 3D Multi Modality tools Reduce the volume opacity by adjusting the position of the Air Noise Boundary in the 3D Multi Modality tools Figure 6 3 3D ROI CEDERE Beem 4 ss Subject Height 19 4mm Perspective 3 Adjust the position of the 3D ROI using the transform tools NOTE It may be helpful to view the surface and or reconstruction results from different perspectives to check the 3D ROI position and size To turn and rotate the surface press and hold the left mouse key then drag the mouse when the hand amy appears a Click the 3D ROI Transform button and select the ROI from the drop down list The first 3D ROI created during a session is named ROI 1 by default A tooltip shows the ROI name when you put the mouse pointer over an ROI 125 Living Image 4 3 Software User s Manual b Click the 3D ROI to begin using the transform tools Chapter 6 3D ROI Tools for Volumetric Data Figure 6 4 explains the tool functions The ROI position is updated in the slice windowpanes coronal sagittal and transaxial views after each adjustment c
4. Drag a column header left or right in the table to reorder the columns 2 Click a column header to sort the table in ascending or descending alphanumeric order 3 Make a selection from the Data Types and Measurement Types drop down lists to change the data and measurements displayed in the table Creating a Custom 3D ROI Table Configuration A table configuration specifies the column headers in the 3D ROI table Several preset configurations are available selected from the Measurements Types drop down list in the ROI table Figure 6 8 You can also create a custom table configuration M NOTE Preset table configurations cannot be edited You can modify a preset configuration and save it to a new name 1 In the ROI Measurements table click Configure The Configure Measurements box appears 130 Living Image 4 3 Software User s Manual Chapter 6 3D ROI Tools for Volumetric Data 131 Figure 6 9 Configure Measurements dialog box F Configure 3D ROI Measurements User Lists photons sec Name photons sec Sort Available Items Average Cells Average pmol Average pmol M cm Center of Mass Max Cell Max pmol Max pmol M cm Min Cell Min pmol Min pmol M crn Quantification ROI Depth mm ROI Height mm ROI Width mm Source Depth mm Source Volume mm Selected Items Total Flux ph 5 Average Flux ph s Stdev Flux ph 5 Min Flux Max Flu
5. Analyze Properties Results DLIT Results DLIT 1 Loaded Key Value Final voxel size mm 1 75 Number of voxels 34 Reduced Chi2 1 71e 02 Starting vsize best 7 00 Kappa best 4 00 Nsurf best 105 Total surf samples 525 Threshold angle 70 00 Kappa limits 0 50 4 00 Nsurface limits 200 00 200 00 x Photon Density Maps E Export Results Save Results Name DLIT_1 v Delete Load Overwrite 197 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Table 11 5 DLIT or FLIT 3D reconstruction results Item Description Final voxel size mm The voxel size length of a side mm that produces the optimum solution to the DLIT or FLIT analysis Number of voxels The number of voxels that describe the light source s Reduced Chi2 A measure of the difference between the computed and measured photon density maps at the optimum solution A smaller X4 value indicates a better quality of fit Index of Refraction Refractive index of light for the imaged subject Angle Limit deg Angle limit of surface normal to optical axis above which data will not be used in the reconstruction Damping reduce The damping parameter is calculated from this reduction factor relative to the maximum singular value of the system matrix Data range For multi view data the image views used in the reconstru
6. Preview picture of the selected data 9 3 DyCE Analysis Automatic or manual DyCE analysis is available Caliper recommends performing an automatic analysis first followed by manual analysis to identify possible additional temporal components Automatic DyCE Analysis 1 Load a DyCE sequence m NOTE The gue icon in the Living Image browser indicates a DyCE sequence Figure 9 10 Load a DyCE sequence Fi RKG20110210131022 SEQ Tool Palette Sequence View Unmixing Composite Image Adjust MUNANG aei Har m gt ROI Tools Units Counts x Use Saved Colors Options Y F Info Rm 7 Spectral Unmixing and DyCE Analyze Results Select Images 00 01 34 4 00 01 56 V 00 02 19 v ae 00 02 41 7 Min 71 Max 541 00 03 43 00 04 45 V 00 05 47 W 00 06 49 V 00 11 51 W 00 16 54 V Min 71 Mina 68 Mins 68 Max 5747 Methods Automatic v Start Unmixing 166 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 9 DyCE Imaging and Analysis 167 2 Click the Analyze tab in the Spectral Unmixing DyCE tools 3 Select Automatic from the Methods drop down list and click Start Unmixing The Auto Unmix Wizard appears and shows the purple data mask that specifies the analysis area Figure 9 11 The data mask includes the entire subject by default 4 If n
7. Preset Animations Presets Spin CW on Z Axis E Frame Factor 1 Animation Setup Time Scale Select a preset animation l 100 3 Key Frame 1 Key Frame 2 Key Frame 3 Key Frame 4 Ad Key Frame 5 Key Frame 6 Key Frame7 Key Frame8 2 Key Frame 9 Key Frame 10 7 Keyframes box Frames Per Second 10 Total Duration secs 5 Creating a Custom Animation To create an animation specify a custom animation setup or edit an existing setup Open an image sequence and load 3D reconstruction results photon density maps Select View 3D Animation on the menu bar The 3D Animation tools appear Figure 11 40 Select properties to display in the 3D View window for example organs sources surface or Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 4 Clear the key frame box if necessary click the w button and select Delete All Figure 11 40 3D Tools Surface Source Registration Animate Preset Animations Presets Spin CW on X Axis wv Frame Factor 1 4 Animation Setup Time Scale 25 E 0 s Play Frames Per Second 10 3 Total Duration secs 5 5 To capture the first key frame click the f button The first key frame is added to the key frame box 6 Aa the position of the reconstruction in the 3D View using an image tool for example 47 or 4 For more det
8. 7 Set the exposure parameters m Bioluminescence DyCE Choose Manual Settings and set appropriate exposure parameter values for your probe m Fluorescence DyCE Choose the Auto Settings option 8 Select a field of view from the drop down list 9 Set the focus by doing either of the following m Fnter a subject height and choose the use subject height focus option OR Choose the manual focus option from the Focus drop down list and set the focus parameters in the Manual Focus Window that appears M NOTE If using the Side Imaging accessory for bioluminescence DyCE set the subject height 0 0 cm and FStop 4 or larger If using the Side Imaging accessory for fluorescence DyCE choose the Manual Settings options and set the subject height 0 0 cm and FStop 2 or larger 10 Specify the time series M NOTE A time series can include up to three intervals Each interval is defined by duration minutes and delay between images seconds The time series can include a maximum of 100 images a Enter the number of intervals b Enter the duration of the first interval and the delay between images The software computes the number of images to acquire during the interval c Repeat step b for each interval 162 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 9 DyCE Imaging and Analysis J NOTE The software alerts you if the number of images in
9. 4 To view the photon density or NTF Efficiency profile at another location on the animal surface drag the cross hairs or click a point on the photon density or NTF Efficiency map Table 11 6 Photon Density Maps window Item Description Image sources A list of images used in the reconstruction Select all images or a particular image number to display Angle of View The thumb wheel position Turn the thumb wheel to rotate the surface on the vertical axis Log Scale Choose this option to display the photon density or NTF Efficiency using a log scale Simulated The photon density or NTF Efficiency computed from DLIT or FLIT source solutions which best fit the measured photon density or NTF Efficiency Measured The photon density or NTF Efficiency determined from the image measurements of surface radiance Horizontal Profile The photon density or NTF Efficiency line profile at the horizontal plane through the subject at the crosshairs location Vertical Profile The photon density or NTF Efficiency line profile at the vertical plane through the subject at the crosshairs location Position mm Horizontal Profile The y axis position of the crosshairs horizontal line Vertical Profile The x axis position of the crosshairs vertical line The x y positions are relative to the center of the FOV where x O andy 0 201 Living Image 4 3 1 User s Manual IVIS Spectrum Chapt
10. AutoCAD DXF dxf Drawing exchange format that is compatible with most DXF file yes yes viewers VRML 1 0 wrl VRML 1 0 wrl Virtual reality modeling language format that yes no is compatible with most VRML viewers Open Inventor iv The ASCII version of the IV file format which is supported by all yes yes IV viewers STL stl or ASCII Stereo lithography binary format compatible with most STL yes yes format viewers binary 11 3D Reconstruction of Sources Overview of Reconstructing Sources Reconstructing Luminescent Sources on page 187 Reconstructing Fluorescent Sources on page 194 3D Reconstruction Results on page 197 Checking the Reconstruction Quality on page 199 Measuring Sources on page 202 Viewing Luminescent and Fluorescent Sources in One Surface on page 205 Comparing Reconstruction Results on page 206 Exporting a 3D Scene as DICOM on page 210 3D Tools Overview on page 213 3D Tools Surface on page 214 3D Tools Source on page 216 3D Tools Registration on page 218 3D Animation on page 225 DLIT FLIT Troubleshooting on page 230 11 1 Overview of Reconstructing Sources The Living Image software provides algorithms which analyze 2 dimensional optical image data to reconstruct 3 dimensional 3D luminescent or fluorescent sources located inside an animal tomographic analysis TIP See the technical note DLIT and FLIT Reconstruction of Sources for more details on the DLIT or FLI
11. Click to display the DICOM file header information 13 9 Viewing RAW Volumetric Data 1 Drag a single RAW file raw or vox from Windows Explorer to the 3D Multi Modality tools Figure 13 24 M NOTE Only single raw or vox files consisting of multiple slices of a 3D volume can be loaded into Living Image f Organize v d Caliper LS d Cakper Date CG jo a Share b CalperlLS Caliper Data Ss ALI20110120171831 SEQ So AutofluorBkgCon D BkgSub CoReg Demo A dam20110306103654 D OUT Datasets emfiters d 1V20110114162356_SEQ d Kathenne Quantum d Kidney J KSA20110305104918_SEQ d LognitudinalData amp lung Si MaAT20090902190433 aneurism bR 2569033aw Daten Open v Bum New folder aneursm Bbit 256x3 raw ech 9 26 2001 442 AM Size 16 0 MB Figure 13 24 Opening RAW volumetric data ml bg Si RAY p sted 2 1 2011 511 PM Toot Palette _ Image Adjust ROI Tools _ Planar Spectral Imaging Surface Topography DUT JD Reconstruction 3V Multi Modality Toots wime Sie Rem ts innmaA qr Dispiay Volene Level Of Octal O 259 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools 2 In the Volume Information dialog box that appears Figure 13 25 enter the m Data width height and the number of slices m Slice row column pixel size and the slice
12. Displays a dialog box that enables you to save the current key frames and animation parameters to an animation setup xkf 226 Living Image 4 3 1 User s Manual IVIS Spectrum Viewing a Preset Animation Chapter 11 3D Reconstruction of Sources 227 Preset animations are factory loaded animation setups They include predefined key frames which are used to generate the animation To view a preset animation 1 2 y Open an image sequence and load 3D reconstruction results Select properties to display in the 3D View window for example organs sources surface or photon density maps Select View 3D Animation on the menu bar In the 3D Animation tools that appear a Clear the key frame box if necessary click the button and select Delete All b Make a selection from the Presets drop down list See Table 11 13 page 226 for a description of the preset animations After a preset animation is selected a list of the key frames appears NOTE You can view multiple animations sequentially For example if you select Spin CW on X Axis and Spin CW on Y axis from the Presets drop down list the animation shows the 3D reconstruction spinning clockwise on the x axis then spinning clockwise on the y axis 5 Click Play to view the animation Figure 11 39 3D Animation tools See Table 11 13 page 226 for details on the animation tools ko Jae IG 3DAnimation
13. G Image Overlay Window Sequence ARW20050826124002_SEQ Photograph Units Radiant Efficiency vv ARW20050826124002_001 Fluorescent Images ARW20050826124002 001 F ARW20050826124002 002 pct fa KI Image Adjust Min B 9 38e7 Color Table mt ColorScale Type Max dJ 1 73e9 Red ri C Palette Label Opacity fal 70 s Reverse Logarithmic Scales per Column 3 2 E fa To overlay all images click the button luminescent image in the list is at the top of the stack The overlay appears The photograph 1s at the bottom of the stack and the last fluorescent or Figure 4 27 Generated overlay C Image Overlay Window Sequence ARW20050826124002_SEQ Photograph Units Radiant Efficiency bi ARW20050526124002 001 v Fluorescent Images V ARW20050826124002 001 V ARW20050826124002 002 mt Bm Image Adjust E sga M Color Table a ColorScale Type Palette Label Max B 4 7469 Green x Scales per Column 3 Opacity P 70 gt E Reverse V Logarithmic eas E a 10 7 10 7 2 10 Radiant Efficiency bani AUR piem Table 4 12 Image Overlay window Item Description Units Choose the type of units for displaying the fluorescent or luminescent image See the concept tech note Image Display and Measurement for more details on me
14. Color Shows the color of the data for the highlighted image Image List Kidney W Brain W Bladder W Photograph Click the color swatch to open the color palette which can be used choose a color for the selected image data Label Bladder Data name for the highlighted image Double click the name to edit it 174 Living Image 4 3 1 User s Manual IVIS Spectrum Correcting Temporal Spectra Chapter 9 DyCE Imaging and Analysis Temporal spectra can be corrected for overlapping spectra for example correcting for tissue autofluorescence l NOTE If correcting for tissue autofluorescence one of the unmixed components of the data set should be tissue autofluorescence signal only 1 Click the fe button in the Unmixing window 2 In the dialog box that appears choose the spectra to subtract Figure 9 20 Figure 9 20 Choose temporal spectra to subtract A x B C Choose spectrum A Choose spectrum B Computed spectrum C pi Fi Compute Pure Spectrum A Mixed Spectrum e g autofluorescence Hlabel Z Normalized 2 Em I i kidney iel 2 Brain BB 3 Bladder E 4 Tissue AF 1 00 0 80 0 60 0 40 o 400 800 1200 Time Point s Ea 1 Kidney 3 Bladder E 4 Tissue AF B Known Spectrum e g autofluorescence Z Normalized 1 00 0 80 0 60 0 40 0 400 800 1200 Time Point s
15. r E Do you want to enable auto saving of acquired data for this session This can be changed anytime from the Acquisition menu Gya a 9 Click Yes in the prompt to enable autosave then choose a location in the dialog box that appears Alternatively click No in the prompt and manually save the image data See page 54 for details Image acquisition begins and the upper area of the control panel changes to red color During acquisition the Acquire button in the control panel becomes a Stop button Click Stop to cancel acquisition The control panel returns to blue color when acquisition is finished and the image window appears Figure 3 13 See Table 3 2 on page 28 for details on the image window 34 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition Figure 3 13 Overlay fluorescent image on photograph in the image window Living Image 43 o a File Eda View Took Window Hate 2471133 Unite Radar Kfficency Display Overlay Optors gt Image TLT200492 17115344 Saree Pluoreecen Background Teste Tue Feb 17 DOA 03 34 57 Doperment 2 female LevelsHigh Emy 5 5 Famy 5 Shig Barnikummabon Bino MI8 tebek 3 Mutia FOV 2 4 f2 is Liig Image Versions 2 50 2 12 2004 restaxheed Carrera MISZ SISMEEV TIP See the tech note Determine Saturation for information on pixel measurements select Help Tech Notes on the menu
16. Figure 8 14 Spectral unmixing results NG ELE y C HX20120419114703_SEQ o ay V Show Labels V Individual Scale 3 amp V Normalized Legend a gl TissueAF 3 Pra zi Spectra plot See page 154 for more y details F 0 50 5 Mins 0 00 e Min 0 00 0 0 Max 9 83e7 x 2 34e8 660 700 740 780 nanaaarya Emission Wavelength nm AF750 Composite Spectrum List See Table 8 3 on oO by x page 155 for details note on this toolbar Select Name Color Pick 1 V TissueAF Mime wigpr 2 V AF680 iz 3 V AF750 We AF A Min 0 00 ax 6 4868 Unmixed images show the extracted signal See page 157 for details on analyzing these images Composite of the unmixed images See page 156 for more details 148 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing Manual Method Sometimes you may want to manually analyze results for example if the explained variance of the principle component analysis of an automatic analysis seems low The example in this section shows how to manually analyze results from a previous analysis 1 Open the image sequence 2 Select the results and click Load Figure 8 15 Open a sequence and select results to load Tool Palette B gt Image Adjust 7G P E Corrections Filtering gt ROI Tools Spectral Unmbcing and DyCE Analyze Results Spectr
17. IVIS Acquisition Control Panel Time Excitation Filter Emission Filter ILJ Auto T sec Medium 1 w Block Open w Jec medium vii v Block Field of View System Status 5 Acquire Lm Sequence Setup Subject height 150 Sem Sequence Setup Focus use subject height Temperature IY Locked M NOTE The options available in the IVIS acquisition control panel depend on the selected imaging mode the imaging system and the filter wheel or lens option that are installed Table A 1 IVIS acquisition control panel from peen o Luminescent Choose this option to acquire a luminescent image Fluorescent Choose this option to acquire a fluorescent image If the Fluorescent option is selected the following options also appear in the control panel Transillumination Choose this option to acquire a fluorescent image using transillumination excitation light located below the stage Normalized This option is selected by default when the Fluorescent and Transillumination options are chosen so that NTF Efficiency images can be produced Ei Photograph Choose this option to automatically acquire a photograph The illumination lights at the top of the imaging chamber are on during a photographic image so that the system can acquire a black and white photograph of the sample s Note You can adjust the appearance of the photographic image using th
18. 00 c eee eee eee eee 194 Image Sequence Requirements eee eee eee eee ees 194 Steps to Reconstruct Fluorescent Sources 194 3D Reconstruction Results i avicce ee ke aud eee and dk oo SE WOO ead 197 ULI or FLIT Results 62660046050 075407 400 Roe See WEW AGA NAG ae eee 197 Checking the Reconstruction Quality 0 00 eee ee ee 199 Viewing Photon Density or NTF Efficiency Maps 200 Measuring SourceS 22 4644 DAWAG KB bbs wee weds Sd bd eg AA ESS e Kag NAG toms 202 Determining the Source Center of Mass 202 Measuring Source Depth es 203 Viewing Location Coordinates 00 cece ees 204 Displaying Slices Through a Reconstruction 204 Viewing Luminescent and Fluorescent Sources in One Surface 205 Comparing Reconstruction Results 0 00 ees 206 Viewing Results in the Longitudinal Study Window 206 Measuring Intensity auacavacee oo AA 208 NIEWING FIOIS 2n autpeneesee hares cans NA APELA DUMADAAN bee ee 209 Exporting a 3D Scene as DICOM cdcccdov ewan deneieiveaed be RED eee 210 Viewing the DICOM Data a4 sa am BABAE uote beet BAKLA ANDAL NA 212 3D Tools Overview a 213 3D Fee a a CA ean Bee en ee eed eae es 214 3D Tools Source 1 ce ee eee eee teens 216 3D Tools Registration s2cehcGedneeataadenbevasce ha DEDE KAMA NGA 218 Displaying Organs With the Reconstructi
19. 52 Tool Palette Bg Sequence View LA 3D View ROI Tools gt po gt Spectral Unmixing and DyCE gt _ Surface Topography gt 3D Multi Modality Tools 3D Optical Tools Coronalfz 16 2 Sagittal x 3 0 Surface Source Registration Registration Tools el 2 v Display Organs dir Gj Opacity Jos E Organ Atlas Female Dorsal Z Organs E ovaries I pancreas E rectum Transaxial 385 17 6 i salivary v skin i spleen I stomach Use Tab key to switch between transformation t Scaling On XYZ Subject Height 26 3 mm ff Perspective Gray surface of the Pink skin surface imaged mouse from the organ atlas Use the transform tools to match atlas organs pink skin in this example as closely as possible to the mouse surface gray 221 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Figure 11 34 Transform tools Use Tab hey to switch belween transformation t oe Tab kany ko verbtcls babaen brareformation t Sening On Kr 4 Ww of the organ s Red W Scales on the z axis Blue W Scales on the x axis Green W Scales on the y axis Use Tab hey to seth between transforination t Click and drag the organ s Click and drag a handle to scale To rotate the organ s on the when the yellow appears inc
20. Choose this option to display the status bar at the bottom of the main window View Tool Palette Choose this option to display the Tool Palette View Activity Window Displays the Activity window at the bottom of the main application window The Activity window shows a log of the system activity View Image Information Displays the Image Information box that shows the label set and image acquisition information for the active data View ROI Properties Displays the ROI Properties dialog box see page 109 View 3D ROI Properties Displays the 3D ROI Properties dialog box see page 128 View ROI Measurements Displays the ROI Measurements table View Volume Data Viewer Enables you to open and view DICOM data View Image Layout Window Opens the Image Layout window that enables you to paste an image of the active data in the window Tools 3D Animation Opens the 3D Animation window that enables you to view a preset animation or create an animation Tools Longitudinal Study Opens the Longitudinal Study window for side by side comparisons of DLIT or FLIT results Tools gt Well Plate Quantification for Opens the Well Plate Quantification window Tools Image Overlay for Opens the Image Overlay window for the active data Tools Colorize Opens the Colorized View tab for the active sequence Tools
21. Display Source Surface ng v Display Voxels Maximum Intensity Projection Threshold a Gradation la 50 Voxel Size 0 31 o Display Voxels As Smoothing 5x5 v Da Texture v Color Scale Min CJ 3 46e6 Color Table BlueYellow Reverse Log Scale rotons sec Source Intensity ae ore Measured Sources Subject Height 26 2 mm Key Value Quantification 6 70e10 photons sec Volume 14 56 mm43 Depth 5 86 mm Center of Mass 3 5 18 1 15 6 Host Organ Unknown Export Voxels Center of Mass Table 11 11 3D Source tools Item Description Select Source A drop down list of available sources Original Results saved with the data lt sequence name SourceVoxelss Pasted voxels Click the bad button to remove pasted voxels from the surface See Viewing Luminescent and Fluorescent Sources in One Surface page 205 for more details on copying and pasting sources from one sequence to another Display Source Choose this option to display the source surfaces reconstructed using DLIT or FLIT A Surface surface will be wrapped around the currently displayed voxels Adjust the voxel display by moving the Threshold slider Drawing styles for the source surface see Display Source Surface 2 php Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Table 11 11 3D Source tools continued Item Descrip
22. Fluorescent emission yield normalized to the incident excitation intensity radiance of the subject illumination intensity NTF Efficiency transillumination fluorescence Fluorescent emission image normalized by the transmission image which is measured with the same emission filter and open excitation filter Image Attributes 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 None Excludes image attributes from the table All Possible Values Includes all of the image attributes for example label name settings and camera settings in the table All Populated Values Includes only the image attributes with values in the table Living Image Universal Includes all Living Image Universal 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 Copies the selected row s in the table to the system clipboard Select All Copies all rows in the table to the system clipboard 120 Living Image 4 3 1 User s Manual I
23. Quantification Plots Copy Select All Export Results Sequence Database WPQUANT_1 w Name WPQUANT_1 X Name Overwrite Overwrite 237 13 3D Multi Modality Tools About the 3D Multi Modality Tools Classifying 3D Volumetric Data on page 239 Volume Display Options on page 242 Smoothing a Volume on page 244 Rendering and Viewing Slices on page 245 Volume Information and Results on page 249 Registering Optical and Volumetric Data on page 250 Volume Data Viewer on page 258 Viewing RAW Volumetric Data on page 259 13 1 About the 3D Multi Modality Tools The 3D Multi Modality tools are used to a Classify volumetric data 3D image data m View slices Refine the appearance of the volume volume processing m Register optical and imported volumetric data for example CT MRI or PET data 3D Multi Modality Tool Requirements The Living Image 3D Multi Modality tools require a separate license Additionally the graphics processing unit GPU must meet the minimum specifications shown in Table 13 1 on page 238 If the appropriate license 1s not installed or the GPU does not meet these specifications the 3D Multi Modality tools will not appear in the Tool Palette Table 13 1 Minimum graphics card specifications Specification Description OpenGL Version Requirement OpenGL 2 0 and above OpenGL Extension Requirement GL EXT texture3D Graphics Card Memory Minimum 256MB Dedicated Sh
24. Spin CW on X Axis Creates an animation from a sequence of 3D views Frame Factor 1 keyframes For example an animation can depict Animation Setup a rotating 3D scene The animation series of key Time Scale frames can be recorded to a movie file Key Frame 1 Key Frame 2 Key Frame 3 Key Frame 4 Key Frame 5 Frames Per Second 10 Total Duration secs 5 Longitudinal Study 206 aS Select Tools Longitudinal Study on the menu D Our nmen pi bar Multiple DLIT and or FLIT reconstruction results at can be viewed side by side in the Longitudinal Study window The Longitudinal Study window provides a gemen gt convenient way to compare different results for example results obtained at different time points _ or results from different types of reporters Voxel intensity within the entire surface or a user selected area can be measured in all results in the Longitudinal Study window a aire aT z Select Tools Well Plate Quantification for ieee Dye molecules Cells lt sequence name gt on the menu bar Measurement Sample Wels 3D 3A O set Generate a database of luminescence or 7 Background Wels 6D 6A fluorescence signal intensities by analyzing images of known serial dilutions of luminescent or fluorescent cells or dye molecules Use the quantification database to extrapolate the number of cells in a DLIT source or the number of dye molecules or cells in a FLIT source 17 Living Image 4 3
25. Surface Source Registration Select Source Original X Display Source Surface iy v 4 Display Voxels Maximum Intensity Projection Threshold CJ radation U Voxel Size 0 31 v Display Voxels As Smoothing 5x5 y Texture v Color Scale Transaxial y 18 1 Min 3 46e6 Color Table on E Reverse Log Scale photons sec Measured Sources Source Intensity Subject Height 26 2 mm Perspective Key Value Quantification 6 70e10 photons sec Volume 14 56 mm 3 Depth 5 86 mm Center of Mass 3 5 18 1 15 6 Host Organ Unknown Export Voxels Center of Mass DLIT 3D Reconstruction 4 Click Center of Mass to obtain the measured source information Note The coronal sagittal and transaxial planes intersect at the center of mass of the selected source see Figure 11 19 on page 203 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 203 Source Description Measurement Quantification The integrated intensity within the selected sources Volume The total volume of the selected sources Depth The perpendicular distance from the source center of mass to dorsal surface Center of Mass The weighted average x y and z coordinates of the selected voxels where the weights are the flux of each highlighted voxel Host Organ The reference atlas organ in which the selected sources ar
26. nG a Image TLT20040217113205 Series Fluorescent Background Tests EK Toolate Image Adjust a a mAg alae Photo Adjustment Color Scale m Tue Feb 17 2004 03 33 20 Experiment 2 female Nu Nu mic NG i e ARN OS Ex Cy5 5 Epi illumination Bin L a 1vS200 Brightness J 100 pr M 8 FOV 12 6 f2 1s Ej abe Living Image Version 2 50 2 12 2004 restructured Comment Contrast J 1 5 Camera IVIS2 SI620EEY x Lan wal Opacity 100 Epi fluorescence Min J 3 048 5 Max Auto Individual Color Table Color Scale Limits Full Manual YellowHat C Reverse Radiant Efficiency t sec cm sy pW iem Color Scale Min 3 04e8 Max 9 75e8 gt Corrections Filtering gt Image Information gt ROI Tools C Logarithmic Scale Detailed information about images is available in the View menu 1 Open an image or sequence 2 Select View Image Information on the menu bar The Image Information window appears 3 Choose an image by making a selection from the Sequences drop down list and the Images drop down list Figure 4 9 Figure 4 9 Viewing image information a maa Show All Sections Image Info Sequence Info Section Drop down list of open sequences Choose Indiv
27. 0 00 0 00 Distance 0 00 gt ROI Tools ke Image Histogram The image histogram plots a frequency distribution of the pixel intensities in an image The software sorts the intensities into groups or bins x axis and plots the number of pixels per bin y axis To view the image histogram 1 Open an image and in the Image Information tools click the Image Histogram button le Figure 4 19 View a histogram of pixel intensities 15 10 i Histogram Window Fa mon 112 Max Bin 19546 Bins 512 TLT20050624145507_006 Overlay lao o Jer 8 104 Tool Palette 53 L gt Image Adjust o Image Information DAP Li Bh unis on Image Binning 8 Width 12 6 cm Height 12 6 cm Image X Y 6 231 6 921 om Image Data 2210 counts TA 00 0 00 Bi 0 00 0 00 Distance 0 00 Go Corrections Filtering A 74 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 75 NOTE By default the Auto min max range of the image data determines the histogram range and bins the software sets the min and max values to optimize image display and suppress background noise To display the histogram using the full intensity range of the image click Full in the Histogram window ell 2 To edit the minimum or maximum bin intensity enter a new value in the Min Bin or Max Bin box or click the
28. 5 Click the Add button Gadd The acquisition parameters appear in the sequence table Figure 3 33 Repeat step 4 to step 5 for each image in the sequence 7 To set a time delay between each acquisition enter a time minutes in the Delay box in the sequence table 8 To save the sequence setup information xsq a Click the Save button mi in the sequence table b Select a directory enter a file name and click Save in the dialog box that appears Figure 3 33 Control panel and sequence table with image settings j IVIS Acquisition Control Panel Imaging Mode Exposure Time i J Auto sec v Medium vii v pao Field of View C v System Status O MIS Subject height 1 50 cm Excitation Filter Emission Filter C Display Photographic Settings Subject Mouse U WE O Seq 1 Mode Exposure Binning FStop Excitation Emission Structure FO Height 1 FESS auto Medium 1 Block 560 Yes C 1 50 Auto Medium 1 Block 580 No 1 50 Auto Medium 1 Block 600 No 1 50 Auto Medium 1 Block 620 No 1 50 1 Auto Medium Block 640 No 1 50 Acquire Sequence Imaging Wizard w Image Setup Focus use subject height v Temperature AN Locked ntie C Number of Segments 1 min Apply to All Ad Update Insert Add Table 3 9 Sequence table Item Description Starts the Imaging Wizard oa Displays a dialog box that enables you to select and open a sequence s
29. C Computed Spectrum e g label Result C A 0 54B Auto Scaling Fit Offset Error Tolerance B Save to Spectrum Index Name New w UMX5 E Normalized 1 00 0 50 Computed spectrum C 0 400 800 Time Point s Table 9 4 Computed spectrum Item Description Normalized Choose this option to normalize the spectra with respect to time Zero Result C A x B The subtraction performed by the software where x is a factor that ensures the residual signal is positive Autoscaling Choose this option to normalize spectra signal on a scale of zero to one Fit Offset If this option is chosen the software computes and removes an intensity baseline from the spectra 175 Living Image 4 3 1 User s Manual IVIS Spectrum Table 9 4 Computed spectrum continued Chapter 9 DyCE Imaging and Analysis Item Description Error Tolerance The software computes a default error tolerance the factor x for A x B such that signal B is maximally removed from signal A with no negative result Moving the slider adjusts the error tolerance and automatically updates the computed spectrum aee AAE DO Choose New to save computed spectrum with the specified 1 name and color Click Apply to add the computed spectrum to the 2 3 line plot and spectrum list in the Unmixing window Choose a spectrum number
30. C Rol Properties mE Label BKG 1 Shape Cirde Type Manual Background ROT C Use as BKG for future ROIs in TLT20050624145507_005 only CO Entire sequence Brightness slider E Lock Fosition A Select Color Xoi pix 117 00218 Basic colors Ye pix 97 93839 Angle deg 0 0000 E Lock Size Width pix 22 06161 Height pix 20 43172 Custom colors Add to Custom Colors Cross hairs in the custom color field Hue 104 3 Red 109 Sat 199 2 Green 255 2 Val 255 Blue 56 Lox cornet 2 To edit the ROI line thickness enter a new value in the Line Size box Alternatively click the arrows 3 To change the ROI line color a Click the Browse button The Select Color box appears b To select a basic color for the ROI line click a basic color swatch and click OK c To define a custom color drag the crosshairs in the custom color field adjust the brightness slider and click Add to Custom Colors d To select a custom color for the ROI line click a custom color swatch and click OK rs bd 115 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data Move or Edit the ROI Label To move the ROI label 1 Put the mouse pointer over the ROI label 2 When the pointer becomes a fh drag the label and then click to release the label a
31. Units Counts E Use Saved Colors opine info FF i a Layout F Display tw Eposure Time Labels Flu Binning Fecter hy ng Lag Ewritation fer Eman Hilter Humbe Field of Whew Select All Chaar all fF erara Tir Dii In this example exposure time and binning factor are displayed on each image Info Click to show or hide the image label information Opens all of the images in the sequence Closes all open images 165 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 9 DyCE Imaging and Analysis Table 9 1 Sequence View window continued Description Opens the Edit Sequence dialog box that enables you to add or remove images from the sequence Enables you to export the active image as a graphic file for example png dcm TEJE Creates a preview picture snapshot of the image or thumbnails that the Living Image Browser displays when the data are selected See page 48 for more details on the browser C Living Image Browser 7mm RKG20110210131022 SEQ Click Number EX Filter EM Filter User ID User Group Experiment Commenti 4 m j r Hide Browse View Close Preview Label Set All Add to List Browse J View Default x Configure Load as Group Load Remove Close Location KATHERINE PC Share Caliper LS Caliper Data CerenkovDyCE RKG20110210131022 SEQ SequenceInfo txt
32. arrows T 3 To edit the number of bins enter a new value in the Bins box or click the arrows Paa M NOTE In the Overlay display mode the histogram plots the luminescent data To obtain a histogram of the photograph select Photograph from the Display drop down list Table 4 7 Histogram window Item Description Full Displays the histogram using the full intensity range of the image Min Bin The lowest intensity bin Max Bin The highest intensity bin Bins The total number of bins p Opens a dialog box that enables you to export the histogram csv Copies the histogram to the system clipboard E Opens the print dialog box Line Profile The line profile plots intensity y axis at each pixel x axis along a user specified line in the image It is particularly useful for inspecting the detailed character of the image data The line profile is automatically updated when you change the line position M NOTE In the Overlay display mode the line profile plots the luminescent data To obtain a histogram of the photograph select Photograph from the Display drop down list To display the line profile 1 Open an image and in the Image Information tools click the Line Profile button A line appears on the image and the Line Profile window appears Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Figure 4 20 View a line pro
33. 121 Copying or Exporting the ROI Measurements Table 122 2 Living Image 4 3 1 User s Manual IVIS Spectrum Contents Chapter 6 3D ROI Tools for Volumetric Data 0 000 eee es 124 ADOUR DRO maang a ann eet adeeaanesee Che DLWA Na AA BA Ka bA 124 Drawing a 3D ROI on ccuageranages Gb NGABA AA MA KAPA ND bas ANG ae 125 ROI Properties ada KAKA BA DI PAPAG AA NM eee ee ww ee GR GAAN 128 Managing the 3D ROI Measurements Table 2 00 eee ee ees 130 Configuring the 3D ROI Measurements Table 130 Copying or Exporting the ROI Measurements Table 132 Chapter 7 image Math irse oh ae erari BOO ee ew we Be A 133 Creating a New Image Using Image Math 0000 cece eee eee 133 Subtracting Tissue Autofluorescence cee ee ees 135 Chapter 8 Spectral Unmixing 0002 ee ee eee ee 138 About Spectral UNMIXINg 2 au aon oak aut See oa Ee ee A ee oe ee 138 Image RequirementS 5 60222 RAK ALL A MAA NBA MAA KAAWAAN MAAN wees 138 Spectral Unmixing Methods 0 00 ccc ee 139 Guided Method aa 86 4 dob od oR ee Re ee Oe ha Gore be ae 139 Liprary Ahihi aatunntdaseceedceudneerana kone bee we eons ee es 142 Automatic Method 145 COMGCUING SPECE caceniceue eae bow ee ene ee eoed seeder es oe ees 152 Spectral Unmixing ResultS xa ics maa Gn na Ah nee PAA AMP bovedswoaee ves 154 SCC PIO waaa PAKA AA bere oreo Geen bees boo eae ee ee oe Ge aces 154 Ana
34. A B if Counts B gt k Useful for fluorescence tomography A B k k Image Math window A user specified scaling factor applied in the results function Compute k from ROI This option is useful for subtracting fluorescence background Draw one ROI in an image on an area considered background In the Compute k from ROI drop down list select the this ROI with Photo from Choose this option to display the new image in overlay mode using the selected photographic image This option is only available if one of the selected images is an overlay Display Result for Opens the image generated by image math in an image window Measuring 7 2 Subtracting Tissue Autofluorescence To remove tissue autofluorescence from image data you can use a subtraction method that uses a second excitation filter which is blue shifted a background filter from the primary excitation filter 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 the signal corrected for background A B x k where A primary image acquired using the excitation filter B background image acquired using the background filter k primary signal background signal The background signal
35. A larger f stop number corresponds to a smaller aperture size and results in lower sensitivity because less light is collected for the image However a smaller aperture usually results in better image sharpness and depth of field A photographic image is taken with a small aperture f 8 or f 16 to produce the sharpest image and a luminescent image is taken with a large aperture f 1 to maximize sensitivity For more details on f stop see the reference article Detection Sensitivity select Help References on the menu bar Excitation Filter A drop down list of fluorescence excitation filters For fluorescent imaging choose the appropriate filter for your application For luminescent imaging Block is selected by default If you select Open no filter is present For systems equipped with spectral imaging capability choose the appropriate emission filter for your application Note On some models with standard filter sets the excitation filter selection automatically sets the emission filter Emission Filter A drop down list of fluorescence emission filters located in front of the CCD lens The emission filter wheel is equipped with filters for fluorescence or spectral imaging applications The number of filter positions 6 to 24 depends on the system For luminescent imaging the Open position no filter is automatically selected by default Living Image 4 3 1 User s Manual IVIS Spectrum Appendix A IVIS Ac
36. C7 TLI20050624145507 sEQ hoa Sequence View b 3D View Tool Palette _ ROI Tools gt Spectral Unmixing and DyCE gt Surface Topography gt 3D Multi Modality Tools 7 3D Optical Tools ty Lh NG BPG s Eg 40 H Help Coronal z 15 6 Sagittal x 3 5 Surface Source Registration Display Subject Surface 4 Apply Simulated Wavelengths 560 photons mm 3 Photon Density Intensity Color Table Rainbow V Reverse Transayial y 18 1 Log Scale p hotons sec Source Intensity Subject Height 26 2 mm Table 11 12 3D Registration tools Item Description at Use this tool to manually adjust the scale of location of organs For more details see 1 page 221 Fits the organs to the surface using a linear transformation that keeps the shape of the H atlas surface A Fits the organs to the surface using linear transformation and volume deformation E ma B a E a After fitting organs to the surface using the H Or pii tool if necessary you can click 3 this button to restore the default fit Display Organs Choose this option to display the organs on the surface Organs that are check marked will be displayed For more details see page 220 Drawing styles for the organs see Display Organs 2 bhh 219 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11
37. Chapter 2 Getting Started Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum continued continued Volume Process slice Results Hor on oO xs VZ Display Volume Level Of Detail U Performance Quality Color Opacity Map 10 opacity ME Ek E revese 0 00 Intensity 6 55e4 V Logarithmic Histogram F Maximum Intensity Projection MIP E Gradient Illumination Set color and opacity values for different intensity ranges of a CT volume so that the color opacity map shows the volume regions you are interested in opaque in the map and hides unimportant regions Co register 3D reconstructions of luminescent or fluorescent sources biological information with a CT volume to provide anatomical context for interpreting biological functional information Note The 3D Multi Modality tools require a separate license Living Image Tools and Functions See Page DyCE Dynamic Contrast Enhancement 159 Analyze Use DyCE to Select Images Type Time hh mm ss m Determine real time pharmacokinetic spatio I temporal biodistribution of a probe or dye signal Extract temporal spectra signal intensity as a function of time from particular anatomical J regions Note DyCE acquisition and analysis tools require a separate license Methods Sure Tapoprabiy iaz ue taala gila al LE Generate 3D
38. Delete ROI Delete All RU Properties ni Lock Position n ergun mm s info mg a ROI BNG 2 bd ROI Propertes o Label exc 3 Shape Orde Type Marua Background RO Subiect NOT Use as EKG for future ROIs in w 33120050630142125_001 orly frre sequerce Lock Posten Kel ox 181 30049 vega 95 17241 Ange eg 0 0000 lock Ste Width po 34 2857 Hegh px 34 25571 107 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data 5 7 ROI Histogram The ROI histogram plots a frequency distribution of pixel intensity The histogram sorts the pixels into groups or bins x axis coordinate and plots the number of pixels in each bin y axis coordinate To view the ROI histogram 1 Open an image that includes measurement ROIs 2 Click the histogram button liu in the Image Information tools 3 Select an ROI or All ROIs from the Plot drop down list of the histogram that appears Figure 5 16 Viewing the ROI histogram E7 11120050624145507_005 oe ss uns Radiance Photons Joey Optens 7 tnt ya 40 Ba 9 7 till E units Subject 1 Binning 8 Width 12 6cm Height 12 6 cm Image X Y 12 495 9 149 am BKG 1 3 495e 05 30 Image Data 1 5621e5 photons Sec on2sr Crop Distance 0 00 0 00 noe 00 0 00 Distance 0 00 2 0 H O W MeasureRos X Apply to Sequence o ye hie Save ROIs Radiance p sec cm js
39. Figure 9 18 The tool palette is available for viewing and analyzing the image a Click the 8 amp 8 button to view the unmixed images as a sequence Figure 9 18 The tool palette is available for viewing and analyzing the sequence The software prompts you to save the sequence when closing the Sequence View window 172 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 9 DyCE Imaging and Analysis Figure 9 18 View an unmixed image in an image window or view all unmixed images as a sequence 7 RKG20110210131022 SEQ P Sequence View Unmixing V Show Labels Individual Scale Normalized Legend a 2 Kidney 3 5 1 00 E A p j 0 30 r Unmixed a fe 0 40 Min 0 00 D 400 800 1200 Max 2 9064 Time Point s Bladder Composite Spectrum List Se x Note Select Name Colgr Pick 1 V Kidney lime A lin 2 V Brain W x Sr 3 V Bladder Bl blue f Min 0 00 Max 2 45e4 re Sm baha Min 0 00 Max 1 55e4 Tool Phere G Ampt Aywan Commies Anra leumg lobrotam ROI Fosh Viewing the Composite Image 1 Double click the composite thumbnail The Composite window opens Figure 9 19 Open the Composite window Select the unmixed images to include in the composite image A RKG20110210131022_SEQ i Sequence View Unmixing V Show Labels V Individual Scale Normalized Legend 5 FE 3 o gi Lol 1 00 amp 0 80 0 6
40. File Browse 3D Volumetric Data Displays the Browse For Folder box so that you can select a volumetric data folder for example DICOM format TIF data The selected folder is displayed in the 3D Browser File Save Saves overwrites the AnalyzedClickInfo text file to update the analysis parameters but the original image data files are not altered File Save As Displays the Browse For Folder box so that you can specify a folder in which to save the image data The original data is not overwritten File Import 3D Surface Opens a dialog box that enables you to import a surface Note This menu item is only available if Show Advanced Options is selected in the Preferences see page 267 File Import 3D Voxels Opens a dialog box that enables you to import a source volume Note This menu item is only available if Show Advanced Options is selected in the Preferences see page 267 File Import Atlas Opens a dialog box that enables you to import an organ atlas iv dxf stl File Export Image Sequence as DICOM Opens the Browse for Folder dialog box that enables you to export the active image data to DICOM format dcm File Export 3D Surface Opens a dialog box that enables you to save the 3D surface of the active data to a file such as Open Inventor format iv File Export 3D Voxels Opens a dialog box that enab
41. IVIS Spectrum Chapter 3 Image Acquisition 42 3 4 Acquire a Sequence Using the Imaging Wizard The Imaging Wizard Figure 3 22 provides a convenient way to set up a sequence for some imaging applications see Table 3 6 and Table 3 7 on page 43 The acquisition parameters for each image in a sequence must be specified The wizard guides you through a series of steps prompting you for the information that the software needs to set up the sequence This section explains how to use the Imaging Wizard and acquire a sequence of luminescent or fluorescent images A sequence can also be set up manually see page 50 for details TIP See the Imaging Wizard tech note for a quick guide select Help Tech Notes on the menu bar Figure 3 22 Imaging Wizard Calc bha sober fer magra bigkermnianient Hi Chairman Creer pah Hi fre her cik baril leies nia o bacteri lucifer Fusrescence magng a Ht Set Up a Sequence M NOTE The IVIS Spectrum should be initialized and the temperature locked before setting up the imaging parameters See page 7 for more details 1 Click Imaging Wizard in the control Panel Figure 3 23 2 If necessary click Restart in the Imaging Wizard to show the first page of the wizard 3 Double click Bioluminescence 4 or Fluorescence imaging four Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition Figure 3 23 Opening the Imaging
42. KA Y Select Analysis Result DLIT_1 Load Unit DLIT Select the UNIts o photons sec of the results cells See Table 11 7 V Display Voxels DLIT for more Color Scale details Min 8 98e 5 3 Max 4 18e6 Rainbow Reverse m Log Scale MIP IV Display Surface Opacity fa 15 5 photo ns sec Display Photon Density Map DLIT Rass For odre details on these Click a surface to select it Voxel color scale display controls see Table 11 7 Living Image 4 3 1 User s Manual IVIS Spectrum Table 11 7 Longitudinal Study window Chapter 11 3D Reconstruction of Sources Display Voxels DLIT Item Description Unit DLIT DLIT Select photons sec or cells results calibrated using a o cea ar quantification database CJ cls FLIT Select pmole M cm or pmoles results calibrated using a Hai pan quantification database pmol M cm 1 pmol cells Voxel display controls Display Voxels Choose this option to show voxels within the surface From the drop down list select a color scheme for the color scale Move the sliders to adjust the color scale minimum and maximum values Reverse Choose this option to apply the colors of the selected color table in reverse order to the photon density scale For example the Red color table represents the source intensity photons sec from low to high using a color scale from transparent to red If Rev
43. Opsons sma Ea Maa E 12 2 Creating a Quantification Database 1 Load the well plate image sequence 2 Select Tools gt Well Plate Quantification for lt name gt SEQ on the menu bar The Well Plate Quantification window appears 3 For fluorescent samples choose the Dye molecules or Cells option Figure 12 2 Well Plate Quantification window Fluorophore Type Dye molecules C Cells gt These options only available for fluorescence data Set For Image EL20090414101005 001 Select the well plate dimensions from the Well Plate Type drop down list The first image in the sequence opens and a grid ROI appears on the image 232 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 12 Quantification Database 233 Figure 12 3 Well plate image with grid ROI rc 7 L20090414101005 001 o amp 23 Units Efficiency v Display Overlay Options info iy o x0 5 Adjust the grid ROI to closely fit the plate wells 6 In the well plate table select the table cells for the samples and click Set Figure 12 4 Clicking a row or column header selects the entire row or column 7 To remove the sample designations from table cells select the table cells and click the button 8 To apply a color to table cells a Select the table cells and click the j button Alternatively ri
44. Restore Threshold Data Adjustment E Select All Cancel Reconstruct The red outline indicates the image selected for data adjustment i Set the Threshold here Note Min Counts 2 aa Threshold Tools Region Selection Tools Adjustment U Draw Erase Reset Min counts 46 Painting size Segment fl Red v Opacity p Cone Ok To select particular regions for reconstruction Open the Data Preview window as shown in Figure 11 7 2 Click Data Adjustment 3 In the window that appears choose the Draw option and put the mouse pointer over the image so that the pencil tool IS appears 4 To automatically select all pixels in a source right click with the region with the pencil tool Alternatively put the pencil over the image and click the mouse key or press and hold the mouse key while moving the pencil over an area of the image M NOTE If the pencil tool markings are applied to the image only the marked pixels are included in the analysis 192 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 193 Figure 11 8 Selecting regions to include in reconstruction TL DataAdjustment Threshold Tools Region Selection Tools Adjustment b 5 5 Draw Erase Min counts 46 Painting size Segment Opacity F Ka Use these tools to select particular image data to include Choose the Draw option t
45. The surface and 3D tools appear in the Tool Palette For more details on the Tool Palette see page 220 Figure 10 4 3D view and 3D tools in the toolbar and Tool Palette 3D View toolbar Fi TLT20050624145507_SEQ Sequence View b 3D View FA Gronal z 13 0 Sagittal x 2 0 1 Transaxial y 13 0 Subject Height 26 2 mm Tool Palette z gt ROI Tools lal L gt Spectral Unmixing and DyCE lel Surface Topography a Optical Surface Reconstruction Orientation Dorsal Subject Nude Mouse v Generate Surface Surface Smoothing Level Low z o Save Results Name SURFACE_4 L gt 3D Multi Modality Tools gt 3D Optical Tools Lo DLIT 3D Reconstruction 179 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 10 Reconstructing a 3D Surface Figure 10 5 3D View toolbar Table 10 1 3D view tools Tool Description Image Tools A drop down list of tools for viewing and working with the surface Select to a Click and display measurement dimensions in the coronal sagittal or transaxial view in the 3D view window a Drag a measurement cursor in the coronal sagittal or transaxial view and display measurement dimensions See page 53 for details on measurement cursors Select pg to zoom in or out on the image use a click and drag operation Select to mov
46. co saa Configure Export The ROI Measurements table displays data for all ROIs created in images or sequences during a session one ROI per row The table provides a convenient way to review and export ROI data For more details on the table see ROI Measurements table page 119 7 Click Yes in the save prompt when closing a data set to save the ROIs with the data 5 3 ROI Tools for Optical Images This section provides an overview of the ROI tools for optical images Table 5 2 The ROI tools that appear in the Tool Palette depend on the type of ROI selected from the ROI Type drop down list and whether an image or sequence is active Some ROI parameters are only available if Show Advanced Options is selected in the General Preferences Figure 5 6 Figure 5 6 ROI tools for optical images Tool Palette 83 Type Save ROIs Name ROI 3 KSA bd Delete Load Save Auto ROI Parameters Threshold 50 Lower Limit These Auto ROI parameters are available if Show Advanced Options is selected in the view Use Bkg Offset General Preferences For more details on setting Restore Defaults Save Load Preferences see Appendix C on page 168 Minimum Size Table 5 2 ROI tools for optical images Item Description Click to select the number of circle ROIs to add to the active image mr 95 Living Image 4 3 1 User s Manual
47. gt Corrections Filtering 3 Options 7 zno 5 R fg a CT maana ia Image Informa 2 ROI Tools aang Nag PALA SNAP PET eae jz Hay a TE 11720050624145507 006 ec t aa Units Counts Display Options 2 Info 8g a Image TLT20050624145507_006 Series Male Nn nu Fri Jun 24 2005 07 57 18 Experiment DOB 03 21 05 Em Filter Open Bin M 8 FOV 12 6 f4 1s Label kidney Living Image Version 2 50 1 5 20 2005 R Camera IVIS 200 Beta II SI620EEV Comment dorsal Luminescence 15000 10000 5000 Counts Color Scale Min 1122 Max 19546 Table 4 3 Image window Item Description Units Select the measurement units for the image display from this drop down list The available units depend on the type of image data See the concept tech note Image Display and Measurement for more details on measurement units select Help Tech Notes on the menu bar Use Saved Choose this option to display the image data using the color table that was specified in the Colors image Preferences at the time of acquisition If this option is not selected image data are sequence displayed using the color table currently specified in the Preferences 61 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 62 Table 4 3 Image window continued Item Description Options Layout Choose a display option for the im
48. iv dxf stl X Open Cancel in the atlas one file per organ and click Open the file name Click Generate Mesh Coefficients Enter a name for the atlas and click Save Organ Atlas In the next dialog box that appears select all of the files iv dxf stl that you want to include In the Select Skin Mesh drop down list select the skin organ file which must include skin in The organ atlas atlas 1s created and is added to the Organ Atlas drop down list in the 3D tools Registration tab 224 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 225 11 14 3D Animation The Living Image software can create an animation from a sequence of 3D views key frames For example an animation can depict a rotating 3D scene Figure 11 38 The animation series of key frames can be recorded to a movie file mov mp4 or avi Use the animation tools to m View a preset animation generated from a factory loaded animation setup page 227 m Create a custom animation created from your custom animation setup page 230 m Save an animation setup page 229 m Record an animation to a movie file page 229 Edit an animation setup page 229 Figure 11 38 Individual 3D views key frames in the preset animation Spin CW on Y Axis Pa 3DAnimation pia Preset Animations Presets Spin CW on Y Axis z Frame Factor 1 Ani
49. or grid shape on the image 93 Free draw Draw line segments that define the ROI 101 97 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data Drawing Measurement ROIs Automatically The Living Image software can automatically identify all of the ROIs in an image or image sequence that meet the auto ROI parameter thresholds or draw one ROI at a user specified location To automatically identify and draw all ROls 1 Open an image or image sequence and in the ROI tools select Measurement ROI from the Type drop down list 2 Click an ROI shape button Circle OJ Square 5 or Contour and select Auto All from the drop down list The ROIs appear on the image or sequence thumbnails The ROI label includes the ROI intensity threshold Threshold and intensity measurement NOTE Auto ROls are created and numbered in order from highest to lowest maximum signal within the ROI ROI 1 contains the highest maximum signal You may want to arrange the ROls in a known order for easier comparison between images To renumber the ROIs ascending order from right to left right click the image and select Sort ROIs on the shortcut menu If the Apply to Sequence option is selected in the ROI tools choose Sort ROIs in Sequence to sort all of the ROIs in the sequence The sort options are only available if the ROIs have not been sorted eli Figure 5 7 Automatically drawing measurement ROls det
50. reconstruct 3D fluorescent sources 194 197 reconstruct 3D luminescent sources 187 193 reconstruct particular pixels 192 Living Image 4 3 1 User s Manual IVIS Spectrum reduced Chi2 198 registering multi modal data fiducial registration 250 loading data 252 254 manual registration 255 258 ROI 90 124 automatically draw 98 99 background corrected signal 105 107 delete 117 edit dimensions 113 free draw 101 histogram 108 managing 109 manually draw 100 101 measurement ROI free draw 101 measurement ROIs 97 99 Measurements table 95 mirror 102 104 move 112 move or edit label 116 quick guide 93 ROI line 115 save 116 subject 105 tools 95 96 ROI Measurements table 119 121 configure 121 122 130 132 copy or export 122 132 ROI properties 109 112 ROI types average background 105 S save ROI 116 segment 51 sequence edit 52 53 sequence requirements DLIT 187 FLIT 194 spectral unmixing 138 slices rendering 245 246 viewing 246 248 smoothing 71 source depth 203 spectral unmixing 138 158 sequence requirements 138 subject ROI 105 surface export or import 183 generate 178 179 manage 182 Index 281 T tag an image 67 Tech Notes 3 4 technical support 4 tile images 60 tissue autofluorescence subtracting with background filters 135 137 tool palette 63 3D tools 213 230 overview 12 18 ROI tools 95 transillumination overview 80 troubleshooting DLIT FLIT 230 U user preferences 267 274 V volume slices inf
51. the Edit Image Labels dialog box see page 25 and other information automatically recorded by the software Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Table 4 3 Image window continued Item Description Opens all of the images in a sequence Ps Closes all open images of a sequence kr Opens the Edit Sequence dialog box that enables you to add or remove images from the Mj sequence display Opens a dialog box that enables you to export the active view as a graphic file Takes a snapshot that is displayed with the data in the Living Image Browser See page 55 for more details on the browser IC Living Image Browser TLT20050624145507 SEQ Click Number EX Filter n EM Filter Illumination Mode User ID gl Experiment Commenti 3EQ CK20100628141050_SEQ i CK CF750 dye in pillows 0 25 pmol uL 2 uL bead 12 BEB CK20050420175030_SEQ CK 3EQ TLT20060510114512_SEQ TLT E TLT20050624145507_SEQ i TLT 4 IN p Hide Browse View Close Preview Label Set All v le Add to List Browse View Default v Configure Load as Group Load Remove Close Location C Share Caliper LS Caliper Data Sample Data IVIS200 data TraserBeadsPC TLT20050624145507_SEQ Sequenceinfo txt Snapshots of an image sequence Tool Palette The Tool Palette appears when you open an ima
52. 0 0000 Rotate Resize ROI 8 BKG lt Ba Copy ROI Copy All ROIs D a Paste ROI Duplicate ROI Lock Size Width cm 0 31500 3 Height em 0 31500 Radiance p secfcm3jsr Color 5cale Min 2 35e7 Max 4 16e8 Set Bkg ROI to none Set Bkg ROI to BKG1 Line Size 2 a Set Subject ROI to none Set Subject ROI to Subject 1 Hide ROI Tag Delete ROI Delete All ROIs Properties Unlock Position Unlock Size Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data see Table 5 6 page 111 Units Radiance Photons v Display Overlay v BKG 1 1 278e 05 ROI 1 BKG 1 25 4 825e 09 ROI 2 BKG 1 25 2 394e 09 Figure 5 18 ROI Properties Background ROI tab LO a Options x Info NG igi Luminescence ay Radiance p sec cm3 sr Color Scale Min 1 46e8 Max 2 54e9 ej TLT20050624145507 006 Units Radiance Photons Display BKG 1 1 278e 05 BUT DR BUTT T Dl ROI 1 BKG 1 25 4 825e 09 ROI 2 BKG 1 25 2 394e 09 o S 8 Options sinfo NG iz Luminescence 2 5 Radiance pisecicm2 sr Color Scale Min 1 46e8 Max 2 54e9 The items in the ROI Properties box depend on the type of ROI selected in the image For more details A ROI Properties o
53. 1 User s Manual IVIS Spectrum Chapter 2 Getting Started Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum continued continued E Use Saved t Options x Info 5 3 ba n Mina 482 N Max 9267 Images of Quantum dot nanocrystals 700 or 800 nm were acquired using different combinations of excitation and emission filters NG aanak Eg D bara Yer ipaa Doras Naa Dae Nar tanga Log Baa Aral Como By L amp Coomap WA Coor tanga Colorize view of the combined images Living Image Tools and Functions See Page IT image Overlay Window cee Image Overlay Window 55 Sequence ARW20050826124002_SEQ Units Radiant Efficiency Cc kg ka ngn Bi Select Tools Image Overlay for lt sequence Cee name gt on the menu ba l v ARW20050826124002 002 View multiple fluorescent or luminescent signals in one 2 dimensional image in the Image Overlay window W w Image Adjust me i N da saa Palette Label Max b 4749 Green Scales per Column 3 Opacity F 70 Reverse V Logarithmic TE toaoo7o420122818 sEQ cae Colorize View 84 CJ Sequence View a Colorize View H wi ter sess Gobrien in Select Tools Colorize for lt sequence name gt Units Counts on the menu bar The colorize tool renders each luminescence or fluorescence image of a sequence in color and combines them into a
54. 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data 106 Measuring Background corrected Signal 1 Draw one or more measurement ROIs on the subject see page 97 for more details 2 Draw an average background ROI on the subject a Select Average Bkg ROI from the Type drop down list b Click the Square H or Circle 7 button and select 1 The ROI is added to the image For more details on adjusting the ROI position or dimensions see page 112 and page 113 M NOTE The average background ROI and measurement ROI do not need to be the same shape or size because the software computes the average intensity signal in each ROI NOTE If the image was acquired using the Side Imager draw a background ROI on each view Figure 5 15 lt Figure 5 15 Draw a background ROI on each view in an image acquired using the Side Imager Permen z 7 IV20120412111353_004 Clo aj Units Radiance Photons v Display Overlay ba Options Y Info NG sj Luminescence ka 4 0 E ROI 2 50 1 931e 06 5 Mirror 1 3 710e 06 ha me Pg eg Mirror 2 2 447e 06 Lo 3 0 RO 1 50 8 593e 05 Pi NG 2 0 thit BKG 2 1 430e 05 Ng il BKG 3 1 293e 05 BKG 1 1 306e 05 i H a Radiance pfseci cm2 sr Color Scale Min 1 29e5 Max 6 13e5 3 Associate each background ROI with a measurement ROI s or mirror ROI s using one of the methods in Table 5 5 Living Image
55. 1s reasonably smooth over the mouse surface Therefore it is recommended that you comb the fur before imaging to eliminate any fluffy areas that may alter the light emission pattern and or trigger artifacts during the surface topography reconstruction In this case it is recommended that you shave the animals or apply a depilatory 3D reconstructions are currently not possible on black or dark colored furred mice Luminescent Exposure vs Luciferin Kinetic Profile It is important to consider the luciferin kinetic profile when you plan the image sequence acquisition The DLIT algorithm currently assumes a stable 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 DLIT Image Sequence Requirements Use the Imaging Wizard to set up the image sequence required for DLIT analysis See page 45 for more details on the Imaging Wizard If you plan to manually set up the sequence the sequence must include m A structured light image m Optical data from at least two different emission filters 560 660 nm at a minimum Emission filter 1 Photographic luminescent a Emission filter 2 Luminescent image Analyzing more optical images usually produces more accurate results Table 11 2 shows the recommended o
56. 253 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools Figure 13 16 3D optical and 3D volumetric data loaded but not registered C k20101119144617 sq Tool Palette Sequence View Des Ain Tools Planar Spectral Imaging 4 o Spectral Unmbong and Pyt Surface Topography 7 3D Hulti Hodality Tools volume process nice Results Hon ki Gan o 1 F Deplay volume Level OF Detal Performance uatr Color Como ty Map 3 a Beverse E 0 00 feces Pipher Hamun Miei Proecten MIP Grackerit Guerre 30 Optical Tooke _ DLT JU Recon tinction Histogram of voxel intensities Registering Multi Modal Data Automatic Fiducial Registration About the Mouse Imaging Shuttle The Mouse Imaging Shuttle Caliper part no 127744 contains the subject during imaging and enables the subject to be transferred between an IVIS Imaging System and the Quantum FX uCT instrument without disrupting the subject s position The Mouse Imaging Shuttle must be correctly docked to the docking station in the IVIS Imaging System and the Quantum FX uCT instrument The docking station in the Quantum FX uCT system 1s marked with a triangle shaped fiducial pattern under the plane where the Mouse Imaging Shuttle docks Automatic fiducial registration is available if both sides of the triangle fiducial pattern are included in the CT images For more details on
57. 3D Reconstruction of Sources Table 11 12 3D Registration tools continued Item Description Shading styles for the organs see Display Organs E Gi 4 Opacity Adjusts the opacity of the organ display Organ Atlas Choose a type of organ atlas Click to select all organs in the database and display them on the surface Click to clear the selected organs and remove all organ diagrams from the surface Displaying Organs With the Reconstruction 1 lt y Load reconstruction results and confirm that the surface is in the perspective view click the toolbar button in the 3D View window or press the R key In the 3D registration tools choose the Display Organs option and select an organ atlas The organs in the selected atlas appear on the surface To fit the organs to the surface click a registration tool H Rigid registration Performs linear transformation but keeps the shape of the atlas surface H Full registration Performs linear transformation and volume deformation F NOTE For an optimum fit when there is a large difference between the orientation or size of the atlas organs and surface first use the transformation tool to manually register the surface and atlas organs then click a registration tool to automatically fit the organs See Manually Adjusting the Scale or Location of Organs page 221 for more details If necessary adjust the opacity of the organs us
58. 4 16 See the tech note Detection Sensitivity for more details on binning select Help gt Tech Notes on the menu bar Smoothing Computes the average signal of the specified number of pixels and replaces the Original signal with the average signal Figure 4 16 Smoothing removes signal noise without changing pixel size Click this button to return the binning or smoothing to the previous setting and update the image 71 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 72 Figure 4 16 Example of binning and smoothing image data Binning at acquisition 8 no smoothing Binning 2 smoothing 5x5 4 7 Viewing Intensity Data and Making Measurements The Image Information tools enable you to view intensity data and measure distance on an image Pixel data can be viewed in different formats Image Information Description See Page x y coordinates and The x y pixel coordinates of the mouse pointer location in the image 74 associated intensity and the intensity counts or photons at that location Histogram Histogram of pixel intensities in an image 74 Line profile Plots a line graph of intensity data at each pixel along a user specified 75 horizontal or vertical line in the image Figure 4 17 Tool Palette Image Information tools Tool Palette gt Image Adjust Image Information lan ci A units cm Image Binnin
59. 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data Table 5 5 Methods for associating measurement or mirror ROIs with a background ROI Method Description Draw a subject ROI See page 105 for details oli as Eamon SST Cl Linets Reia Phom oF Disney Oweley T pioa 7 info o wj Luminescence 24 i 15 s1 la OS Radiance tpat ir Optone Y info mg a liwwenmence E wmennnsss om Inte Redange Photons Deplsy Overlay X Patama pl secfon ir Right click a measurement ROI and select an average background ROI from the shortcut menu EF JOST ON sila Unit Radar Photons or apay Oei 7 Onion nio Ca wj birma h AUN Copy ROI Copy Al Rots Duplecte ROI 100 2a fka ROI la None Ca Bka ROl t0 EG 1 Set Blg KOI lo BG 7 bal Sula RIH bo nana NGO Sat Subject RID bo Subject I Has ROH Ting Dene RA l Counts eee SN HO Dakr Seale Properbes i Unlock Pagan Mara a NE 1 Right click a background ROI and select Properties on the shortcut menu 2 Inthe ROI Properties box that appears click the Background ROI tab and puta check mark next to Use as BKG for future ROls in 3 Choose the image name or the Entire sequence option CG ua lt a E Nt20080620142125 001 Units Radance Photons v Deplsy Overlay X MG 15 ber Optons Retate Copy ROI Copy All ROls Dupdicate ROL Hide ROI Tag
60. 7 Photon Density Maps Export Results Save Results Name DLIT_1 2 To copy user selected results a Select the results b Right click the selection and choose Copy from the shortcut menu 11 5 Checking the Reconstruction Quality Comparing the measured and simulated photon density plots 1s a useful way to check the quality of a 3D reconstruction The photon density is closely related to the measured radiance Photon density is the steady state measure of the number of photons in a cubic millimeter Light sources inside the tissue contribute to photon density in other portions of the tissue The reconstruction algorithm first converts the luminescent or fluorescent image of surface radiance to photon density just inside the animal surface because this 1s what can be observed The algorithm then solves for intensity values at locations inside the tissue which would produce the observed photon density near the surface 199 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 200 For fluorescence reconstructions using NTF Efficiency data the photon density of the fluorescence image is divided by the photon density of the transmission image giving the NTF Efficiency The NTF Efficiency values are the data just inside the animal surface for this type of data set Viewing Photon Density or NTF Efficiency Maps 1 After the reconstruction is finished or results
61. 8 599e 03 Min Count Max Count 1 723e 02 4 730e 02 2 209e 04 3 042e 03 2576e 04 3 301e 02 9 393e 02 4 280e 04 6 048e 03 The ROI Measurements table displays data for all ROIs created in images or sequences during a session one ROI per row The table provides a convenient way to review and export ROI data For more details on the table see Managing the ROI Measurements Table page 119 To automatically draw an ROI at a user specified location 1 Open an image 2 Click an ROI shape button Circle CJ Square Gj or Contour and select Auto 1 from the drop down list The create tool appears on the image Figure 5 9 ROI create tool G a AN NAN linda Ca AGA Minh TE TLI20050624145507 006 Units Radiance Photons v Display Overlay v Done A baeo Options z Info NG KO Luminescence 25 Radiance pisec cm2 sr Color Scale Min 1 46e8 Max 2 54e9 3 Use the ring to move the create tool to the area where you want to draw the ROI then click Create The ROI appears on the image and the ROI label displays the intensity signal 4 To draw another ROI on the image repeat step 2 to step 3 For information on how to save ROIs see page 116 99 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data 100 Drawing Measurement ROIs Manually 1 Open an image or image sequence and in the RO
62. Confirm Password 4 Enter and confirm a master password Click Close The master password will be required to delete users To unlock user accounts 1 In the Security tab click Unlock User Accounts 2 Enter the master password and click Unlock Click Close Figure 2 12 User Settings Security p A User Settings adduser 2 change Password 24 Delete User Currently User Accounts are Locked Change Master Password Old Password New Password Confirm Password Security fae Unlock User Accounts ge E User Settings 2 Add User 2 change Password amp Delete User Security Currently User Accounts are Locked Enter Master Password Enter the Master Password to Unlock the User Account settings Master Password 21 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 2 Getting Started 22 2 6 Tracking System and User Activity Activity Window The Activity window shows the imaging system activities Figure 2 13 The software creates and saves a log of the system activities related to data acquisition This information may be useful for Caliper field service engineers to understand the imaging system behavior over time or for troubleshooting The activity log is located at C Program Files Caliper Life Sciences Living Image The software tracks user time on the system hr
63. Datasets Ab miners gt je ANION anbes6 SEC Mane Sei Camension j SEF Moused 3 Day 28 145522 C Shaew Calper LS Caliper Oata Cofteg ero S12 756x256 ICT maeno ox coe a sai j k3 z j 512 Pie vaid E Beene Dala wil ba kush i a rem tirdi 3D Volumetric Browser M NOTE The next time you start the Living Image software and open the Browse For Folder box the software automatically returns to the last folder visited The 3D Volumetric Browser automatically previews a playback of the data along with other information about the data Figure 13 15 BEM DICOM file tzF TIFF file 3 Load the volumetric data with the optical data a Confirm that the Load in a new window option is not selected If this option is selected the volumetric data are loaded in a new window b Double click the data row in browser Alternatively select the data row and click Load The 3D volumetric data appears in the 3D View window of the optical data Figure 13 16 The software converts loaded volumetric data into an 8 bit representation to reduce memory overhead and for easier color mapping The 3D Multi Modality tools provide an 8 bit color opacity map for volume visualization which maps each voxel to an RGB color or a color and opacity value A histogram of voxel intensities appears in the Multi Modality tools and the software sets a default air noise boundary Living Image
64. E ROI BKG 1 M Label BKG 1 Shape Cirde Type Manual Background ROI SubjectROI la 71720050624145507_006 only Use as BKG for future ROIs in _ Entire sequence E Lock Position Xc cm 6 22835 Ye cm 4 41437 Angle deg 0 0000 E Lock Size Width cm 0 96816 2 Height cm 0 94369 Line Size 2 MG KS Ta a T rotproperies ROI ROI 1 v Label ROI 1 Shape Type Background ROI SubjectROI Image Number TLT20050624145507 006 Label Contour Auto 25 V Lock Position Angle deg 0 0000 Width cm 0 57750 Height cm 0 57750 2 Line Color Saad saa Line Size ROI selected in the image Label of the ROI selected in the image Double click to edit Selected image 110 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data Figure 5 19 ROI properties Subject tab The items in the ROI Properties box depend on the type of ROI selected in the image For more details see Table 5 6 page 111 Square Manual E Lock Position Xe pix 122 2144 Yc pix 127 40694 O Lock Size Line Size Done Subj ROI tab Subject 1 Width pix 78 65097 Height pix 197 84919 Line Color EY p 5 Drop down list of subject ROIs in the image ROI label name Edit the label here Enter information about the selected ROI optional Table
65. ID User entered information about a subject ROI Label Label name of the selected subject ROI Lock Position Choose this option to lock the position of the ROI selected in the image Xc x axis coordinate at the center of the ROI selected in the image Yc y axis coordinate at the center of the ROI selected in the image Lock Size Choose this option to lock the dimensions of the ROI selected in the image Width Width pixels or cm of the ROI selected in the image for more details on setting the units see ROI Dimensions page 120 Height Height pixels or cm of the ROI selected in the image Line Size Specifies the ROI line thickness To change the line thickness enter a new value or click ak the up down arrows Line Color Specifies the color of the ROI line To select a line color click the Browse button Ri Done Click to close the ROI Properties box and apply any new settings including m Linkage between a measurement ROI and subject ROI for more details see Drawing ROIs Using the Free Draw Method page 101 ROI size dimensions or position s Subject ROI ID information Moving an ROI To move an ROI on an image select it and do one of the following m Press a keyboard arrow key a Drag the ROI a Edit the settings in the ROI Properties box NOTE An ROI cannot be moved if it was created using the auto ROI tool or if the ROI position is locked To drag an ROI 1 Put the mouse pointer over the RO
66. Image Binning Photographic Auto Photographic Luminescent Luminescent 1 00 sec Il Fluorescent 1 00 CO Manual z S BE Auto Save Folder cao Restore Defaults Apply Table B 4 Camera settings Item Description Default Image Exposure Sets the default exposure settings that appear in the IVIS acquisition control panel Default Image Binning Standard Binning choices include Small Medium and Large These are predetermined factory loaded binning values that depend on the imaging system camera Manual Allows the user to choose a binning value 1 2 4 8 or 16 Auto Save Specifies the folder where images are automatically saved Click the button to select a folder Restore Defaults Click to apply the default settings B 4 Theme Figure B 6 Image view preferences FS E Preferences exa General Options Acquisition Theme Optical Properties Image View 3D View Color Palette Luminescent Rainbow v V Reverse Fluorescent YellowHot v Reverse Use saved color palette while loading datasets Restore Defaults Background amp Text Color ROI Color Background Color Luminescent a Text Color a Fluorescent m Text Size 8 Restore Defaults Restore Defaults Cancel Apply Living Image 4 3 1 User s Manual IVIS Sp
67. Location Charr Caliper LG Caliper Dala Senne Cente fiii 200 data treed TL a0 S06 4145507 E0 Gener tro tet wj Creates a preview picture snapshot of the image or thumbnails that the Living Image Browser displays when the data are selected For more details on the browser see page 55 Figure 3 29 Control panel IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning Excitation Filter Emission Filter Field of View System Status O mis Imaging Wizard 1 50 Er Sequence Setup use subject height v Temperature IAN Locked j IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter Display Photographic Settings Subject None v ag pao Shee W nedm sl Ni Field of View System Status Acquire Sequence O MIS Idle Imaging Wizard Subject height 1 50 Image Setup Focus use subject height w C Number of Segments p Delay o o min Apply to All Ad Update Insert Add Batch Sequences option 3 To set up the first sequence do either of the following m Click Imaging Wizard and step through the wizard see page 42 for details OR 48 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition m Set up the sequence manually see page 50 for details 4 To set up the next sequ
68. Measurements table click Configure The Configure Measurements box appears 121 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data 122 Figure 5 30 Configure Measurements dialog box E Configure Measurements User Lists Name Counts Update Delete A Sort Available Items Selected Items Analysis Comment Kami Analysis User ID a customize Avg Counts Move Up Angle Stdev Counts Animal Model Add gt Min Counts Move Down Max Counts Animal Number Animal Strain Area ccd Pixels Area cm7 Avg Dark Charge Counts Avg Efficiency Avg Fluorescent Bkg Counts Avg Radiance p s cm sr Avg Radiant Efficiency p s crm Binning Cell Line 4 FI Remove lt Column headers in the active ROI table 2 Select a configuration from the User Lists drop down list and click Customize 3 To add column header to the ROI table make a selection from the Available Item list and click Add 4 To remove column header from the ROI table select the item that you want to remove in the Selected Items list and click Remove To reorder an item in the Selected Items list select the item and click Move Up or Move Down The columns in the ROI Measurements table are updated 6 Enter a name for the custom configuration in the Name box and click Save To delete a custom table configuration Select the configuration from the User Lists drop down list and
69. O Solid Color Gradient Colors Color Theme Midnight r Top o P Bottom 4 is Restore Defaults Color Palette Source Vowels Bluevellow BAT E Reverse Restore Defaults Optical Properties SuFace amp Text Color Surface Color i Text Color Mm Text Size Restore Defaults m 272 Living Image 4 3 1 User s Manual IVIS Spectrum Appendix B Preferences Table B 6 3D view preferences Item Description Color Theme Predefined color schemes available for the 3D View window shown here Click the button to restore the defaults for the selected color theme E TLT20050624145507_5E0Q f o CI Sequence View De gt po P GP amp Ng Background Color Settings that modify the appearance of the background in the 3D View window Solid Color Choose this option to apply a non gradient background color to the 3D view in the image window Gradient Color Choose this option to apply a gradient background color to the 3D view in the image window Top the color at the top of the window Bottom the color at the bottom of the window Surface amp Text Color Settings that modify the display of the surface and text in the 3D View window Color Palette Source voxels Choose a color table for voxel display Reverse Choose this option to reverse the min max colors of the selected color table Restore Defaults Clic
70. Pin Count Maa Coum TL re PEAS IP a FU Dreslay KB ALAA KUA WA MA TL OSes RU Derly M H A MWA H A Hia eee Pali KO 22 Jed ea Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data 93 5 2 Quick Guide Drawing Measurement ROIs on an Optical Image or Sequence These steps provide a quick guide on how to apply a measurement ROI on an optical image or image sequence See page 97 for details on measurement ROIs 1 Open an image or sequence and click ROI Tools in the Tool Palette 2 Select Measurement ROI from the Type drop down list 3 Click the button and select Auto All on the drop down list igure 5 2 Select a type of K alta Da ta tat te ra ta ta tag ta ta ta va ta ta rag Ya ta ra va ta ta va a ta ra Ya ta Da va Ya ta ra Va ta ta Ya ta Ta ra Ya ta ra Ta tal ra TI HE n R Saville AA ai Delete Auto ROI Parameters Threshold o 4 Click the Contour button and select Auto All from the drop down list The software automatically draws measurement ROIs on all images The ROI label shows the total intensity in the ROI and the Threshold Figure 5 3 M NOTE Auto ROIs are created and numbered in order from highest to lowest maximum signal within the ROI ROI 1 contains the highest maximum signal You may want to arrange the ROIs in a known order for easier comparison between images To renumber the ROIs ascending order fr
71. Tissue AF Z o O o 1 00 S 0 50 Min 0 00 00 Max 9 91e7 660 700 740 780 AF750 Emission Wavelength nm Spectrum List U bper Xx Note Select Name Color Pick 1 V TissueAF lime yL 2 V AF630 Brea z f 3 V AF750 We W A Min 0 00 ax 2 3768 Composite Value No SelectedImages 8 SpectrumType Emission Method Guided AnalysisBinning 8 ImageWidth 240 ImageHeight 240 DataUnit Radiant Efficiency SPUM Ver 2 0 4 3 1 0 15582 Livinglmage Save Results Name SPUM 4 v Delete Load Save Spectra Plot The spectra plots shows the unmixed spectra Figure 8 22 Spectra window Normalized El Legend r 1 00 0 50 0 0 660 700 740 TAN Emission Wavelength nm Spectrum List Note Select Name Color Pick 1 Tissue AF 2 AF680 3 E AF750 154 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing Table 8 3 Spectra window Item Description Normalized Choose this option to display signals normalized on a scale from zero to one Legend Choose this option to display a key for the spectra plot Opens a dialog box that enables you to export the spectra plot data to a csv file Opens a dialog box that enables you to select and load a spectrum library AC No mi Opens a dialog box that enables you so save spectral unmixing results as a reference spectrum library for
72. User ID ma a Ci Click No CK20090729114835 SEQ AG o a aaa s MU Dataset Type Sequence Dataset E No of clicks 16 Aa Aa Location Ci Share Caliper LS Caliper Data Sample Data FLIT CK20090729114835 SEQ Sequencelnfo txt HEQ TLT20050624145507 SEQ 4 1 J Close Preview Label Set Al v Add to List Browse View Default Configure Load as Group Load Remove Close Location C Share Caliper LS Caliper Data Sample Data FLIT CK20090729114835 SEQ SequenceInfo txt a Ss To preview data click a row Note A preview snapshot is automatically taken at the time of image or sequence acquisition A snapshot can also be captured manually see page 26 for more details 3 To load data do one of the following a Double click the data row m Right click the data name and select Load on the shortcut menu m Select the data row and click Load Double click the thumbnail The image s and Tool Palette are displayed Green rows in the browser indicate loaded data Figure 4 3 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Figure 4 3 Image sequences opened loaded Multiple data sets can be loaded at the same time f Living Imago Browser lt Fle Edt Wew Took Amiin Window Help saut RB R tite Reset Eey v C Appby toa TLT20040322002427
73. User s Manual IVIS Spectrum Chapter 2 Getting Started 10 2 3 Overview of Image Acquisition The control panel provides the image acquisition functions Figure 2 6 See Appendix A on page 262 for details on the imaging parameters in the control panel NOTE The control panel is only available on the PC workstation that controls the instrument The items available in the control panel depend on the selected imaging mode luminescent fluorescent and acquisition mode Image Setup or Sequence Setup Figure 2 6 IVIS Acquisition Control Panel E WIS Acquisition Control Panel o 83 Imaging Mode Exposure Ti Time F Stop Excitation Filter Emission Filter Imaging modes mto sce vedm wi foc Jopen See Table 2 1 on page 11 for an overview of imaging modes medium vle Field of View System Status _ or Acquire TC Mouse Imaging Shuttle fia i Imaging Wizard Subject height 1 50 om Sequence Setup ppa Setup Auto Exposure Feature The Auto exposure setting is useful in situations where the signal strength is unknown or varies widely for example during a time course study If Auto exposure is chosen Figure 2 6 the system acquires an image at maximum sensitivity then calculates the required settings to achieve as closely as possible an image with a user specified target max count If the resulting image has too little signal or saturated pixels the software adjusts the par
74. User s Manual IVIS Spectrum Chapter 3 Image Acquisition Quick Guide Acquire a Fluorescent Image With Epi IIlumination Figure 3 7 Quick Guide Acquire a fluorescent image with epi illumination 1 Start the ping Image software 3 and initialize the IVIS Spectrum page 7 Note See the V S Spectrum Hardware Manual part no PN121450 Rev00 for more information on the instrument 2 Place the anesthetized subjects in the imaging h b dol tha d Imaging Mode Exposure Time inning F Stop Excitation Filter Emission Filter am geramd CER MEAE 3 Puta check mark next to d Fluorescent and select Auto exposure 4 Select an excitation and emission filter 5 Choose Photog ra ph n a nd Field of View System Status TEN Overlay O ms 6 Select Use subject height 2 8 LL tmagrgweard and enter the height in subject height 1 50 jem centimeters Focus use subject height v Temperature AN Locked 7 Click Acquire 8 When prompted select a location for the image data optional Image data acquired during the session will be automatically saved to this location 9 Enter experiment and subject information in the Edit Image Labels dialog box that appears optional The image window and tool palette appear when acquisition is finished Image Window Tool Palette E nrmonanssa Cha Tool Palette 5 nine Radiant Efoency Oasi Overlay wre 7 il sko iy m image s NT 2004
75. User s Manual IVIS Spectrum Chapter 9 DyCE Imaging and Analysis Figure 9 12 DyCE results showing three temporal components E RKG20110210131022 SEQ C sequence View Unmixing V Show Labels V Individual Scale Normalized Legend a Kidney 1 00 A j B ri Bilis Unmixed images Each Temporal spectra 0 80 7 i image is a representation show signal time J of a temporal spectrum at course of different pp West 7 SR the peak signal time point anatomical regions 0 40 l Min 0 00 Min 0 00 800 1200 Max 2 90e4 Max 1 58e4 Timo Point e Bladder Composite Spectrum List Sh X ins Composite of the Select Name Color Pick unmixed images 1 V kidney Bime br 2 V Brain mig 3 V Bladder BB O ze Min 0 00 Max 2 45e4 Table 9 1 Spectrum list toolbar Item Description SOO Enables you to view and save the unmixed images as a sequence data set The image adjust corrections filtering image information or ROI tools are available for the images Enables you to subtract one spectrum from another see page 175 Adds a temporal component to the spectrum list when performing a manual analysis See page 169 for more details on manual analysis Deletes the last component in the spectrum list Click Unmix after deleting a spectrum to view updated DyCE results 9 To save the results a Enter a name in the Results tab of
76. Wew Era ribs Cons or Cl Use Saved Cos CUA M p e Tt me Delay Fly Gapan Ime p Labels Winning Factor Excitetion filter Emiswon filter Number Fred of Vaasi Select All n this example exposure time and binning factor are displayed on each image Info Click to show or hide the image label information Figure 3 28 Opens all of the images in the sequence Closes all open images Opens the Edit Sequence dialog box that enables you to add or remove images from the sequence Enables you to export the active image as a graphic file for example png dcm 47 Living Image 4 3 1 User s Manual IVIS Spectrum Table 3 8 Sequence View window continued Chapter 3 Image Acquisition Item Description BEB CK2O00628141050 560 F gt ER CKINGLAIGLADOSE SOO j EE Seg TLTAMGOSTMISI ISE f AT Preview picture of the selected data 3 5 Acquire Multiple Sequences in Batch Mode Use the batch mode to set up multiple separate sequences which will be automatically acquired one after another without manual intervention To setup and acquire sequences in batch mode 1 Click Sequence Setup in the control panel 2 Choose the Batch Sequences option Figure 3 29 a AA AY igea TLS SEQ iTLT i C ana iy PNET ary Fide Became View Chobe Preview Laba Gat Ail Add to Lest Browse Wi Tett 7 Gonfigure Load ss Group
77. Wizard Click to start the Imaging Wizard Sequence Setup Click to open the sequence table Image Setup Click to close the sequence table Initialize Click to initialize the IVIS Spectrum See page viii for more details on initializing the system Table A 2 Field of view FOV settings FOV Setting FOV cm A 4 B 6 5 C 13 D 22 5 A 2 Manually Setting the Focus The IVIS Imaging System automatically focuses the image based on subject height If you do not want to use the automatic focus feature you can manually set the focus 1 In the control panel choose Manual Focus in the Focus drop down list The Manual Focus window appears Figure A 2 Opening the Manual Focus window IVIS Acquisition Control Panel il C Marsal Focus Window ol ed Fhstop Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter v ube Maeght pao Field of View System Status Acquire CJ mis 2 8 Imaging Wizard Subject height I Sequence Setup 2 To mark the center of the camera in the window put a check mark next to Display CCD Center 3 Select the size of the step increment that the stage moves Coarse Normal or Fine Living Image 4 3 1 User s Manual IVIS Spectrum Appendix A IVIS Acquisition Control Panel 266 4 Click Up or Down to move the stage and change the focus 5 If necessary select another F stop setting from
78. a PR i NG a Min 9 70e6 Max 1 87e8 93 TP Hx20120410114019sEQ Se Tool Palette gt Image Adjust Corrections Filtering ROI Tools Spectral Unmixing and DyCE Analyze Results Select Images Type Emission 660 7 680 7 700 V 720 9 740 F 760 7 Emission wavelengths of the sequence Methods Cen gt Start Unmixing Excitation wavelength Select Library 2 Click the Analyze tab of the Spectral Unmixing and DyCE tools By default all wavelengths are included in the analysis Remove the check mark next to wavelengths that you want to exclude from the analysis 3 Select Library from the Methods drop down list and click Start Unmixing 4 Select a reference spectral library in the dialog box that appears and click Apply Figure 8 7 The software identifies pixels with spectral characteristics that match the spectrum library The Unmixing window shows the analysis results which include unmixed spectra unmixed images and a composite of the unmixed images Figure 8 8 See Spectral Unmixing Results page 154 for information about the results 143 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing Figure 8 7 Select a reference spectral library Available spectrum libraries CG Load Spectrum Library Spectrum Libraries Informat
79. absolute calibrated or in terms of efficiency calibrated normalized Note See the concept tech note Image Display and Measurement for more on quantifying image data select Help 5 Tech Notes on the menu bar sae eer NG masagana ra kabad mx Ea Ipa mg o w Arae Oes bet i d Fluorescent image Sree Overlay Fluorescent image on photograph Photograph A short exposure of the subject illuminated by the lights located in the ceiling of the imaging chamber The photographic image is displayed as a grayscale image ira Cora ee ega Cpe T fe egg 2 4 Overview of Living Image Tools and Functions The Living Image tools are organized in the Tool Palette or under Tools in the menu bar Figure 2 7 Some tools are for use with a single image others require an image sequence Table 2 2 provides an overview of the tools available for data acquired on the IVIS Spectrum If analyzing data acquired on a different type of VIS instrument say for example the IVIS Spectrum CT please see the Living Image Software User s Manual specific for the IVIS Spectrum CT M NOTE The tools available in the Tool Palette or menu bar depend on the active image data 12 Living Image 4 3 1 User s Manual IVIS Spectrum Figure 2 7 Living Image tools are located in the menu bar and Tool Palette Living Image 4 2 4 Fie Edit View Acquisition Window Help 3D An
80. adds slices to the volume If the processing performance is impacted at the original resolution you may want to reduce the resolution to improve performance Reducing the resolution down samples the data and fewer slices are displayed To adjust the image resolution 1 Move the Level of Detail Slider to the left or right Figure 13 5 The color opacity map is updated 2 To return the resolution to 1x click the Reset button a 242 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools 243 Figure 13 5 Level of Detail slider 4 Display Volume Level Of Detail Os Quality Resolution 0 5x 1x 2x 3x Table 13 2 Example volume with 512 slices at 1x resolution Volume Resolution No of Slices Displayed 0 5x 256 1X original resolution 512 1 5 768 2X 1024 2 5X 1280 3X 1536 Adjusting Volume Opacity Adjust the volume opacity using the slider in the 3D Multi Modality tools Figure 13 6 Adjusting the volume opacity Tool Palette _ BR0111027132749 seq Cl ops L gt ROI Tools lal gt Spectral Unmi ng andoy o gt Surface Topography f 3D Multi Modality Tools Gronal z 10 7 Sagittalfx 2 5 E laces Volume Process Slice Results Ho RK DR ok IV Display Volume Level Of Detail p le Performance Quality Color Opacity Map 1 0 Opacity X o Md Rever
81. bar 3 3 Fluorescent Imaging With Transillumination Fluorescent imaging captures signals from fluorescent molecular reporters Transillumination excitation light source located below the stage is recommended if the fluorescent source is deep relative to the imaged side of the animal Acquisition with transillumination includes a Normalized Transmission Fluorescence NTF Efficiency image in which the fluorescent emission image is normalized by the transmission image measured with the same emission filter and open excitation filter Figure 3 14 TIP See these tech notes for helpful information and quick guides select Help Tech Notes on the menu bar a Transmission Fluorescence m Transmission Fluorescence Raster Scan m Transmission Fluorescence Normalized Transmission Fluorescence m Transmission Fluorescence Well Plates Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition Figure 3 14 Fluorescent images acquired with transillumination The NTF Efficiency image in this example highlights the presence of fluorescence in the animal while the Radiant Efficiency image shows signal ambiguous with autofluorescence ands AT Tery na Deeply yaa This section explains how to acquire a single fluorescent optical image with transillumination See page 42 for information on acquiring a fluorescent sequence To acquire a fluorescent image with transillumination NO
82. browser see page 55 D Living Imaget Browser ko C fats TL ANI SN6 2415807 SE Chek Miers EX Pilbrx Eh Filler Burrurasheres kigdir Lhner M Lhe imap Erpin BED CKAN00628141050 SCO ck ICF750 dye in pillows LE CKO SLOSS SOG EE Irena LAOS DUT subjects phantom S09 TLASILA SE Tt SEA TLTANOSNG2AJ45507 EQ F ALT ka HG Bra oa Chose Preview Label Set AI mid add toust Browse view Dein Configure Los aso abad reme ose Location C Bharr Caliper Liian baa hamak baa NASAN deta ina TT a S06 4h 145507 E0 Gran tot Preview picture of the data selected in the browser blue row Color Scale Provides a reference for the pixel intensities in a luminescent or fluorescent image Pixels less than the color scale minimum do not appear in the image Pixels greater than the color scale maximum are displayed in the maximum color 3 2 Fluorescent Imaging With Epi Illumination Fluorescent imaging captures signals from fluorescent molecular reporters This section explains how to acquire a single fluorescent optical image with epi illumination excitation light source located above the stage a Quick guide See Figure 3 7 on page 30 m Detailed instructions See page 25 See page 42 for information on acquiring a fluorescent sequence TIP See the concept tech note Fluorescent Imaging for more about fluorescence imaging theory select Help Tech Notes on the menu bar Living Image 4 3 1
83. composite image to the system clipboard Click to export the composite image to a graphic file for example jpg be oo Opens the Print dialog box 156 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing Analyzing Images Do either of the following Click the 89 button toolbar button to view all images as a sequence a Double click a particular unmixed image The image s appear in a separate window and the tool palette is available for image analysis When closing the window the software prompts you to save the sequence or image Figure 8 24 View unmixed images as a sequence rr CO HX20120419114703_SEQ p C Sequence View Unmixing V Show Labels V Individual Scale NAALALA UG V Normalized T Legend fa o j g Ei 1 00 0 50 Mins 0 00 p N 6 Mins 0 00 0 0 Max 9 83e7 ax 2 34e8 660 700 740 780 Emission Wavelength nm AF750 Spectrum List amp 2 pbb kb Xx Note Select Name Color Pick 1 V TissueAF Mime vifr 2 V AF680 Hired z p 3 V AF750 YA AW dba Mins 0 00 axs 6 4968 Managing Spectral Unmixing Results Figure 8 25 Spectral unmixing results Information about the analysis method and analysis inputs C7 HX20120419114703_SEQ bodka sci GD sequence View Unmixing Senra Unmiring nod OVCE ee sisal l Anal ze Results V
84. data folder Cll Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 207 2 To show particular results a Select a sequence in the upper box b Select one or more analysis results in the lower box To choose multiple adjacent results press and hold the Shift key while you click the first and last result To choose non adjacent results press and hold the Ctrl key while you click the results c Click Load 3 To show more results repeat step step 2 4 To remove results from the Longitudinal Study window right click a surface and select Remove on the shortcut menu Alternatively select a surface click the Remove button Xx Remove and choose Selected Result To remove all results click the Remove button X Remeve and choose All Results 5 To view a particular image in a sequence a Click the surface b For DLIT results make a selection from the Wavelength drop down list For FLIT results make a selection from the Image drop down list Figure 11 24 DLIT and FLIT results in the Longitudinal Study window Choose an image to display Use the thumb wheel to rotate the surfaces from the selected results NG as 3 E Longitudinal Study Window abais 5 soe ofve IFTTT TOOTH xa Select a Sequence 3D View Plots DUT EL20100601160926 sEQ BD burt EL20100608105326 sEQ Gl DLIT EL20100616161949 sEQ EJ DLIT EL20100621094608 sEQ Wavelength 580 XE Remove
85. data to remove electronic noise before any measurements For more details see page 105 Replace ROIs If this option is chosen all auto ROIs are replaced when new ROl s are created Restore Defaults Restores the factory set defaults for the auto ROI parameters Save Load Click to display or hide the tools that enable you to save load or delete auto ROIs in the active data Note The save function saves parameters the not actual ROls This means that when you load saved auto ROI parameters the software draws a new ROI using the saved values Threshold Lower Limit Minimum Size 5 4 Measurement ROIs This section explains in detail how to draw a measurement ROI on an optical image to obtain the intensity signal in a user specified area Table 5 3 lists the three methods for drawing measurement ROIs on an image M NOTE See page 93 for a quick guide to drawing measurement ROIs on an optical image or sequence Table 5 3 Methods for drawing a measurement ROI Drawing Description See Page Method Automatic The software automatically locates and draws an ROI s on the image To do 98 this the software locates the peak pixel intensities in the image and searches the neighborhood around a peak pixel A pixel is included in the ROI if the pixel intensity is greater than the threshold a user specified percentage of the peak pixel intensity Manual Places one or more ROls circular square
86. each voxel summed or integrated over the 3D ROI Average Flux ph sec Total flux Number of voxels in the 3D ROI Stdev Flux Standard deviation of the flux of the voxels inside the ROI Min Flux The smallest flux value of a voxel Max Flux The largest flux value of a voxel Source Voxels cells Note This measurement type requires a quantification database See Chapter 12 on page 231 for more details Total Cells The number of cells in the 3D ROI Average Cells Total number of cells Number of voxels in the 3D ROI Stdev Cells Standard deviation of the number of cells in the 3D ROI Min Cell The smallest number of cells in a voxel included in the 3D ROI Max Cell The largest number of cells in a voxel included in the 3D ROI Source Voxels pmol M cm measurements Total pmol M cm The fluorescence yield summed or integrated over the 3D ROI Average pmol M cm Total fluorescence yield Number of voxels in the 3D ROI Stdev pmol M cm Standard deviation of the fluorescence yield of the voxels in the 3D ROI Min pmol M cm The smallest fluorescence yield in the 3D ROI Max pmol M cm The largest fluorescence yield in the 3D ROI Source Voxels pmol measurements Note This measurement type requires a quantification database See Chapter 12 on page 231 for more details Total pmol Total picomoles of fluorescent probe within the 3D ROI Average pmol Total picomoles N
87. from the drop down list to overwrite that spectrum with the computed spectrum when you click Apply 176 10 Reconstructing a 3D Surface Generating a Surface Managing Surfaces on page 182 Export or Import a Surface on page 183 A surface is a 3D reconstruction of the animal surface topography derived from a structured light image A surface is a required input to DLIT or FLIT analyses Figure 10 1 You can also import a surface or export a surface for viewing in other 3D viewer applications Figure 10 1 Example surface Generate a surface for DLIT Analysis FLIT Analysis 3D reconstruction of luminescent 3D reconstruction of fluorescent sources displayed as voxels sources displayed as voxels page 160 page 167 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 10 Reconstructing a 3D Surface 178 10 1 Generating a Surface 1 Load the image sequence for the reconstruction For example a sequence that was acquired for DLIT analysis Select an orientation dorsal or ventral and subject in the surface topography tools Select a smoothing level NOTE The default Low smoothing level is sufficient in most cases but it may be necessary to modify this if there are tufts of hair on the animal which disrupt the surface smoothness Click Reconstruct The Tomography Analysis box appears By default the entire subject is selected for the reconstruction Figure 10 2 Figure 10 2 D
88. i these images 0 mission Wavelength nm Mace 1 00e8 y nob Sic Spectrum List See Table 8 3 on fb Hsk X f fth page 155 for detailson t Composite of the this toolbar ning Name Color a unmixed Images 1V TissueAF Mime zZ See page 156 for 2 V aF6so Be Bid more details 3 V AF750 We t Library Method The library method uses a user generated spectrum library to analyze a data set If you plan to analyze data by this method the data must be acquired using the same or a subset of the excitation emission filter pairs of the spectrum library The probe depth in the data set being analyzed and the spectrum library data set should be similar for optimum analysis results For example do not use a spectrum library generated from in vivo data to analyze in vitro data m NOTE Use the guided method to generate a spectrum library of known probes with known locations see page 139 for more details on the guided method 1 Load the image sequence In Figure 8 6 the fluorophores are Alexa Fluor 680 and Alexa Fluor 750 Images were acquired using a 605 nm excitation filter and emission filters from 660 to 800 nm in 20 nm increments Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing Figure 8 6 Sequence for spectral unmixing Sequence View Units Radiant Efficiency E Use Saved Colors Options x Info j
89. image Excitation and Emission filters will be specified for fluorescent images and the Emission nm ak i os i i Open filter for Emission will be specified for bioluminescent images Extinction Coeff A measure of excitation photon absorption interaction with the well plate samples based on a base 10 logarithmic derivation The quantum efficiency factor of the conversion of the absorbed photon to the emission wavelength is also included Cross Section A measure of excitation photon absorption interaction with the well plate samples based on a natural logarithmic derivation The quantum efficiency factor of the conversion of the absorbed photon to the emission wavelength is also included Bioluminescence 235 Living Image 4 3 1 User s Manual IVIS Spectrum Table 12 1 Quantification results continued Chapter 12 Quantification Database 236 Item Description Total Flux cell quantification A measure of total flux photon sec emitted from a single cell This number can be used to estimate the number of cells from the total flux in the 3D 12 3 Managing Quantification Results The quantification results can be saved with the image sequence and as a calibration database that is made available in the DLIT or FLIT 3D reconstruction tools in the Properties tab When you define the properties for performing a 3D reconstruction and a calibration database is specified the 3D reconstr
90. list that appears a If analyzing a reflectance epi illumination fluorescent image go to step 4 otherwise go to step 5 4 For reflectance epi illumination fluorescent images only a Confirm the purple data mask in the dialog box that appears Figure 5 13 The data mask includes the entire subject by default and defines the area of excitation light projection onto the animal If you do not want to analyze the entire subject select the Data Mask option and mask a particular area using the data mask options Table 5 4 b Click OK The mirror ROIs and intensity measurements appear on the image Figure 5 14 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data Figure 5 13 Excitation Projection Setup dialog box For fluorescent images only fa sx 7 3 re en Excitation Projection Setup Confirm Excitation Light Projection Area Data Mask Options Photograph Threshold lel Draw Mask 3 Rectangle TIO EE Table 5 4 Data mask options Option Description Photograph If this option is chosen the software automatically draws the data mask by using higher intensities in the photograph The mask selects high valued photograph image pixels which are located continuously and centrally in the photograph image The photograph mask works best with light colored subjects Threshold If necessary use the threshold slider or mi arrows to ad
91. on creating a sequence from individual images see page 88 TIP See the tech note Image Math for a quick guide select Help gt Tech Notes on the Help menu 7 1 Creating a New Image Using Image Math 1 Load an image sequence 2 Select Tools Image Math for lt name gt _SEQ on the menu bar Figure 7 1 Opening the Image Math window O Fle Edt View Took Window Help wo 9 w Pa 3D Animation wal Longitudimal Stucky Well Plate Quantification for TLT20060S10114512 SEQ Image Overlay for TLTA0060510114512_SEQ Colonze bmage Math for TLT20060510114512 SEQ N Memory Tracker E iitmo60510114512 SEQ A C Sequence View spectra TLE20060510114512_001 a Units Radkant kEhaeney TIT20060510114512 002 L TIT2006051011451 2 003 z TIT200E0510114512 004 FUZ0060910114512 005 ILIZ0060510114512 006 IT WNS INI 1451 T ANT b TUZ0060510114512 001 TIKINO60510114512 002 TIT2006051011452 7_O0F TIT20060510114517 004 T20060510114512 005 TL 20060510114512 006 LTUTWWSOSINIIASS AAT o Color Scale Limits for A and B o fu Auto Resu Color Scale Lumits er Auto Result A O Kk k 1 00 Compute K from ROL 7 Vi mith Photo from A Deplay Result For Measuring 3 In the Image Math window that appears select an image from box A and from box B The Image Math window shows a thumbnail of image A image B and the new image Living Image 4 3 1 User s Manual IVIS Spectrum Chap
92. related to animal subjects m Concept Tech Notes Background information on in vivo imaging topics Table 1 2 Tech Notes Tech Notes Title 1 Adaptive Fluorescence Background Subtraction 2 Auto Exposure 3 Determine Saturation 4 Bioluminescence Tomography DLIT m 4a Setup and Sequence Acquisition 4b Topography 4c Source Reconstruction and Analysis 5 ROls optical data a 5a Drawing ROIs m 5b Subtracting Background ROI from Sequence a 5c Subject ROIs 6 Fluorescence Tomography FLIT m 6a Setup and Sequence Acquisition a 6b Topography 6c Source Reconstruction and Analysis 7 High Resolution Images 8 Image Math 9 Image Overlay 2D 10 Image OVerlay 3D 11 Imaging Wizard 12 Load Groups of Images 13 Spectral Unmixing Living Image 4 3 1 User s Manual IVIS Spectrum Table 1 2 Tech Notes continued Chapter 1 Welcome 14 Transillumination m 14a Transillumination Fluorescence m 14b Raster Scan m 14c Normalized Transmission Fluorescence a 14d Well Plates 15 Well Plate Quantification Biology Tech Notes Title 1 d luciferin Prep Sheet 2 Kinetic Analysis of Bioluminescent Sources 3 Imaging Protocol Guide 4 Imaging Procedure 5 Intraperitoneal Injections Concept Tech Notes Title 1 Luminescent Background Sources and Corrections 2 Image
93. spacing in millimeters Figure 13 25 Volume information Ga C Volume Information Volume Information File C Share aneurism_8bit_256x3 raw Header Offset 0 bytes Number Of Slices Images 1 F Slice Information Pixel Spacing Row Column 0 1000 0 1000 5 mm Slice Spacing 0 1000 mm Slice Resolution Low Down samples volume data It conserves memory and improves performance Full Loads and renders volume data using the original resolution Low Y Full Memory Requirement Status Good Select a data type Enter the 7 Width height and number of slices z Slice row column pixel spacing and the slice spacing in millimeters 3 If loading the data will cause low memory you are prompted to down sample the data Figure 13 26 Decrease the slice resolution by moving the Slice Resolution slider to the left until the Memory Requirement Status is Good P Volume Information i Slice Information Pixel Spacing Row Column 0 1000 0 1000 mm Slice Spacing 0 1000 34mm Slice Resolution Low Down samples volume data It conserves memory and improves performance Full Loads and renders volume data using the original resolution Low Figure 13 26 Down sample 3D volumetric data to improve memory and performance 2 x Changing the Orientation of RAW Volumetric Data Occasionally RAW files raw or vox may be loaded with the orienta
94. the animal surfaces derived from VIS data Organ files must be segmented from MRI or CT 3D volumetric data in third party medical imaging analysis software M NOTE The imported atlas must include a surface skin file which delineates the animal surface The file name must include the word skin for example rat skin iv 1 Load a DLIT or FLIT image sequence that is associated with the mouse comprising the organ files in iv dxf or stl format 2 Select File Import Organ Atlas on the menu bar 3 In the dialog box that appears click Add Organ Files Figure 11 37 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Figure 11 37 Import Organ Atlas dialog box cE Import Organ Atlas Ea Organ Files Select Skin Mesh Generate Mesh Co efficients Select Organ Files Look in organ atlas IY files e amp ee E ie stomach iv uterus iv Organ At My Recent Documents Add Organ Files Save Organ Atlas My Network Places File name Files of type Import Organ Atlas Organ Files Select Skin Mesh skin iv ka Generate Mesh Co efficients uberus iv bladder iv bones iy brain iv colon iv eves iv Fat iv heart iv kidneys iv liver iv lungs iv muscle iy Organ Atlas Name 45507 Add Organ Files Save Organ Atas uterus iv bladder iv bones iv brain iy c Y Al Mesh Formats
95. 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 See Table A 2 for more details on the calibrated FOV positions Service Moves the stage to a position for cleaning the imaging chamber below the stage Load Moves the stage from the cleaning position back to the home position MIS Choose this option if the subject will be contained in the Mouse Imaging Shuttle during image acquisition Subject height cm Sets the position of the focal plane of the lens CCD system by adjusting the stage position The subject height is the distance above the stage that you are interested in imaging For example to image a mouse leg Joint set the subject height to a few mm To image the uppermost dorsal side of a mouse set the subject height to the 1 5 2 0 cm The default subject height is 1 5 cm IMPORTANT The IVIS instrument has a protection system to prevent instrument damage however always pay close attention to subject height 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 Ima
96. the drop down list and adjust the light level using the arrows 6 Click Update to apply the settings The resulting focal plane cm above the stage is automatically entered in the Subject height box 7 Click OK when the image is focused Appendix B Preferences General Preferences Options on page 269 Acquisition on page 270 Theme on page 271 Optical Properties on page 274 You can manage user IDs and specify defaults for some parameters that are associated with the user ID selected at the start of a new session After you log on select Edit Preferences on the menu bar to view the user modifiable preferences M NOTE Any changes made to the Preferences are implemented at the start of the next session The Acquisition tab is only available in the Living Image software that controls the IVIS Imaging System B 1 General Preferences Figure B 1 General preferences C7 Preferences General Options Acquisition Theme Optical Properties Start Up Defaults EF Dock Tool Palette Show Activity Window on Warnings l Errors Left Right Save Settings Window Size Save float corrected image aan W Color Selections Width 86 55 F Restore Defaults W Folder Locations Height 65 IM Window Size amp Position Most Recently Used Dataset Histor Apply Individual Color Scale For Sequences UU d 5 Show Transillumination Locations Display ROT Label As Measure
97. the tool palette Figure 9 13 b Click Save 168 Living Image 4 3 1 User s Manual IVIS Spectrum Figure 9 13 DyCE results Tool Palette Selectedimages Spectrum Type Method Automatic AnalysisBinning 16 ImageWidth 120 ImageHeight 120 DataUnit Radiance SPUM_Ver 2 0 Livinglmage 4 3 1 0 15621 Save Results Name SPUM 4 Results name Delete Load Manual DyCE Analysis Chapter 9 DyCE Imaging and Analysis 1 Load a DyCE image sequence Alternatively load DyCE results obtained from an automatic analysis Figure 9 14 l NOTE This section illustrates manual analysis of DyCE results obtained from an automatic analysis Figure 9 14 Load DyCE results Sequence View Unmixing Min 68 Max 57193 7 RKG20110210131022_SEQ Flo fms Tool Palette 63 F Analyze Results Spectral Unmixing Results Item Value DataMask Yes SelectedImages 10 SpectrumType Time Method Manual AnalysisBinning 16 ImageWidth 120 ImageHeight 120 DataUnit Radiance SPUM_Ver 2 0 Livingimage 4 3 1 0 15526 Save Results Name SPUM_1 Delete gt Image Adjust gt 2 ROI Tools E Units Counts x Use Saved Colors Options vY Info 2 iy NG a L i Spectral Unmixing and DyCE A Select DyCE results from the Name drop down list and click Loa
98. to generate a spectrum library a set of reference spectra for probes with known spectra and known locations Library This method requires a user generated spectrum library The library 139 method identifies pixels in the data with spectral characteristics that match the spectrum library Note The data being analyzed must be acquired using the same or a subset of the excitation emission filter pairs of the spectrum library The probe depth in the data being analyzed and the spectrum library data set should be similar for optimum analysis results For example do not use a spectrum library generated from in vivo data to analyze in vitro data and vice versa Automatic Use this method when the probe locations are unknown 145 Manual If necessary perform a manual analysis after an automatic analysis to 149 identify additional probe locations Guided Method Use the guided method m When the probe locations are known and probe signals do not overlap m To generate a spectrum library for probes with known spectra and known locations 1 Load the image sequence In Figure 8 2 the fluorophores are Alexa Fluor 680 and Alexa Fluor 750 Images were acquired using a 605 nm excitation filter and emission filters from 660 to 800 nm in 20 nm increments Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing 140 Figure 8 2 Sequence for spectral unmixing D wamo seo LI Segre Views n
99. tools page 68 Corrections Filter tools page 70 Image Information tools page 64 ROI Tools page 90 24 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition 25 Acquire a Luminescent Image This section provides detailed instructions for image acquisition NOTE The IVIS Spectrum should be initialized and the temperature locked before setting the imaging parameters in the control panel See page 7 for more details 1 Put acheck mark next to Luminescent and select Auto exposure click the arrows in the control panel The software automatically determines the binning and F Stop settings TIP See the tech note Auto Exposure for helpful information select Help gt Tech Notes on the menu bar Alternatively manually set the exposure binning and F Stop See Appendix A on page 262 for details on these parameters Figure 3 2 Control panel l IVIS Acquisition Control Panel Imaging Mode Exposure Time Excitation Filter Emission Filter Auto sec v Medium vil v Field of View System Status Acquire C MIS Idle 2 8 Imaging Wizard Subject height 1 50 3 cm w Sequence Setup Focus use subject height v Temperature EI Locked 2 Put a check mark next to Photograph 3 Select a Field of View size of the stage area to be imaged TIP See the technical note Detection Sensitivity for more information about the Field of V
100. will generate 20 images If the raster scan option is selected the software takes all of the images from the transillumination locations and adds them together into one image The raster scan option may be helpful when trying to determine the optimal excitation and emission filters for a particular fluorescent probe Grid Type 9x19 grid Update Photograph Click to acquire a new photographic image If the chamber door is opened during transillumination setup you are prompted to acquire a new photograph Clear Selections Clears selected highlighted transillumination locations on the grid 5 Confirm that the Lamp Level is set to High in the control panel Living Image 4 3 1 User s Manual IVIS Spectrum NOTE The lamp may be set to Low for certain applications such as long wavelength data through thin tissue Chapter 3 Image Acquisition 6 Select a Field of View size of the area to be imaged Table 3 5 Field of View FOV settings FOV Setting FOV cm A 4 B 6 5 C 13 D 225119 5 E 22 5 26 Some IVIS Spectrum instruments may have the FOV in parentheses FOV 19 5 and 26 were replaced by FOV 22 5 7 Select a focus option Figure 3 10 The focal distance to the camera is set a stage z 0 for each field of view To focus at the top of the animal the stage moves down so that the top of the animal is at z 0 You can enter the height of the animal and select the
101. 0 0 40 0 00 Min 0 00 0 400 _ 800 1200 904 Max 1 58e4 Time Point s Spectrum List wo by Xx Note Select Name Color Pick 1 V Kidney Mime aL 2 Brain i E v f 3 V Bladder blue ze Min 0 00 Max 2 45e4 I i CE RKG20110210131022 SEQ T Kidney V Brain V Bladder Photograph Image Adjust Min 3 98e3 3 Brightness Sequence View Unmixing Composite anrima Hers Max 3 5864 Color Logarithmic Scale Label Bladder Close 173 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 9 DyCE Imaging and Analysis 2 Add or remove the check mark next to an image to include or exclude the data from the composite image 3 Use the image adjust tools at the bottom of the Composite window to adjust the appearance of the composite image Table 9 3 Composite window Item Description Sends the composite image to the top of the image cube Click the Image Cube tab in the Unmixing window to view the image cube See Figure 9 15 on page 170 for more details on the image cube ImageCube Image Cube Viewer V Overview Composite image displayed on top of the image cube Image Cube Viewer V Overview Copies the Composite window to the system clipboard Opens a dialog box that enables you to export the composite image to a graphic file for example png Wo gh y Opens the print dialog box
102. 0 Emission Filters 1 00 y 0 50 0 0 400 500 600 700 800 900 Cancel sj Back J Restart Wizard Next Select the type of imaging subject Figure 9 5 161 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 9 DyCE Imaging and Analysis Figure 9 5 Imaging Wizard Bioluminescence DyCE Fluorescence DyCE Gg A Imaging Wizard Bioluminescence DyCE Imaging Subject Mouse Exposure Parameters Auto Settings Manual Settings Exposure Binning F Stop Luminescent 1 00 mM Photograph Auto sec l2 Sa dama sec x is Si Geet Field of View C 13 4cm z Focus Subject Height 1 50 cm Focus use subject height w Options Time Series Total number of Intervals 3 Interval Duration min Delay sec Max Images 1 4 5 2 5 45 3 2 5 48 6 Ab d1 ab dh 24 Cancel Back Next Restart Wizard La A Imaging Wizard Fluorescence DyCE Imaging Subject Mouse KA Exposure Parameters o Auto Settings Manual Settings m Fluorescent sA V Photograph Field of view C 13 4cm v Focus Subject Height 1 50 gt cm Focus use subject height w Options Time Series Total number of Intervals 3 Interval Duration min Delay sec Max Images 1 4 is 48 2 5 45 all 3 al 5 24 Restart Wizard Cancel Back Next
103. 00 1 0000 0 0000 0 0000 0 0000 PatientsBirthDate 20111027 Save Results eee Name MULTIMODALITY_1 v Load 3D Optical Tools DLIT 3D Reconstruction Saving the registered and classified data provides a convenient way to share data The software saves the following m Level of detail setting m Color tables for the opacity map and slices m Histogram tool control settings and the resulting color opacity map Crop settings Managing Results Saving Registered Results Multi modal registration settings 1 In the Results tab confirm the default name in the Name drop down list or enter a name 2 Click Save The registered 3D volumetric data along with the color opacity settings appear in the 3D View window i NOTE The results are saved in XML format in the optical data set location The results can only be accessed from the same optical data set Loading Results 1 Select the results from the Name drop down list 2 Click Load 249 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools 250 Deleting Results 1 Select the results from the Name drop down list 2 Click Delete 3 Click Yes in the confirmation message that appears 13 7 Registering Optical and Volumetric Data Registering multi modal data optical and volumetric data provides an anatomical context for interpreting biological functional informati
104. 02 17413344 Sores Fuxexent Background Tests Tue Feb 17 200403 34 57 Greek 2 ferele Nu Mu ree Level regh Em eCy5 5 Ex CyS Shing Ex Auraton Sn 00S t FOW 12 4 12 16 Living image Werson 2 50 2 42 2004 restructured Communi Camera V15 2 51620EEV The Tool Palette includes the Image Adjust tools page 68 Corrections Filter tools page 70 Image Information tools page 64 ROI Tools page 90 See Table 3 2 on page 28 for more details on the image window 30 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition 31 Acquire a Fluorescent Image With Epi IIlumination This section provides detailed instructions for image acquisition M NOTE The IVIS Spectrum should be initialized and the temperature locked before setting the imaging parameters in the control panel See page 7 for more details 1 Puta check mark next to Fluorescent and select Auto exposure click the arrows in the control panel The software automatically determines the binning and F Stop settings TIP See the tech note Auto Exposure for helpful information select Help gt Tech Notes on the menu bar Alternatively manually set the exposure binning and F Stop See Appendix A on page 262 for details on these parameters Figure 3 8 Control panel 4 IVIS Acquisition Control Panel Imaging Mode Exposure Time Binnin J FiStop 7 Excitation Filter Emissi
105. 114 50 Sequence Setup CCD Temperature The IVIS Acquisition Control Panel indicates the temperature status of the charge coupled device CCD camera Figure 2 4 After the system is initialized the temperature box turns green when the temperature is locked at the 90 C demand temperature The green temperature box indicates that the instrument is ready for operation and image acquisition The demand temperature for the CCD camera is preset and generally should not be changed Electronic feedback control maintains the CCD camera temperature to within a few degrees of the demand temperature The instrument is ready for imaging after the system is initialized and the operating demand temperature of the CCD camera is reached locked Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 2 Getting Started Figure 2 4 Instrument temperature status in the control panel IVIS Acquisition Control Panel o 83 Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter Fe a Cx eS Co loom IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter medum gt a Field of View System Status E Mouse Imaging Shuttle Demand Measured Camera Temp 90 s gno Service 13 4 xt SHS Stage Temp 37 0 3 Subject height 1 50 2 ge Temp 37 0 Ji Field of View c 7 E Mouse Imaging Shuttle 13 4 o Subject height 1 50 2 cm
106. 3 4 In the dialog box that appears select a folder for the file tfn and enter a file name Click Save Loading a Color Opacity Map 1 2 Click the Open button a Figure 13 4 In the dialog box that appears navigate to the map file tfn and click Open 241 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools Figure 13 4 Save or load a color opacity map Tool Palette gt ROI Tools a Spectral Unmixing and DyCE le gt Surface Topography Volume Process slice Results AO os Level Of Detail Y Performance _ Quality Color Opacity Map 1 0 Opacity _ X o W Reverse All Click a button to save or load a color opacity map a Ni 0 00 Counts v V Logarithmic Histogram Maximum Intensity Projection MIP F Gradient Illumination gt 3D Optical Tools DLIT 3D Reconstruction 13 3 Volume Display Options Adjusting the Image Quality By default the color opacity map displays the volumetric data at original 1x resolution This means for example if the volume comprises 512 slices then all of the 512 slices are displayed You can increase or decrease the resolution of the data display from 0 5x to 3 0x resolution see Table 13 2 for examples If the resolution is increased the software interpolates the data and
107. 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools Figure 13 15 3D Volumetric Data Browser Click a row to preview data playback Double click a row to load the data Click a column header to sort the browser contents in ascending alpha numeric order Click the column header again to sort in descending alpha numeric order i 7 Browse 3D Volumetric Data Folder Path V Add to list V Load in a new window To view a particular slice stop playback then move the slider or enter a slice number iw Starts data playback p la Stops data playback 1 p TS 517 FE To select a range of slices for playback move the left and right sliders or enter the first and last slice numbers Table 13 5 3D Volumetric Data Browser Item Description Add to List If this option is chosen the data selected in the Browse for Folder box is added to the 3D Volumetric Data Browser If this option is not chosen the data selected in the Browse for Folder box replaces the contents of the 3D Volumetric Data Browser except for loaded data Browse Opens the Browse For Folder box Load in a new window If this option is chosen multiple data sets can be loaded each in a separate window If this option is not chosen only one data set can loaded at a time Load Click to open the data selected in the 3D Volumetric Data Browser
108. 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 81 4 9 Overlaying Multiple Images The image overlay tool provides a convenient way to view multiple reporters in one image You can use the image overlay tool to display multiple luminescence or fluorescence images on one photographic image TIP See the technical note Image Overlay 2D for a quick guide select Help Tech Notes on the menu bar To coregister multiple images 1 Acquire an image sequence using the appropriate filters for each reporter Alternatively create a sequence from images acquired during different sessions For more details see page 88 2 Load the image sequence Figure 4 25 Image sequence IG amama tO LA quence Few Linits Puackend Eco oF Use Saved Colors 3 Open one of the images and optimize the image display using the color scale Min and Max sliders in the Image Adjust tools To view all images in the sequence click the Display All button to open each image overlay mode in a separate image window 4 Select Tools Image Overlay for lt sequence name gt _SEQ on the menu bar The image overlay window appears and shows the first photograph in the sequence To view a different photograph make a selection from the photograph drop down list Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Figure 4 26 Image Overlay window
109. 5 6 ROI Properties Item Description ROI A drop down list of ROIs in the active image or image sequence To select an ROI double click the ROI in the image or make a selection from the drop down list Shape The shape of the ROI circle square grid or contour selected in the image Type Indicates the method that was used to draw the selected ROI automatic manual or free draw ROI Label Click to edit the selected ROI label name Image Number A drop down list of open images Background ROI tab The Background ROI tab shows a drop down list shows all average background ROIs in active image that can be linked to a user specified measurement ROI or subject ROI selected from the drop down list at the top of the dialog box 111 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data 112 Table 5 6 ROI Properties continued Item Description Subj ROI The Subject ROI tab shows a drop down list of all subject ROIs in the image number selected above that can be linked to a user specified measurement ROI or average background ROI selected from the drop down list at the top of the dialog box The Background ROI tab shows a drop down list of all average background ROls in the click number selected above that can be linked to a user specified measurement ROI or subject ROI selected from the drop down list at the top of the dialog box
110. 5000 Counts Color Scale Min 1122 Max 19546 Paaa ee EERE REE RE RE RR TT M TTT dd a a a ee a a a a a a 3 Adjust the ROI position a Place the mouse pointer over the ROI When the pointer becomes a click the ROI b Drag ROI s m NOTE To move multiple ROIs at the same time press and hold the Shift key while you click the ROls and then drag them to a new location Contour ROIs cannot be moved using this method 4 Adjust the ROI dimensions a Place the mouse pointer over the ROI When the pointer becomes a click the ROI b Place the mouse pointer over an ROI handle so that it becomes a Drag the handle to resize the ROI Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data J NOTE You can also change the ROI position or size using the adjustment controls in the ROI Properties box see Moving an ROI page 112 and Editing ROI Dimensions page 113 Click the Measure button Wf MeasureROls The ROI measurements and table appear For more details on the table see Managing the ROI Measurements Table page 119 For information on how to save ROIs see page page 116 Drawing ROIs Using the Free Draw Method 1 2 3 Open an image and in the ROI tools select the type of ROI that you want to draw from the Type drop down list Click an ROI shape button Circle Square 0 or Contour and select Free Draw from
111. 50624145507 006 Units Counts AE KATE GN Cole aj nG a x Display Overlay z Options v Info Luminescence 15000 10000 5000 Counts Color Scale Min 1122 Max 19546 Tagging an Image An image tag displays the x y pixel coordinates of the location and the pixel intensity z counts or photons You can apply a tag at a user selected location in an image To apply a tag 1 Right click a location in the image 2 Select Insert Tag on the short cut menu Figure 4 13 Insert a tag on an image left move the tag label right a Units Counts v Display Luminescence 10000 Insert Tag Insert Comment Remove Tag Remove Comment Remove All Tags emove All Co ents Remove All Comments Colon Stale Min 586 Copy All ROIs Max 10348 Paste ROI Hide ROI Tags Delete All ROIs Sort ROIs Zoom Area Zoom In Zoom Out ab Tr Reset Zoom TC TLI20050624145507 005 E TLI20050624145507 005 Units Counts v Display Overlay v Options rj Info nG igj Options zji Info nG wj Luminescence 10000 8000 6000 4000 2000 Counts Color Scale Min 586 Max 10348 67 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data To move a tag 1 Position the mouse pointer over the tag 2 When the hand tool appears
112. 5507 006 DEAR Units Counts Display Overlay Fino a i i gt Image Adjust n n gt Corrections Filtering Luminescence Image Information lan S lii 1 BB Units Cm NG Image Binning 8 Width 12 6 cm Height 12 6 cm Image X Y 8 569 12 077 cm Image Data 16 count Crop Distance 6 69 9 16 0 002 493 Distance 2 43 om gt ROI Tools Counts Color Scale Min 1122 Max 19546 2 To change the cursor position or size drag the A or B end of the cursor to a new location on the image The measurement information in the Tool Palette is updated 3 To hide the cursor click the Ka button Table 4 10 Measurement cursor position and length Item Description A Pixel x y coordinates of position A on the cursor Note Measurements are report in pixels or cm whichever is selected from the Units drop down list in the Image Information tools Figure 4 23 Pixel x y coordinates of position B on the cursor Length of the cursor from A to B number of pixels vertical distance from A to B number of pixels Distance Length of the cursor from A to B number of pixels Living Image 4 3 1 User s Manual IVIS Spectrum Measurements are report in pixels or cm whichever is selected from the Units drop down list in the Image Information tools Figure 4 23 To measure distance using the crop box 1 Chapter 4
113. 660 700 740 780 Max 9 8267 Emission Wavelength nm 7 AF750 Composite Spectrum List s amp h x Note Methods Select Name Color Pick 1 Tissuear OCE Select Manual 2 V AF680 E S 3 V AF750 Bb 4 Min 1 58e2 4 Select Manual from the Methods drop down list and click Start Unmixing The Unmixing window appears Figure 8 3 Figure 8 17 Unmixing window Image cube showing a pseudo color image composite of colorized sequence images man e HX20120419114703 sEQ Sequence View Unmixing Z Normalized Legend G ImageCube No data available Spectrum List See Table 8 3 on ____LL d betx page 155 for Note details on this Select Name Color Pick toolbar i TissueAF Dime v 4 r 3 V AF750 Wifbue JZE Image Cube Viewer M Overview Close List of the spectral components to unmix If the Imaging wizard was used to set up the sequence the list includes the probes selected in the wizard The default list also includes a tissue autofluorescence background component 150 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing 151 The image cube represents a stack of the sequence images sorted according to the spectral axis When the Overview option is selected the image cube shows a pseudo color image that is a composite of the stack images which have been colorized to encode spectral informatio
114. Avg Counts Total Counts Number of pixels or super pixels Stdev Counts standard deviation of the pixel counts inside the ROI Min Counts lowest number of counts in a pixel inside the ROI Max counts highest number of counts in a pixel inside the ROI 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 Tip See the tech note Image Display and Measurement for more details on count units select Help Tech Notes on the menu bar Radiance Photons fluorescence Total Flux photons sec the radiance photons sec cm steradian 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 photons sec cm sr Stdev Radiance standard deviation of the pixel radiance inside the ROI Min Radiance lowest radiance for a pixel inside the ROI Max Radiance highest radiance for a pixel inside the ROI Tip See the tech note mage Display and Measurement for more details on photon units select Help Tech Notes on the menu bar Radiant Efficiency fluorescence Epi fluorescence Fluorescence emission radiance per incident excitation intensity p sec cm sr UW cm Transillumination fluorescence Fluorescence emission radiance per incident excitation power p sec cm sr mW Efficiency epi fluorescence
115. Counts Apply to all HX 20070420121444_SEO a Sequence View gt Image Adjust gt 4 gt ROI Tools p its Counts v Use Saved Colors gt Spectral Unmixing Units O FES oS aa Pace ns Kag NA l MESIS Max2311 26 2 Select Tools Colorize on the menu bar The software renders each luminescent or fluorescent image in color and combines them into a single image Figure 4 29 Figure 4 29 Colorize view 00 042m naaa seq Cla Cl bagane iew Sperra Corine View CH a Colormacc NI Color Range Riker Range t Log Scale Seal Color Ga iy 84 Living Image 4 3 1 User s Manual IVIS Spectrum Table 4 13 Colorize tools Chapter 4 Working With Optical Image Data Item Description Colorize View Color Map Color Range Filter Range NIR A special camera setup that extends the color response into the near infrared range Near infrared fluorophores appear red to purple using the NIR camera setup VIS Regular camera setup that mainly renders color in the visible range It is similar to the color response of a commercial digital camera NIR fluorophores appear dark red to invisible using the VIS camera setup The color map indicates the color range of the selected camera setup from short to long wavelength The two sliders determine the lower and upper limits of the color range that is used to render color The parts of the color map outs
116. D Reconstruction of Sources 2 Select File Export 5 3D Scene as DICOM on the menu bar 3 In the dialog box that appears set the export options and click Export For more details on the 3D Scene Exporter see Table 11 8 Figure 11 28 3D Scene Exporter dialog box E 3D Scene Exporter Save DICOM as Single Frame DICOMs Slice Orientation Transaxial Slice z Export voxels using original resolution Parameters Slice Resolution Low Total slices 256 Slice spacing mm 0 3977 Pixel spacing mm Hollow mesh Approx size 2 9 MB Solid mesh 0 2891 73 Export 4 In the Browse For Folder dialog box that appears choose a folder for the DICOM files and click OK During the export operation the 3D View window displays the each slice in the export For example if Transaxial Slice is selected for export then the transaxial windowpane cycles through a display of each exported slice Table 11 8 3D Scene Exporter dialog box Item Description Save DICOM as Multi Frame DICOM Exports a single file that contains multiple frames Note Choose the Single Frame or Multi Frame DICOM option depending on the third party software you will use to import and view the 3D scene Some applications cannot reconstruct multi frame DICOM files Single Frame DICOMs Exports multiple files that contain a single frame each 211 Living Image 4 3 1 User s Manua
117. Delete ROls from the system Tool Palette gt Corrections Filtering PROT oC LI HE h Measure ROIs ba Apply to Sequence Name ROI 1 KSA 118 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data ee a GA ee ee a a ee ee ee eee a a ee GG a a a igure 5 Removing ROls from the system ee a A EAEE Tool Palette Image Information O O Uf Measure ROls x Apply to Sequence te JAG JB ha Ja JJ JAG JAG SW a JA MG JAG SA a O JAG JB Ja JA GING SB a a JAG JA Ja JA MG JM UM a JA Di 5 9 Managing the ROI Measurements Table The ROI Measurements table shows information and data for the ROIs created during a session The ROI measurements can be displayed in units of counts radiance Radiant Efficiency Efficiency or NTF Efficiency depending on the type of image data See the technical note Quantifying Image Data for more details select Help Tech Notes on the menu bar Viewing the ROI Measurements Table Click the Wf Measurerots button to display the ROI measurement table Alternatively select View gt ROI Measurements on the menu bar Figure 5 28 ROI Measurements table r A ROI Measurements o llas ROI Measurements Grid ROI Measurements Refresh Image Number ROI Image Laye Total Coun Avg Count Stdev Cour Min Count Max Count ROI Pixels Area Xc Yc Widt ccd Pixels pixels pixe
118. Display and Measurement Detection Sensitivity Fluorescent Imaging DLIT and FLIT Reconstruction of Sources 1 4 Caliper Technical Support For technical support please contact Caliper at Telephone E mail Fax 1 877 522 2447 US 1 508 435 9500 tech support caliperls com 1 508 435 3439 Caliper Life Sciences US Corporate Headquarters 940 Winter Street Waltham Massachusetts 02451 USA 4 2 Getting Started Starting the Living Image Software Initializing the System and Checking Temperature on page 7 Overview of Image Acquisition on page 10 Overview of Living Image Tools and Functions on page 12 Managing User Accounts on page 19 Tracking System and User Activity on page 22 2 1 Starting the Living Image Software The Living Image software on the PC workstation that controls the IV IS Spectrum includes both the acquisition and analysis features The Living Image software on other workstations includes only the analysis features For information on installing the software see the Installation Guide included on the Living Image CD ROM Table 2 1 shows the default software installation locations Table 2 1 Living Image software installation locations Living Image Software Operating System Installation Location 32 bit version 32 bit Windows C Program Files Caliper Life Sciences Living Image 64 bit Windows C Program Files x86 Caliper Life Sciences Living Image 64 bit vers
119. Focus lusesubject height v Temperature Click the temperature AN box to display the White System is not initialized demand and measured temperatures Temperature box color Ef Red System is initialized but CCD camera temperature is out of range and not ready for imaging Ka Green System is initialized and CCD camera is at or within acceptable range of the demand temperature and locked The system is ready for imaging NOTE The options available in the control panel depend on the selected imaging mode and the installed filter wheel or lens option For more details on the control panel see Appendix A on page 262 Stage Temperature The stage is temperature controlled to keep subjects warm during imaging The temperature control is enabled after the instrument is powered on and initialized from the Living Image software The default temperature is 37 C and is self monitoring after the system is initialized The imaging stage may be set to a temperature from 20 40 C Figure 2 5 Set the stage temperature in the control panel IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Field of View System Status Acquire E Mouse Imaging Shuttle Demand Measured makababa 3r Imaging Wear BI la li HP spamgiin Pa bade API nma nas GxabraIGiht tapes ses Living Image 4 3 1
120. I so that it becomes a 4 arrow 2 Drag the ROI 3 Release the mouse button when the ROI is properly positioned To move an ROI using the ROI Properties dialog box 1 Double click the ROI in the image The ROI Properties box appears and displays the position and dimensions of the selected ROI Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data 113 Figure 5 20 ROI Properties dialog box CC kO Popeia rika ROI BKG L Z Label BKG 1 Shape Cirde Type Manual Badyomdror B 7 Use as BKG for future ROIs in TLT20050624145507_005 only CO Entire sequence E Lock Position Xo pix 117 00218 Position of the ROI selected in the image Yc pix 97 93839 Angle deg 0 0000 E Lock Size Width pix 22 06161 Height pix 20 43172 Line Size F Line Color aaa l Done 2 To set ROI position enter new coordinates for the center of the ROI Xc pix or cm and Yc pix or cm values in the ROI Properties box 3 To rotate the ROI clockwise enter the degrees in the Angle deg box and click outside the box 4 To lock the current ROI position choose the Lock Position option M NOTE The ROI position cannot be changed until the Lock Position option is cleared Editing ROI Dimensions There are two ways to resize a circle or square ROI m Drag a handle on the ROI m Edit the settings in the ROI Properties box M NOTE You cannot chang
121. I tools select Measurement ROI from the Type drop down list 2 Select the ROI shape a Click the Circle OJ Square OJ or Grid J button The grid shape is useful for drawing a grid of ROIs on an image of a well plate b On the drop down list that appears select the number of ROIs that you want to add to the image or the grid ROI dimensions The ROIs and intensity measurements appear on the image l NOTE Manual ROls are numbered in the order they are created You may want to arrange the ROls in a known order for easier comparison between images To renumber the ROIs ascending order from right to left right click the image and select Sort ROIs on the shortcut menu If the Apply to Sequence option is selected in the ROI tools choose Sort ROIs in Sequence to sort all of the ROIs in the sequence The sort options are only available if the ROls have not been sorted A EG GG a A igure 5 10 Placing two circular ROIs on the image a ba nt a a a a a a a a Ree a a ee ee ee Ya AS a a OS a a OO Ya a O Ya TA O Ya YA a a O a a AS a O OS a a AS a TA O Ya a O Ya a ee a a AS YA AS a YA OS a TA O a a O Ya YA YA TA eed a PAP BP EET E S E ot I TLI20050624145507 006 CE ag Toot Palette Units Counts v Display Overlay ba Options v Info nG ig Luminescence 15000 Auto All Auto 1 10000 Free Draw
122. IT Choose the data to display in the photon density or NTF Efficiency map Images FLIT Intensity Set the maximum intensity of the photon density or NTF Efficiency map using the slider or by entering a value Color Table Color scheme for the photon density or NTF Efficiency map Reverse Choose this option to apply the colors of the selected color table in reverse order For example the Red color table represents the mapped intensity from low to high using a color scale from transparent to red If Reverse is chosen the mapped intensity from low to high is represented using the color scale from red to transparent Log Scale Choose this option to apply a logarithmic scale to the photon density or NTF Efficiency scale 215 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 216 11 12 3D Tools Source Use the Source tools to a Adjust the appearance of sources in DLIT or FLIT reconstructions m Make source measurements page 202 m Export voxel measurements csv Figure 11 31 Source tools and example DLIT reconstruction Tool Palette gt ROI Tools _ Spectral Unmixing and DyCE _ Surface Topography 3D Multi Modality Tools 7 3D Optical Tools A TLT20050624145507_SEQ o amp amp Sequence View LE 3D View Gronal z 15 1 Sagittal x 3 5 Surface Source Registration Select Source Original
123. IVIS Spectrum Chapter 5 ROI Tools for Optical Data Table 5 2 ROI tools for optical images continued Item Description Click to select the number of square ROls to add to the active image Click to specify the grid pattern for a measurement ROI that you want to add to the active image This tool is useful for an image of a multi well culture plate or microplate kai Click and select Auto All to automatically draw ROls in the image using the auto ROI parameters Click and select Auto 1 to automatically draw one ROI at a user selected location using the auto ROI parameters For more details on using the auto ROI features see page 98 Y Measure ROIs Click to display the ROI Measurements table or compute intensity signal in an ROI ng Click to display a drop down list of options to delete an ROI s in the active image For more details see page 117 Note These commands do not delete the ROls that are saved to the system listed in the Menu Name drop down list Apply to Choose this option to apply the selected ROI to all images in a sequence Sequence Type Choose the ROI type from the drop down list Measurement Measures the signal intensity in an area of an image Average Bkg Measures the average signal intensity in a user specified area of the image that is considered background Subject ROI Identifies a subject animal in an image The software automatically associates a
124. Image Adjust tools Item 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 Cmd click for Macintosh users Click this button to incrementally zoom in on the image incrementally magnifies the image ey in the image window 68 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Table 4 4 Image Adjust tools continued Item Description Click this button to magnify the area inside a rectangle that you draw using a click and drag operation Sets the dimensions of the magnified area equal to image window dimensions Click this button to return the image to the default display magnification Click this button to move a magnified image pan in the image window For more details see page 70 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 Photo Adjustment Brightness Click and move the slider left or right to adjust the brightness of an image displayed in overlay or photograph mode Alternatively enter a brightness value Contrast Click and move the slider left or rig
125. Image Math for Opens the Image Math window for the active data Acquisition Background gt Measure Dark Charge Opens a dialog box that enables you to acquire a dark charge measurement Acquisition Background gt Add or Replace Dark Charge Opens a dialog box that enables you to select an instrument luminescent background This background measurement is subtracted from luminescent images 276 Living Image 4 3 1 User s Manual IVIS Spectrum Appendix C Menu Commands Toolbars and Shortcuts Table C 1 Menu bar commands and toolbar buttons continued Menu Bar Command Toolbar Description Button Acquisition Background gt Measure and Replace Dark Charge Measures the dark charge under the same conditions as the currently selected image When the measurement is complete the newly acquired dark charge image will be included in the dataset of the current image replacing any existing dark charge image that may be present in the dataset Acquisition Background gt View Available Dark Charge Opens a dialog box that enables you to view the dark charge measurements for the system Acquisition Background gt Clear Available Dark Charge Clears all dark charge images from the system Acquisition Background gt Auto Background Setup Opens a dialog box that enables you to acquire background images or schedule or disable automatic back
126. Modality Tools 3D Optical Tools Surface Source Registration Select Source Original Ng Display Source Surface i v Maximum pte Projection Threshold 1 33e 1 Gradation lel 50 Voxel Size 0 31 vile Display Voxels As r T r TO ee overwe Transavialfy 10 5 Smoothing 5x5 3 Texture Transaxial y 10 5 7 Color Scale 7 3 2 mm m gi Min f mi pm 8 U ese tam olor Table Blue Yellow x j T Reverse laf P Log Scale EE REE Subject Height 26 3 mm Key Value Quantification 0 00 photons sec Volume 0 00 mm43 Depth 0 00 mm Center of Mass 0 00 0 00 0 00 Host Organ Unknown gt DUT 3D Reconstruction L Subject Height 26 3 mm Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 204 Viewing Location Coordinates Click a location in the reconstruction slice in the Coronal Sagittal or Transaxial windowpane The coordinates mm of the position are displayed Figure 11 20 The coordinates are updated when you press and hold the mouse button while you drag the cursor Slice Plane Displays Coronal The x y coordinates of a position Sagittal The y z coordinates of a position Transaxial The x z coordinates of a position Figure 11 20 Viewing y z coordinates in the sagittal plane Saqgithalfx 3 5 Displ
127. No C 1 50 1 1 4 PJ auto Medium 1 Block 620 No Field of View System Status s E auto Medium 1 Block 640 No Acquire Sequence O ms 2 8 cm Imaging Wizard Subject height 1 50 cm Image Setup Focus use subject height w Temperature NAAN Locked C Number of Segments 1 min Apply to All A Update Insert Add 6 To clear the sequence click the Remove button and select All 44 Living Image 4 3 1 User s Manual IVIS Spectrum Acquire the Sequence Chapter 3 Image Acquisition 1 Confirm that the IVIS Spectrum is initialized and the CCD temperature is locked See page 7 for details Click Acquire Sequence in the control panel when ready to begin acquisition Enter information about the image in the Edit Image Labels box that appears optional Click OK Figure 3 26 l NOTE You can enter image label information at any time during or after acquisition Click Cancel if you do not want to enter image information Figure 3 26 Enter information to include with the image optional 7 Edit Image Labels UserID ary Living Image Universal Saved Labels LABELS_1 Ab User 7 Group Information entered here appears in the image label see Figure 3 28 on page 46 V Experiment 87 MG uc2 Intracranial Implantation v Male nu nu day 8 Mouse 4 Spectrum CT v Commenti v Comment2 v Time Point V Animal Numb
128. Overlay 1 425e 06 4 167e 02 6 726e 02 8 462e 00 2 545e 03 3420 2 189e 05 4 141e 02 1 020e 03 2 87 HX20070420121444 001 ROI2 Overlay 1 489e 06 3 309e 02 6 061e 02 8 642e 00 2 545e 03 4500 2 880e 05 4 168e 02 9 001e 02 2 822 HX20070420121444 001 ROI3 Overlay 1 676e 06 3 637e 02 5 988e 02 1 056e 01 2 545e 03 4608 2 949e 05 4 168e 02 1 152e 03 2 846 HX20070420121444_002 ROI1 Overlay 7 261e 06 2 123e 03 3 445e 03 2 181e 01 1 031e 04 3420 2 189e 05 4 141e 02 1 020e 03 2 87 HX20070420121444 002 ROI2 Overlay 7 572e 06 1 683e 03 3 107e 03 2 181e 01 1 031e 04 4500 2 880e 05 4 168e 02 9 00le 02 2 82 _ 4 LU r Customized Selections Measurements Types Image Attributes ROI Dimensions Copy Select All Counts v All Possible Values x Pixels M Configure Export Close 3 Make a selection from the Image Attributes drop down list to include image information in the ROI table 4 Select units Pixels or cm from the ROI Dimensions drop down list to include ROI dimensions in the table Creating a Custom ROI Table Configuration A table configuration specifies the column headers in the ROI table Several preset configurations are available selected from the Measurements Types drop down list in the ROI table Figure 5 29 You can also create a custom table configuration l NOTE Preset table configurations cannot be edited You can modify a preset configuration and save it to a new name 1 In the ROI
129. PatientsBirthDate 20101018 4 IN p Save Results Name MULTIMODALITY_7 v Delete Load Save M NOTE Registration information is saved with the results for the volumetric data and is specific for a particular optical data set Manual Registration To manually register data use the 3D Multi Modality tools to translate scale or rotate the 3D volumetric surface so that features common to both surfaces are matched and aligned in the x y and z planes Examine the matched surfaces in the 3D slice views to help you fine tune the registration 255 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools 8 256 Figure 13 19 Example surfaces before and after registration 3D volumetric surface 3D structured light surface Surfaces before registration Registered surfaces To manually register data 1 Load the data that you want to register for more details see page 252 The software determines a default air noise boundary for the 3D volumetric data Figure 13 20 If you need to remove noise from the 3D volumetric data move the air noise boundary to the right in the histogram tool Figure 13 20 Adjusting the air noise boundary in the histogram tool Default air noise Example noise in boundary the 3D volumetric data Adjust the air noise boundary to reduce noise in the 3D volumetric data Reduced noise 3 If the volumetric data nee
130. Press the Tab key to switch between the transformations tools d Turn off the transform tool when you finish positioning the ROI click the 3D ROI Transform button x Figure 6 4 3D ROI transformation tools Click and drag the 3D ROI when the yellow appears Click and drag a handle to scale increase or decrease the ROI size Red W Scales on the z axis Blue W Scales on the x axis Green W Scales on the y axis To rotate the 3D ROI on the x y or z axis click the blue green or red circle and drag the mouse arrowin the direction of interest NOTE The 3D ROI location x y or z coordinates and dimensions width height or depth can be viewed and modified in the 3D ROI Properties dialog box See page 128 for details 4 Click the 3D ROI Measurement button W s jn the tool palette to view the intensity measurements Figure 6 5 Figure 6 5 3D ROI measurements Name 3D ROL 3 KSA x E Spectral Unmbang and Dy 2 30 multi modality Tools 3D opticalToos DD ROI Measurements 3D ROI Measurements Data Types 3D Volumetric Data v Measurement Types A Refresh Sequence Number ROI Voxels Total Counts Average Counts Min Counts Max Counts BI20111027132749 SEQ ROI1 54015 6 668e 08 1 234e 04 0 000e 00 5 500e 04 126 Living Image 4 3 Software User s Manual Chapter 6 3D ROI Tools for Volumetric Data 127 Table 6 1 3D ROI Measurements tabl
131. Printing Images AK 5565550486665 KAKA gee eee he NG 85 Editing an Image Sequence ees 87 Creating an Image Sequence from Individual Images 88 ROI Tools for Optical Data 04 0a es 90 ADOUCROIS 2bacsvagtune se adie ed LOW AHE NLA DAN RW kee eke LAAN oe oso 90 Quick Guide Drawing Measurement ROls on an Optical Image or Sequence 93 ROI Tools for Optical IMAGES 4a a GB BAWA Wa KA KAN WG KG KA 95 Measurement ROIS 0 ee ee eee eee 97 Drawing Measurement ROIs Automatically 2 00 cee ee ees 98 Drawing Measurement ROIs Manually 0 000 eee eee eee 100 Drawing ROIs Using the Free Draw Method 101 NITTO THOUS om hoes 5s ooo cece oe bce gee eo oe A Be ae Oe bn AAO eG 102 Measuring Background Corrected Signal 2 200 cece ee eee eee 105 SUDICCUINOIG serere nang aai kama Khim se beeen enn AG AA es eee es 105 ROI Histogram 22 684 entender AWA GRABA LL KA ends ced aah es wears 108 Managing ROI Properties 0c ccc ete teens 109 Viewing ROI Properties 0 000 ce eee 109 Moving an ROI ERE PAY 112 Editing ROI DimensionS 2aaha a Gam kh KAAGAD SE MAAN Bk MG Bhd Ah WG 113 Save Load or Delete ROIS 27 0 xa 6554282 bu 05440 eek wee DD KK AWA ee wes 116 Managing the ROI Measurements Table 00 aa 119 Viewing the ROI Measurements Table 00 eee eee eee 119 Configuring the ROI Measurements Table
132. SEQ Cd PED bi aan apa a N E MTS KO ny Traser tests Soruff and abdominal D 25 1120040922022427 EQ i i Fucrescerk Vackground Tests 2 Female Nulu mic tee 11720030206124438 EX Fite EM Fitter Hummakion Mode User ID Sete DsRed OsRedn Belin tive PTIR Rudy PKHDG init PTIR273 ng 4 TLT 200506724145907 Sta osa Prvan Labot Set A Locations Pihikan pubic NN igea TampleDak af AukoPhaorking Cor TITINDANITIN92427 SEQ Seganala kit Tool Palette Pn e r S Ca Table 4 1 Living Image Browser Item Description Hide Browse View Closes the browser table Close Preview Closes the image preview box Label Set A drop down list of the available label sets which specify image information column headers that is displayed in the Living Image Browser Add to List If this option is chosen the data selected in the Browse for Folder box is added to the Living Image Browser If this option is not chosen the data selected in the Browse for Folder box replaces the contents of the Living Image Browser except for the loaded data Browse Opens the Browse For Folder box View The name of the Living Image Browser configuration the column headers and their order in the browser Configure Opens a dialog box that enables you create and save custom Living Image Browser configurations Note To reorder a column in the browser click the column header then press the mous
133. Show Labels Y Individual Scale NG amp ig sey ani Dad dte V Normalized T Legend a aj TissueAF j 3 v 1 00 E Emission Guided g AnalysisBinning 8 E ImageWidth 240 0 50 ImageHeight 240 DataUnit Radiant Efficiency SPUM_Ver 2 0 Min 0 00 RA Min 0 00 LivingImage 4 3 1 0 15582 o0 Max 9 91e7 ax 6 5268 660 700 740 730 4F750 Composite Emission Wavelength nm Spectrum List Save Results ol apy x Name SPUM_4 Note Select Name Color Pick 1 V TissueAF Dime A dag 2 V AF680 rec z 3 V AF750 Wue ae Min 0 00 ax 2 3768 157 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing 158 Items in the Results Tab Description Name The name for the active spectral unmixing results Select results from this drop down list Delete Deletes the selected results Load Opens the selected results in the Unmixing window Save Saves the active results using the selected name The results are saved to the sequence click number folder and are available in the Name drop down list Overwrite If you reanalyze results saves the new results and overwrites the previous results 9 DyCE Imaging and Analysis About DyCE Dynamic Contrast Enhancement Acquire a DyCE Sequence on page 160 DyCE Analysis on page 166 DyCE Results on page 172 9 1 About DyCE Dynamic Contrast Enhancement M NOTE The DyCE acquisition and analysis feature
134. Spectrum List Note Select Name Color 1 7 TissueaF B ime 2 V AF680 E 3 0 AF750 We 660 700 740 780 oe by x T Hemann KO paka CI Sequence vew Unm Composite J AF680 T ARD J Photograph Min 0 00 ax 6 4668 image Adast Unghiress y s Logaritiwac Scale Label TasurAF Table 8 2 Composite window Item Description Units The type of data displayed in the composite image Image list A list of the images that comprise the composite background component s probe s and a photograph Min Max Sets the minimum and maximum count to display in the image Brightness Adjusts the brightness of the component signals Logarithmic Scale Choose this option to display signals using a logarithmic scale This may be useful when probe signal strengths differ significantly for example a bright source and a dim source Color Shows the color of the figure legend for the image selected in the image list Click the color swatch to open a color palette that enables you to select a new color for the figure legend Label The name of the image selected in the image list To edit the name double click the name in this box Right click the label name to show a short cut menu of edit commands for example Cut Copy Paste Sends the composite image to the top of the image cube This helps improve the pseudo color visualization of the image cube Copies the
135. T algorithm select Help s Tech Notes on the menu bar 3D Reconstruction Description See Page Algorithm Diffuse Tomography DLIT provides a complete 3D reconstruction of the luminescent 188 DLIT source distribution within the subject DLIT places no constraints on the geometry or spatial variation of the source strength throughout the volume DLIT is well suited for analyzing complex and spatially extended luminescent sources The 3D reconstruction is presented as voxels If a luminescent quantification database is available the number of cells per source can be determined in addition to source intensity photons sec Fluorescent Tomography FLIT provides a complete 3D reconstruction of the fluorescent 194 FLIT source distribution within the subject The 3D reconstruction is presented as voxels If a fluorescent quantification database is available the number of fluorophore molecules or cells per source can be determined in addition to the total fluorescence yield Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 185 Reconstruction Inputs M NOTE Use the Imaging Wizard to set up the DLIT or FLIT image sequence See page 42 for more details DLIT The input data to the DLIT algorithm for a 3D reconstruction of luminescent light sources includes a A surface topography of the subject generated from a a structured light image m A sequence of two or more ima
136. T or FLIT results Load Opens the selected reconstruction results in the 3D View 198 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Item in the DLIT 3D Reconstruction Results Tab Description Save Saves the active DLIT or FLIT results to the selected name The results are saved to the sequence click number folder and are available in the Name drop down list Overwrite If you reanalyze saved results saves the new results and overwrites the previous results Export Results Saves the results to a csv file Copying Results to the System Clipboard 1 To copy all results a Right click the results and chose Select All from the shortcut menu b Right click the results again and select Copy from the shortcut menu Figure 11 15 Select and copy results Tool Palette _ ROI Tools gt Spectral Unmixing and DyCE lo _ Surface Topography gt 3D Multi Modality Tools gt 3D Optical Tools DLIT 3D Reconstruction Analyze Properties Results DLIT Results DLIT_1 Loaded Key Value Final voxel size mm 1 75 Number of voxels 34 Reduced Chi L71e 0 Starting vsiz Copy Kappa best Nsurf best Select All Total surf sa Threshold a Export Results m Kappa limits 0 50 4 00 Nsurface limits 200 00 20 00
137. T or FLIT sequence using the Imaging Wizard see page 45 Acquire and load the sequence 2 Generate or load a surface using the Surface Topography tools See Chapter 10 on page 177 for more details Tool Palette Tog Palette gt Image Adjust lel sa Tania Al jet H Corrections Filtering l2 2 Corrections Filtering i gt ROI Tools gt ROL Took 3 Inthe DLIT or FLIT 3D Reconstruction tools 2 Spectral Unasising and DyCE eane Daere mad INE ll Surface Topography m Ha lace lopagraghey select the 30 Mul Hodaity Tools i 30 Haiti Modality Toots Wavelengths or excitation point images to me an ng Er Sequence 5720111027132749_SEQ Tagos Properties Mouse Tignan ana yze Select ites ping T l irie paiia Prti m Tissue and source properties enn voor 62 68 4 Reconstruct sources Psv s See page 188 for detailed DLIT steps See page 194 for detailed FLIT steps NNS o v Start E 11720080624145507 SEQ DA Sequence View lt WD View 5 View source measurements see page 202 Sabie Heidi 263 ten Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 187 11 2 Reconstructing Luminescent Sources General Considerations Animal Requirements The best surface topography reconstruction is obtained from nude mice It is possible to perform 3D imaging on white or light colored furred mice if the fur
138. TE Use only the Single Mouse Anesthesia Manifold when imaging with transillumination The Dual Mouse or Five Mouse manifolds cannot be used with transillumination el 1 Puta check next to Fluorescent and Transillumination in the control panel M NOTE The Normalization option is selected by default so that NTF Efficiency images can be produced Figure 3 15 Control panel IVIS Acquisition Control Panel 1 00 Se w vedun wfe v Field of View System Status CJ ms Subject height 150 lem Focus use subject height w Temperature ANN Locked 2 Select an excitation and emission filter from the drop down lists The instrument has 18 narrow band excitation filters that span 490 850nm with a 20nm bandwidth enabling spectral scanning over the blue to NIR wavelength region Figure 3 16 36 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition 37 Figure 3 16 IVIS Spectrum excitation and emission filters Excitation Filters Transmission 96 Wavelength nm Emission Filters 550 600 750 800 Wavelength nm Transmission 3 Click Setup Click Yes if prompted to acquire a subject photograph 4 Choose the location click a square for transillumination and image acquisition in the Transillumination Setup box that appears Figure 3 17 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition 38 F
139. Tools which are used to modify the source display parameters Load data in new Ifthis option is selected DICOM data are opened in a new 3D View window when you If this option is not selected DICOM data are loaded in the active 3D View window custom animations and record an animation to a movie file 3D Tools Functions See Page Surface Tools Adjust the appearance of the reconstructed animal surface and See below photon density or NTF Efficiency maps Source Tools Adjust the appearance of reconstructed sources make source 216 measurements export voxel measurements Registration Tools Display organs on the reconstructed surface adjust the location or 218 scale of organs on the surface import an organ atlas Animate Tools Display preset animations of the 3D View scene Enables you to create 225 213 Living Image 4 3 1 User s Manual IVIS Spectrum 11 11 3D Tools Surface Use the Surface tools to adjust the appearance of the reconstructed animal surface and photon density maps Chapter 11 3D Reconstruction of Sources Figure 11 30 Surface tools and example DLIT reconstruction with photon density or NTF Efficiency maps ree C fsa A TLT20050624145507_SEQ Sequence View EL 3D View ty KG Be Y Y ED ig Sagittal x 3 5 Cronal z 15 1 x Y Transaxial y 22 8 Subject Height 26 2 mm Tool Palette gt ROI Tools gt Spect
140. Type Dye molecules Cells Measurement Measurement Sample Wells 3D 3A C Set 6 Sample Wells 3D 3A C Set 7 Background Wells 6D 6A 7 Background Wells 6D 6A V Apply to Sequence V Apply to Sequence WellPlate Quantification Plots Results Well Plate Quantification Plots Results Cick a pe Pie Quentifeotion Results Unsaved Excitation Emission Extinction Coeff Cross Section 5 nm nm Qe M cm 1000 Qo mm pr aaa aa pren ii a a 165 520 2919exo7 111508 2 465 540 1 262e 07 4 821e 09 3 465 560 5 894e 06 2 251e 09 4 465 580 2 230e 06 8 518e 10 4 2 o o Sequence Database A xl 0 0 1 0 20 3 0 40 50 Name WPQUANT_1 w Name WPQUANT_1 v well plate population Delete Load Delete Load 16 Check the linear fit of the data for each image in the quantification plot A good fit to the straight line gives confidence to the results values Large deviations of individual points from a straight line could indicate possible issues with the dilution series or errors when entering sample dilution values 17 To export the quantification plot values a Click the g button b In the dialog box that appears select a folder for the file csv and click Save 18 To copy the quantification plot values to the system clipboard click the button Table 12 1 Quantification results Item Description Fluorescence Excitation nm The excitation and emission filter wavelengths for the
141. VIS Spectrum Chapter 5 ROI Tools for Optical Data Table 5 7 ROI Measurements table continued Item Description Refresh Updates the ROI Measurements table for example after you draw new ROls move an ROI and close or open image data Configure Displays the Configure Measurements box that enables you to specify and organize the data categories column headers for the table Export Displays the Save Measurements box so that the data can be saved to a txt or csv file Note Grid ROI measurements exported to a csv file can be opened in a spreadsheet application like Microsoft Excel Close Closes the ROI Measurements table Configuring the ROI Measurements Table You can customize the data and information column headers in the ROI Measurements table Several preset categories are available in the Measurement Types Click Attributes and ROI Dimensions drop down lists 1 Drag acolumn header left or right in the table to reorder the columns 2 Make a selection from the Measurement Types drop down list to change the measurement units Figure 5 29 ROI Measurements table 7 ROI Measurements o 23 ROI Measurements Grid ROI Measurements Refresh Image Number ROI Image Laye Total Coun Avg Count Stdev Cour Min Count Max Count ROI Pixels Area Xe Ye Widt ccd Pixels pixels pixels pixe HX20070420121444 001 ROI1
142. Wizard C MIS Exposure Time Auto sec v Medium Y Field of View Service 12 8 IVIS Acquisition Control Panel System Status Idle cm Subject height 1 50 A S cm Focus use subject height w Temperature Locked Excitation Filter Emission Filter Imaging Wizard Sequence Setup E brgy Piccard ae magang Hode Bipkumi nao ian Calact the opie for imaging bolannawani o Bephorerericnene Chimki nata Gch ad feet lucerne cik beste ioes pani w bacteri licence If this screen does not appear when the wizard starts click Restart Wizard on the wizard screen to restart the wizard 4 Click Next in the wizard and choose the type of image sequence to acquire See Table 3 6 and Table 3 7 on page 43 for more information on the imaging options Figure 3 24 Choose the type of image sequence Open Filter Spectral unmixing Imaging Wizard Bioluminescence options Imaging Wizard Bioluminescence Bioluminescence Open Filter For bioluminescent imaging an open filter image gives the most sensitivity since none of the emission light is being filtered This measurement technique is the most commonly used bioluminescent measurement Imaging Wizard Fluorescence options j Imaging Wizard Fluorescence ilter Pair Spectral Unmixing 4 Filter Scan Fluorescence Filter Pair Imaging T
143. Working With Optical Image Data Open an image and in the Image Information tools click the Image Crop button Figure 4 23 Using a crop box to make measurements Image X Y 12051 11 831 an Image Data 15 counts 15000 Crop Distance papa a AE a 6 81 8 10 1 62 1 48 Distance 2 19 am 10000 5000 Counts Color Scale Min 1122 Max 19546 Too Palette Ca YU YY images oo Wo Counts v Display Overlay Options smo iy ic gt Corrections Filtering lal a AB FE units cm x Image Binning amp Width 12 6 cm Height 12 6 cm 2 When the mouse pointer changes to a draw a rectangle on the area of interest 3 To change the size or position of the crop box drag a handle at a corner or side of the box 4 To delete the crop box from the image click the 77 button Table 4 11 Crop box position and dimensions Item Description x y coordinates at the upper left corner of the box x y coordinates of lower right corner of the box Box width and height Distance Length of the diagonal from the upper left to lower right corner of the box 79 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 80 4 8 Creating a Transillumination Overview The transillumination overview tool combines the images of a FLIT sequence a fluo
144. Yr Caliper Living Image Software User s Manual Version 4 3 1 For the IVIS Spectrum 2012 Caliper Corporation All rights reserved PN CLS5135291 Caliper Life Sciences US Corporate Headquarters 940 Winter Street Waltham Massachusetts 02451 USA 1 877 522 2447 US 1 508 435 9500 Fax 1 508 435 3439 E mail tech support caliperls com Discovery in the Living Organism IVIS Imaging System Living Image DLIT and FLIT are either registered trademarks or trademarks of Caliper Life Sciences Inc The names of companies and products mentioned herein may be the trademarks of their respective owners Apple Macintosh and QuickTime are registered trademarks of Apple Computer Inc Contents Chapter 1 Chapter 2 Chapter 3 Chapter 4 WOIGOIMG a aa Sede he ee ERS ERE Se ee oO ee es ee oa ee esas 1 About this Manual ssm si Rodos oe Geese oe MA KA AA ee Ae ee ees a 1 What s New in the Living Image 4 3 1 Software 0 000 c eee ees 2 Living image Help 2 448 cucaeewed Tan Geet et bA aan nE Rea eed one PEE WA NANAMAN 3 Caliper Technical Support 0 iiiten gawa od oe Ga we a ee ee ee 4 Getting Started 0 2 02 ee es 5 Starting the Living Image Software 0 ccc eas 5 Initializing the System and Checking Temperature 7 CCD Temperature cap cA 8 Ra 4 KA DAA SoS Ow Ew wD OA BKA DOGO ee 8 Stage Temperature 0 cc ees 9 Overview of Image ACQUISITION 2 0 0 eee e
145. a See the concept tech note Image Display and Measurement for more details on color tables select Help Tech Notes on the menu bar Reverse Choose this option to reverse the selected color table Logarithmic Choose this option to apply a log scale to the relationship between numerical data and the color range in the color table A log scale improves the visibility of dark areas in an image Palette label To include a brief line of text next to the color scale enter text in the palette label box then press the Enter key To remove the text from the image window delete the text in the palette label box and press Enter Scales per Column Sets the number of color scales to display in a column 83 Living Image 4 3 1 User s Manual IVIS Spectrum 4 10 Rendering Intensity Data in Color Chapter 4 Working With Optical Image Data The colorize tool renders luminescence or fluorescence data in color enabling you to see both intensity and spectral information in a single view The tool provides a useful way to visualize multiple probes or scale probe signals that are not in the visible range To view colorized intensity data 1 Load an image sequence Figure 4 28 Microplate images Images were acquired using different combinations of excitation and emission filters The samples are quantum dot nanocrystals 700 or 800 nm File Edit View Tools Acquisition Window Help o amp aa R Units
146. a colorize 84 85 open 59 image histogram 74 image layout window 86 87 image math 133 135 image overlay tool 81 83 image sequence create from individual images 88 89 edit 87 88 image window 61 imaging acquire a sequence 42 48 acquire a sequence batch mode 48 49 acquire a sequence manual 50 53 fluorescent epi illumination 29 35 fluorescent transillumination 35 41 fluorescent quick guide epi illumination 30 luminescent 23 29 luminescent quick guide 24 imaging modes 11 12 import organ atlas 223 Index 280 surface 183 information about an image 64 L line profile 75 Living Image browser 58 Longitudinal Study window 206 luminescence reconstruct 3D sources 187 193 luminescent imaging 23 29 quick guide 24 M manual focusing 265 266 maximum intensity projection 244 measurement ROI automatically draw 98 99 measurement ROIs 97 99 free draw 101 measurements 78 mirror ROI 102 104 Mouse Imaging Shuttle 254 multiple reporters per photograph 81 83 N NTF Efficiency 120 262 O open image data 59 optical image data browse 55 59 organ atlas import 223 organ display 220 223 organ registration tools 218 224 overlaying images 81 83 P photon density 199 photon density map measured 201 simulated 201 photon density maps 200 preferences 267 274 print images 85 87 O quantification database 231 236 create 232 235 manage results 236 samples 231 R RAW volumetric data 259 261
147. a mask on a particular area Select the Data Mask option and the using the mouse Rectangle or Ellipse option Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing Table 8 2 Data mask options Option Description Photograph If this option is chosen the software automatically draws the data mask so that it includes the entire photograph Threshold If necessary use the threshold slider or we arrows to adjust the mask so that it matches the underlying subject photograph as closely as possible without including any area outside the subject image Draw Mask Choose this option to manually draw a data mask on an area of the photograph Rectangle Specifies a rectangular shape for the manual data mask Ellipse Specifies an elliptical shape for the manual data mask 5 Choose an imaging subject and background signal s Figure 8 12 Auto Unmix window p TE Hx20120419114703_SEQ Ser x Sequence View Auto Unmix ks Select Image Mask Choose Components To Unmix Tips 8 wavelength pairs selected Choose the number of components to unmix Pick the significant background signals first then add the probe information to the table if it is not listed If you are unclear about the probe or it is not in the library choose Unknown Imaging Subject Mouse v Select a su bj ect Background Signals 7 Tissue Autofluo
148. a yee 54 Working With Optical Image Data 0 2a 55 Loading Optical Image Data ak bA NG kA KA KAG KALMA KAKA NG MAA 55 Loading Optical Images From the Living Image Browser 55 Living Image 4 3 1 User s Manual IVIS Spectrum Contents Chapter 5 Opening Data from the Menu or Toolbar 4 eee eee es 59 Organizing ImageS APA AA AA 60 About the Image Window and Tool Palette 2 cee eee eee 61 Image Window 22 24 amma be SAGES bebe abe wee eee twee bH4 ba beeen ds 61 Tool Palette REA ee ee ee a a ee a ee 63 Viewing Image Information cee ee 64 Editing the Image Label 00 aaa 65 Adding Comments or Tags to an Image ee 66 Adding COMMERIS 2x tccitsenr ese BIDENERRLD ene see beebse ceweeess 66 tagging am iMag 246265665 AG be ten ee ewes sob SS LAMA NBA HG KAG Soon ew 67 Adjusting Image Appearance 2 eee 68 Correcting Optical Image Data 0 0 cc 70 Viewing Intensity Data and Making Measurements 72 Image Histogram 4 55 64 e086 od re oe a a oe eee ee oe ee 74 LING PrOUle sa2avenuee ede deeeeneaetse bac NMEN DAMA PANA Bee oes 75 Viewing 3D Signal Intensity 0 0c eee 77 Making Measurements 2 ees 78 Creating a Transillumination Overview saana anaana annann 80 Overlaying Multiple Images 0c ccc eee ee eee eee 81 Rendering Intensity Data in Color 0 0 00 ccc eens 84 Exporting or
149. age TLT20050624145507 005 Series Male Nn nu Fri ng si cog bone 56 46 a R Experiment DOB 03 21 05 Em Filter 640 Bin M 8 FOV 12 6 f2 1s Label kidney Camera IVIS 200 Beta II SI620EEV Comment dorsal Luminescence 10000 8000 6000 4000 2000 Counts Color Scale Min 586 Max 10348 Edit an entry For example revise the comment 3 Edit the label information To add information to the image label 1 Click the H toolbar button Alternatively select Edit Image Labels on the menu bar 2 In the Edit Image Labels box that appears select information and or enter a comment Figure 4 11 M NOTE If a single image is active changes are applied to that image only If a sequence is active changes are applied to each image of the sequence 65 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 66 Figure 4 11 Edit Image Labels E Edit Image Labels x UserID B Living Image Universal Saved Labels LABELS 1 Ab V User X v Group v Experiment 87 MG uc2 Intracranial Implantation v Male nu nu day 8 Mouse 24 V Commenti es v T Comment2 v 7 Time Point x V Animal Number 4 T Animal Strain F Animal Model E Sex v M View Na Cell Line X Reporter Treatment v Luc Injection Time IACUC Number m 3 Click OK when finished The im
150. age software oe initialize the IVIS Spectrum page 7 Note See the V S Spectrum Hardware Manual part no PN121450 Rev00 for more information on the instrument 2 Place the anesthetized subjects in the imaging chamber and close the door 3 Puta check mark next to Luminescent and select Auto exposure 4 Choose Photograph optional Selecting Photograph automatically selects Overlay 5 Select Use subject height and enter the height in centimeters O mis 6 Click Acquire Service Field of view Subject height cm Excitation Filter P i F Stop TE Emission Filter System Status Acquire Imaging Wizard Sequence Setup Focus luse subject height v Temperature M Locked Image Window E EF TLT20050624145507_001 ALIES Options Info ify ig Units Counts v Display Overlay X Luminescence 3000 2000 1000 Counts Color Scale See Table 3 2 on page 28 for more details on the image window 7 When prompted select a location for the image data optional Image data acquired during the session will be automatically saved to this location 8 Enter experiment and subject information in the dialog box that appears optional The image window and tool palette appear when acquisition is finished Tool Palette 3 Tool Palette The Tool Palette includes the Image Adjust
151. age information is updated 4 Save the image to save the updated image label select File Save or File Save As on the menu bar 4 4 Adding Comments or Tags to an Image Adding Comments Comments can be added to an image and saved with the image 1 Open an image 2 Right click the image and select Insert Comment on the shortcut menu Enter comments in the yellow box that appears Figure 4 12 To reposition a comment 1 Position the mouse pointer over the comment Th 2 When the hand tool appears use a click and drag operation to move the comment box then click the mouse to set the location To remove a comment s a To remove a comment right click the comment and select Remove Comment on the shortcut menu a To remove all comments right click the image and select Remove All Comments on the shortcut menu Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Figure 4 12 Add a comment to an image fo TE TUT20050624145507 006 v Display Overlay v See Info nG KU Units Counts Options z Insert Tag Luminescence Insert Comment N Remove Tag Remove Comment Remove All Tags 15000 Remove All Comments C opy All ROIs Paste ROI 10000 Hide ROI Tags Delete All ROIs Sort ROIs 5000 Zoom Area Zoom In Zoom Out Counts Reset Zoom Color Scale Min 1122 Max 19546 PETE NN NAAN ge oe TU TLI200
152. ages in a sequence Default Dynamic or Film image Strip For example here is Film Strip mode sequence TE TUT20050624145807 seo O sequence View Spectra Units Counts E use saved Colors options 7 sino a Layout gt Default SortBy gt i Display gt Labels Sort by Options for ordering images in the sequence window a Default Order in which the images are stored in the folder TimeStamp Ascending order of the image acquisition time a UserID Ascending alphanumeric order of the user ID Display Choose the types of information to display with each image TE Ter200s06241ass07 seo Hla O Sequence View Ceca units Coan Z Dies aai oF iy ms Duplay gt y Epose Time N Labels y Binning Factor Excitation filter Emiswon fiter 1 Number Field of View Select All Clear AS In this example exposure time and binning factor are displayed on each image C7 TUI20050624145507 005 Cleo las Units Counts v Display Overlay v Cano aQ Image TLT20050624145507 005 Series Male Nn nu Fri Jun 24 2005 07 56 46 Experiment DOB 03 21 05 Em Filter 640 Bin M 8 FOV 12 6 f2 1s lab ab Ima ge label Camera IVIS 200 Beta II SI620EEV Comment dorsal Luminescence 10000 8000 6000 4000 2000 Counts Color Scale Min 586 Max 10348 Info Click to show or hide the image label The image label includes information you enter in
153. aging Animation Setups To save an animation setup 1 Click Save 2 Select a directory and enter a file name xkf in the dialog box that appears To record the animation to a movie 1 Click Record 2 Choose a directory enter a file name mov mp4 avi and click Save in the dialog box that appears To edit an animation setup 1 Open an image sequence and load a reconstruction 2 Open an animation setup To select a predefined setup make a selection from the Preset drop down list To select a saved user defined setup a Click Load b Select an animation setup xkf in the dialog box that appears Figure 11 42 List of key frames in the selected animation KG 7 3DAnimation ES Preset Animations Presets Spin CW on Y Axis x Frame Factor 1 Key Frame 2 Key Frame 3 Key Frame 4 Key Frame 5 3 To add a key frame Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 230 a Aa the position of the reconstruction in the 3D view using an image tool for example 47 or 4 For more details on the image tools see page 190 b Click the button c To reorder a key frame in the sequence select the key frame and click the or FF arrow To update a key frame a Select the key frame and adjust the 3D view b Click the J button To delete a key frame a Select the key frame that you want to remove b Click the am
154. ails on the image tools see page 190 7 Click the fl button The second key frame is added to the key frame box Figure 11 41 Example key frames for a custom animation 3DAnimation o Preset Animations Presets Spin CCW on X Axis Frame Factor 1 Animation Setup Time Scale 0 Key Frame 1 i Key Frame 2 Key Frame 3 Key Framed ee Key Frame 1 t F Play Record Frames Per Second 10 Total Duration secs 5 Load Save Key Frame 3 Key Frame 2 Key Frame 4 8 Repeat step 6 to step 7 until all of the key frames are captured For details on how to edit the key frame sequence see page 229 228 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 229 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 9 Confirm the defaults for FPS frames per second and Total Duration length of animation or enter new values FPS x Total Duration No of frames generated to create the animation The number of generated frames should be to the number of key frames Otherwise the frames may not be properly animated 10 To view the animation click Play To stop the animation click Stop An animation setup series of key frames can be saved xkf or recorded to a movie mov mp4 avi mpg Man
155. al Unmixing Results G A a r 14 HX20120419114703_SEQ O Sequence View Units Radiant Efficiency M F Use Saved Colors Options z Info PB 3 Y NG Ko Item Value Min 1 25e7 Mins 7 16e6 Min 1 41e7 Mans 1 00e8 42 Maxs 1 39e8 P3 Max 2 71e8 gt Select the results and click Load Save Results Name SPUM_Auto Min 9 4966 Min 6 50e6 om 1 55 0368 Max 1 0168 6 Max 2 8 Bis Delete Load 3 Click the Analyze tab of the Spectral Unmixing and DyCE tools All wavelengths are selected by default Remove the check mark next to wavelengths that you want to exclude from the analysis In Figure 8 16 the fluorophores are Alexa Fluor 680 and Alexa Fluor 750 Images were acquired using a 605 nm excitation filter and emission filters from 660 to 800 nm in 20 nm increments 149 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing Figure 8 16 Unmixing window showing loaded results T HX20120419114703_SEQ fo Sequence View Unmixing Tool Palette Spectral Unmixing and DyCE Analyze Results Show Labels V Individual Scale nG 3 Select Images Type Emission V Normalized T Legend lo TissueAF AF680 Excitati on o g 7 wavelength GI 1 00 E 7 v F J Emission E 73 wavelengths of 7 the sequence v v 0 0 Min 0 00
156. ameters and takes another image In most cases the default auto exposure settings provide a good luminescent or fluorescent image However you can modify the auto exposure preferences to meet your needs See page 270 for more details Living Image 4 3 1 User s Manual IVIS Spectrum Imaging Modes on the IVIS Spectrum Chapter 2 Getting Started Table 2 1 briefly explains the types of images that can be acquired on the IVIS Spectrum Table 2 1 Imaging modes on the IVIS Spectrum Imaging Mode Description Example Luminescent optical imaging A longer exposure of the subject taken in darkness to capture low level luminescence emission from the surface of the subject The optical luminescent image data is displayed in pseudocolor that represents intensity ra ma ay Lee 5 Optom E retwomenyesser o gt IPAD ors m Luminescent image Overlay Luminescent image on photograph 11 Living Image 4 3 1 User s Manual IVIS Spectrum Table 2 1 Imaging modes on the IVIS Spectrum continued Chapter 2 Getting Started Imaging Mode Description Example Fluorescent optical imaging An exposure of the subject illuminated by filtered light The light source is located above the stage epi illumination The target fluorophore emission is captured and focused on the CCD camera The optical fluorescent image data can be displayed in units of counts or photons
157. anes as you move the line Figure 11 22 Moving the transaxial plane Transazialfy 22 0 Subject Height 26 2 mm 11 7 Viewing Luminescent and Fluorescent Sources in One Surface When an experiment includes luminescent and fluorescent reporters DLIT and FLIT reconstructions can be displayed in one surface if the luminescent and fluorescent imaging is done in the same imaging session without moving the animal M NOTE If the DLIT and FLIT image sequences are acquired during the same session the generated surfaces are nearly identical 1 Load a DLIT reconstruction and a FLIT reconstruction 2 Choose one of the reconstructions click the Ey button and select Copy source voxels 3 In the other reconstruction click the button and choose Paste source voxels 205 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 206 NOTE Pasted voxels can be measured For more details on measuring sources see page 202 ell 11 8 Comparing Reconstruction Results Multiple DLIT or FLIT reconstruction results can be viewed side by side in the Longitudinal Study window Voxel intensity within the entire surface or a user selected area can be measured in all results in the Longitudinal Study window The Longitudinal Study window provides a convenient way to compare different results for example results obtained at different time points or results from different types of
158. are loaded click Photon Density or NTF Efficiency Maps in the Results tab The photon density maps for all wavelengths are displayed Figure 11 16 2 To rotate the surface and view it from a different angle move the thumb wheel to the left or right Figure 11 16 Photon density maps or NTF Efficiency maps for fluorescence Use the thumb wheel to rotate the surfaces E Photon Density Maps EA Wavelength All Waves Angle of View j F Log Scale Close 3 Select a wavelength from the drop down list The photon density or NTF Efficiency profiles at the crosshairs location are displayed In a good reconstruction the simulated photon density or NTF Efficiency curves red closely resemble the measured photon density or NTF Efficiency curves blue Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Figure 11 17 Simulated red and measured blue photon density or NTF Efficiency plots 640 nm wavelength selected E Photon Density Maps Horizontal Profile ag Photon density photons mm gt o BOB PANG Position Wavelength 640 X Angle of View photons mm 4 4 3 5 3 5 3 0 3 0 2 5 2 5 2 0 2 0 1 5 1 5 1 0 1 0 0 5 0 5 ete 0 0 8 7 lenis Log Scale Gg g W pee Vertical Profile m Measured E HEN ao Simulated N Photon density photons mm 40 20 0 20 Position mm Position 4 2 40
159. ared Recommended 1GB Dedicated Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools 239 Table 13 1 Minimum graphics card specifications continued Specification Description Consumer Graphics Cards Desktop Supported Mobile Windows Mac a NVIDIA GeForce 8 Series and above 8 9 100 200 300 and 400 series m ATI Radeon HD 4000 Series and above 4000 and 5000 series Recommended m Desktop NVIDIA GeForce GT 240 and above a Mobile NVIDIA GeForce GT 230M and above Workstation Graphics Cards Desktop Supported Mobile Windows Mac a NVIDIA Quadro NVS Series and Above NVS and FX series m ATI FireGL V5600 and Above FireGL FirePro and CrossFire series Recommended m Desktop Quadro FX 1800 and above a Mobile Quadro FX 880M and above f these specifications are not met the 3D Multi Modality tools do not appear in the Tool Palette 13 2 Classifying 3D Volumetric Data The 3D Multi Modality tools provide a histogram based method to classify the 3D volumetric data The histogram represents the distribution of voxel intensities in the 3D volumetric data and their color opacity values The goal of classification is to set color and opacity values for different intensity ranges so that the color opacity map shows the volume regions that you are interested in opaque in the map and hides unimportant regions transparent in the map For example F
160. ary Voxels below this threshold are not displayed The color table is mapped to voxels above threshold V Logarithmic Histogram Maximum Intensity Projection MIP E Gradient Illumination m To change the color table for the color opacity map make a selection from the Color table Opacity Map drop down list To apply the reverse color table select the Reverse option a To view the histogram in a separate window click the Al button m If the histogram intensity range appears narrow or suppressed choose the Logarithmic Histogram option This option enhances the histogram display by magnifying the smaller regions of interest in the histogram while keeping noise and air related intensity peaks high It helps bring out hidden regions visible in the histogram for easier identification of interesting intensity ranges Managing Control Points Use the control points to edit the 3D volumetric data color opacity map During volume rendering the color opacity map is used to map color and opacity to the corresponding intensity value as well as interpolate color and opacity for all data between adjacent control points 1 Place a control point on the histogram by clicking anywhere on the histogram between the point represents the lowest intensity in the volume and O point represents the highest intensity in the volume 2 Drag any control point up or down to set the opacity level that is
161. associated with the intensity value represented by the point Drag a user added control point left or right to change the Intensity associated with the opacity specified by the point When you add delete or modify a control point the color opacity map and the rendering of the volume data are updated in real time 240 Living Image 4 3 1 User s Manual IVIS Spectrum M NOTE The minimum and maximum intensity levels associated with the and O control points cannot be changed The opacity level associated with these points can be changed Chapter 13 3D Multi Modality Tools Figure 13 3 Histogram tool Color Opacity Map L0 Opacity O Y gg Reverse all Each control point specifies a particular Double click a control point to open the color palette Perspective TOCCU ioe TTL ee Trill HT TT ET IT aaan opacity intensity color 4 To select a color for particular data double click a control point In the color palette that appears choose a color and click OK The software interpolates the color range between adjacent control points To delete a control point right click the point To delete all control points click the x button M NOTE The and O control points cannot be deleted from the histogram Saving a Color Opacity Map A color opacity map can be saved and applied to any volumetric data set 1 2 3 Click the Save button mj Figure 1
162. asurement units 82 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Table 4 12 Image Overlay window continued Item Description Photograph A drop down list of the photographs in the image sequence Fluorescent or Luminescent Images The sequence images Copies the overlay to the system clipboard a Click to export the overlay to a graphic file 8f Click to include all fluorescent or luminescent images in the overlay 8 Click to remove all fluorescent or luminescent images from the photograph Image Adjust Tools for adjusting the appearance of the highlighted fluorescent or luminescent image Adjustments can only be made on one image at a time Min The minimum pixel intensity associated with the color scale for an image Pixels less than the minimum value are not displayed Max The maximum pixel intensity associated with the color scale for an image Pixels greater than the maximum value are displayed in the maximum color Opacity Controls the opacity of the fluorescent or luminescent image Color Table Tools for selecting and modifying the color scale associated with an image Color Scale Type Choose BlackLevel to show black at the low end of the color scale Choose WhiteLevel to show white at the low end of the color scale Red Click the drop down arrow to select a color table for the image dat
163. at appears Restart Wizard select Corea J cose Jl Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing 139 TIP See the Imaging Wizard tech note for a quick guide on sequence acquisition select Help gt Tech Notes on the menu bar If you do not use the Imaging Wizard to set up the image sequence it 1s recommended that the sequence include images acquired using several filters that sample the emission and or excitation spectra at multiple points across the entire range Make sure that the band gap between the excitation and emission filters is sufficiently large for example 535 nm so that the excitation light does not leak through the emission filter where it can be detected by the CCD If a data set includes multiple excitation and emission filter scans the software automatically unmixes signal according to the filter type with the most entries For example a data set acquired using three excitation filters and four emission filters will be unmixed by emission wavelength 8 2 Spectral Unmixing Methods The Living Image software provides four spectral unmixing methods Table 8 1 Table 8 1 Spectral unmixing methods Method Description See Page Guided Use this method when 139 m Probe locations are known m Probe signals are mixed with background signal but not other probe signals Note This method is not recommended if probe signals are overlapping Use this method
164. ata About ROIs Quick Guide Drawing Measurement ROls on an Optical Image or Sequence on page 93 ROI Tools for Optical Images on page 95 Measurement ROls on page 97 Mirror ROIs on page 102 Measuring Background Corrected Signal on page 105 ROI Histogram on page 108 Managing ROI Properties on page 109 Managing the ROI Measurements Table on page 119 5 1 About ROIs This chapter explains how to draw and measure signal within a region of interest ROI on an optical image Four types of ROIs are available for optical data Table 5 2 Table 5 1 Types of ROls for optical images ROI Name Description Shape See Page Measurement ROI Measures the signal intensity in an area ofan Circle square 93 for optical data optical image grid or contour Quick Guide 97 detailed steps ROT 1 25 J 1 073e 06 ROT 2 25 J 5 324e 05 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data Table 5 1 Types of ROls for optical images continued ROI for optical data ROI Name Description Shape See Page Mirror ROI for left or Circle or square 102 right views of optical data obtained using the Side Imager three views left right and center Note Use mirror ROls to measure signal in the left or right views which are reflected from the mirrors Use measurement ROls to measure signal in the direct non reflected center view only Average Background Measures th
165. ata selected for surface reconstruction pink Tool Palette imagen II gt Spectral Unmixing and DyCE mg Crop image Optical Surface Reconstruction Orientation Draw a region of interest subject rectangle to proceed Surface Smoothing Level 0 Restore Save Results TLT20050624145507 001 Next gt Valid crop region 5 If you want to reconstruct only a particular region of the subject resize the rectangle drag a green handle M so that it includes only the area of interest 6 Click Next The purple data mask appears The mask is an overlay on the subject image that defines the area of interest for the surface topography reconstruction The mask should match the underlying photograph of the subject as closely as possible without including any area outside the subject Image Living Image 4 3 1 User s Manual IVIS Spectrum Figure 10 3 Data mask purple O A Single View Surface Topography Analysis Threshold image Use left and right TLT20050624122348 001 Threshold 2 Chapter 10 Reconstructing a 3D Surface 7 If itis necessary adjust the threshold value so that the mask fits the subject image as closely as possible To change the threshold do one of the following m Press the left or right arrow keys on the keyboard Move the Threshold slider left or right a Click the arrows or enter a new value in the box 8 Click Finish
166. average background ROI 105 B background corrected signal 105 107 batch mode 48 49 binning 71 browse optical image data 55 59 browser optical image data 58 volumetric data 252 C Caliper Corporation technical support 4 cascade images 60 classifying 3D volumetric data 239 control points 240 colorize data 84 85 color opacity map 240 composite image 133 135 control panel 262 265 copy ROI measurements 120 correction filtering tools binning 71 cosmic correction 71 dark background subtraction 71 flat field correction 71 smoothing 71 cosmic correction 71 crop box 79 D dark background subtraction 71 Data Preview window 191 193 DICOM Viewer 212 258 DLIT sequence requirements 187 DLIT results 197 199 manage 198 DLIT FLIT troubleshooting 230 DyCE 159 176 E edit image label 65 sequence 32 53 87 88 export images 54 85 87 surface 183 Living Image 4 3 1 User s Manual IVIS Spectrum F fiducial registration 250 255 258 flat field correction 71 FLIT sequence requirements 194 FLIT results 197 199 manage 198 fluorescence reconstruct 3D sources 194 197 fluorescent imaging quick guide epi illumination 30 fluorescent imaging epi illumination 29 35 fluorescent imaging transillumination 35 41 focus manually 265 266 G gradient illumination 244 H histogram ROI 108 Image adjust appearance 68 69 cascade 60 export 54 85 87 information 64 measurements 78 print 85 87 tag 67 tile 60 image dat
167. awing a 3D ROI on page 125 Managing the 3D ROI Measurements Table on page 130 6 1 About 3D ROIs A 3D region of interest ROI can be drawn on a a DLIT reconstruction of a luminescent source a FLIT reconstruction of a fluorescent source a CT volume NOTE The 3D Multi Modality tools see page 238 are required to load IVIS Spectrum CT volumetric data or import volumetric data PET MRI or CT data from instruments other than the IVIS Spectrum CT A 3D ROI measures the signal intensity within a user specified bounding box Figure 6 1 Example 3D ROI IC B0111027132749 sEQ fo e jes Sequence View L 3D View Ea e H pe gy ns SME The Living Image software records information about the ROIs you create during a session and computes statistical data for the ROI measurements The ROI Measurements table displays the data and provides a convenient way to review or export ROI information Figure 6 2 If a data set includes ROIs on both optical and volumetric data the measurements for the two types of ROIs are displayed in separate tabs of the ROI table Living Image 4 3 Software User s Manual Chapter 6 3D ROI Tools for Volumetric Data Figure 6 2 3D ROI measurements table 3D ROI Measurements Data Types 3D Volumetric Data v Measurement Types ROI Voxels Total Counts Average Counts Min Counts Max Counts BI20111027132749 SEQ ROI1 39304 5 181e 08 1 318e 04 0 000e 00 5 500e 04
168. aying Slices Through a Reconstruction ngo nfa 1 Click a location on a source Alternatively click the toolbar button draw a box around a source then click Center of mass in the 3D Source tools 2 Click the gp toolbar button The Coronal Sagittal and Transaxial windowpanes show a slice through the surface taken by the associated plane Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Figure 11 21 Planes cutting a reconstruction Tool Palette a E TLI20050624145507 sEQ oo ea gt ROI Tools lo Sequence view L 3D View z_ Spectral amiina nod Ove z M ge er epe _ Surface Topography J GE KA pana gt 3D Multi Modality Tools Gronal z 15 1 Sagittal x 3 5 7 3D Optical Tools Surface Source Registration Select Source Original z Display Source Surface 4 iE Opacity Display Voxels 100 G Color Scale Transaxial y 22 8 Color Table Measured Sources Subject Height 26 2 mm ff Perspective Key Value Quantification 6 70e10 photons sec Volume 14 56 mm43 Depth 5 86 mm Center of Mass 3 5 18 1 15 6 Host Organ Unknown Export Voxels DLIT 3D Reconstruction 3 To move a plane put the mouse cursor over a line in the coronal sagittal or transaxial windowpane When the cursor becomes a ji or arrow drag the line The view is updated in the windowp
169. be locations Figure 8 4 Mark the probe locations for unmixing on the image cube E HX20120419114703 sEQ Sequence View Unmixing Normalized Legend el H o ImageCube Background subtracted spectra 3 at the probe 3 ig locations marked MI AA on the image cube Ke 6 60 700 740 Emission Wavelength nm 780 Spectrum List by xX Note Background Name Color Pick 1 V TissueAF J ime AT 2 AF680 W af Image Cube Viewer iv Overview Next Cancel 8 Click Next after you finish marking the probe locations The Unmixing window shows the analysis results which include unmixed spectra corrected for tissue autofluorescence unmixed images and a composite of the unmixed images Figure 8 8 See Spectral Unmixing Results page 154 for information about the results 9 To save the results as a spectrum library a Click the pe button in the Spectrum List toolbar Figure 8 5 b Enter a file name in the dialog box that appears and click Save Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing 142 Figure 8 5 Spectral unmixing results O HXx20120419114703 sEQ Ces al Sequence View Unmixing Composite Y Show Labels V Individual Scale ag 5 F Normalized E Legend a 10 i Unmixed images show Spectra plot See 5 the extracted signal page 154 for more E See page 157 for details details on analyzing
170. bject Height 19 4mm Perspective 4 To change the color of the 3D ROI a Click the Browse button The Select Color box appears Figure 6 7 Select a 3D ROI color 7 3D ROI Properties o For Sequence BI20111027132749_SEQ ROI Name ROI 1 Location i Brightness slider Yc Cross hairs in the custom color field Val BEBE EES ko BEBE SESS b To select a basic color for the ROI line click a basic color swatch and click OK c To define a custom color drag the crosshairs in the custom color field adjust the brightness slider and click Add to Custom Colors d To select a custom color for the ROI line click a custom color swatch and click OK Living Image 4 3 Software User s Manual Chapter 6 3D ROI Tools for Volumetric Data 6 3 Managing the 3D ROI Measurements Table Configuring the 3D ROI Measurements Table You can customize the data and information column headers in the 3D ROI Measurements table Several preset categories are available in the Measurement Types drop down list Figure 6 8 3D ROI Measurements table p ROI Measurements e r Data Types 3D Volumetric Data x Measurement Types Counts hd sro Sequence Number ROI Voxels Total Counts Average Counts Min Counts Max Counts BI20111027132749 SEQ ROI1 54015 6 668e 08 1 234e 04 0 000e 00 5 500e 04 Configure Export
171. click Delete M NOTE Preset table configurations cannot be deleted Copying or Exporting the ROI Measurements Table To export the table 1 Click Export in the ROI Measurements table 2 In the dialog box that appears a Select a folder and enter a name for the file b Select a file type txt or csv and click Save To copy the table to the system clipboard m Copy selected rows Select the rows of interest and click Copy Alternatively select the rows then right click the table and choose Copy on the shortcut menu Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data a Copy all rows Click Select All and click Copy Alternatively press Ctrl A then right click the table and choose Copy on the shortcut menu Figure 5 31 Copy all rows in the ROI Measurements table to the system clipboard ROI Measurements Kinetic ROI Measurements Plot Kinetic ROI Measurements TLT20050624145507 002 ROI2 Overlay 2653ex04 6 982e 02 _ 1 402e 02 TLT20050624145507 003 8 290e 05 2 Copy Ctrl C TLT20050624145507 003 ROI4 Overlay 2 687e 05 Select All Ctri A TLT20050624145507 004 ROIS 1621e 06 4 053e 04 8 599e 03 TLT20050624145507 004 ROI6 Overlay _ 7 273e 05 8 456e 03 1 784e 03 6 007e 03 1199e 04 Cee as Na r Customized Selections Measurements Types age Attributes ROI Dimensions 123 6 3D ROI Tools for Volumetric Data About 3D ROIs Dr
172. ction Mirror XOffset For multi view data the mirror location from the x center line Starting voxel size The voxel size at the start of the analysis The length of the side of the voxel cube in mm units for the coarsest initial grid size in the adaptive gridding scheme Total of data pts The total number of data points used in the reconstruction Median Filter Indicates whether or not a median filter was applied to the data Image Threshold The percentage of the minimum radiance at each wavelength DLIT or source location FLIT is of the maximum radiance This defines the minimum intensity included in the data Samples of Image The data in each image is sampled This parameter shows the number of pixels sampled from each image Tissue Properties The tissue properties for modeling the photon propagation Source Spectrum The emission spectrum of the type of luminescent source Quantification Selection A user selected quantification database used in the reconstruction to convert reconstruction voxel units to cells or picomoles units Sequence name Image data sequence name Version Living Image software version Managing 3D Reconstruction Results Item in the DLIT 3D Reconstruction Results Tab Description Name The name for the active DLIT or FLIT results Select results from this drop down list Delete Deletes the selected DLI
173. d 2 Click the Image Cube tab Figure 9 15 169 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 9 DyCE Imaging and Analysis The image cube represents a stack of the DyCE sequence images If the Overview option is selected the image cube shows a composite of all images To view a particular image remove the check mark next to Overview and move the slider or enter an image number Figure 9 15 Image cube The temporal spectrum at the mouse pointer location is shown in gray Image cube tab 7 RKG20110210131022 SEQ Sequence View Unmixing 4 Normalized Legend E a Qh ImageCube Temporal spectra time plots 0 400 200 1200 Image Time Point s cube Spectrum List She Xx Note Select Name Color Pick Temporal 1 Kane fine zs spectra 2 r a rly names and 3 V Bladder W l 4 bal color codes Image Cube Viewer V Overview Unmx Close Overview shows a composite of all images in the DyCE data set Remove the check mark to view individual images Move the mouse pointer over the image cube to see the temporal spectrum at a particular location The temporal spectrum at the pointer location is updated as you move the pointer l NOTE If analyzing DyCE results the Normalized option for the spectrum plot must be checked to see all of the temporal spectra when the mouse pointer is over the image cube 4 To add another componen
174. d by the software For example a threshold of 80 will exclude pixels with intensities that are less than 80 of the highest pixel intensity in the image M NOTE After the ROIs have been created right click an ROI to view a shortcut menu of ROI commands Ctrl click for Macintosh users The shortcut menu provides easy access to many functions for managing ROIs and viewing ROI properties 6 Click the Measure button eueROls in the ROI tools to show the ROI Measurements table 94 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data Figure 5 5 ROI Measurements table A ROI Measurements o amp z ROI Measurements amp Refresh Image Number ROI Image Laye Total Coun Avg Count Stdev Cour Min Count Max Count A TLT20050624145507_001 ROI 1 Overlay 6 536e 03 2 334e 02 4 395e 01 1 723e 02 3 301e 02 E TLT20050624145507_002 ROI2 Overlay 2 653e 04 6 982e 02 1 402e 02 4 730e 02 9 393e 02 TLT20050624145507 003 ROI3 Overlay 8 290e 05 2 961e 04 5 807e 03 2 209e 04 4 280e 04 TLT20050624145507 003 ROI4 Overlay 2 687e 05 4 405e 03 9 302e 02 3 042e 03 6 048e 03 TLT20050624145507_004 ROI5 Overlay 1 621e 06 4 053e 04 8 599e 03 2 976e 04 5 899e 04 _TLT20050624145507 004 ROIG Overlay 7 273e 05 8 456e 03 1 784e 03 6 007e 03 1199c 04 z Customized Selections Me ts Types Attributes ROI Dimensions vesatenens Tips mate Abibnesm
175. d images show the extracted signal See page 157 for details on analyzing these images Composite of the unmixed images See page 156 for more details 144 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing Automatic Method Use the automatic method to analyze data when the probe locations are unknown 1 Load the image sequence In Figure 8 9 the fluorophores are Alexa Fluor 680 and Alexa Fluor 750 Images were acquired using a 605 nm excitation filter and emission filters from 660 to 800 nm in 20 nm increments Figure 8 9 Sequence for spectral unmixing ad Tool Palette gt Image Adjust gt IC HX20120419114703 sEQ misa Sequence View Units Radiant Efficiency v 3 E Use Saved Colors Options z z Info PB R NG a _ Corrections Filtering gt ROI Tools Spectral Unmixing and DyCE Analyze Results Select Images Type Emission Excitation wavelength Mins 1 41e7 Max 2 7166 Emission wavelengths of the sequence Min 1 25e7 Max 1 00e8 92 Methods Automatic gt Start Unmixing Select Automatic 2 Click the Analyze tab of the Spectral Unmixing and DyCE tools By default all wavelengths are included in the analysis Remove the check mark next to wavelengths that you want to exclude from the analysis 3 Select Aut
176. details on the control panel see Appendix A on page 262 a b c d NOTE The Living Image software has optional password protection for user accounts See page 20 for more details Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 2 Getting Started 7 Figure 2 2 Living Image main window and IVIS Acquisition Control Panel Menu b ar for more File Edit View loos Acquisition Window Help details see Appendix C eG B5 RES NW on page 275 Toolbar IVIS Acquisition Control Panel F Stop Excitation Filter System needs initialization maging Wizard 13 4 Jom Click on Initialize button to proceed mr E Focus use subject height Temperature Unlocked Type Description 5 Information Living Image 4 3 0 15291 Jan 92012 10 16 03 ka D Information gt gt K5A Logged IN lt lt Activity window D Information IVIS configuration file found and loaded D Information Reading user preferences User KSA NOTE The Living Image software on the PC workstation that controls the IVIS Spectrum includes both the acquisition and analysis features The Living Image software on other workstations includes only the analysis features Macintosh users have access to the analysis features only 2 2 Initializing the System and Checking Temperature The IVIS Spectrum must be initialized each time Living Image software is started or if the power has been cyc
177. drag it a distance greater than its width To dock the Tool Palette in the main window drag the palette to the right or left side of the window and release 268 Living Image 4 3 1 User s Manual IVIS Spectrum Appendix B Preferences Figure B 2 Main application window Q Sequence View Diver specta ng Gants Use Sawed Coors Type Dewnpton Actwty x f 4 information 75KSA Logged N lt lt D efermaten Reading user preferences D fle fet Yew koh Window Help sagut AS K um las M area 2 4 ax D tefermation Liang imege 43019006 6 ba Development Build Mow P2911 18 31 22 User ESA B 2 Options Figure B 3 User preferences IC Preferences Options Acquisition Theme Optical Properties User Defaults Label Set Living Image Universal Edit Label Choices Table B 2 User preferences Item Description Edit label Choices Opens a dialog box that enables you to edit the Living Image Universal label set Default Units Choose counts or radiance photons for image display 269 Living Image 4 3 1 User s Manual IVIS Spectrum Appendix B Preferences 270 B 3 Acquisition Figure B 4 Acquisition preferences Auto Exposure C7 Preferences Acquisition Theme Optical Properties Auto Exposure Camera Settings Luminescent Fluorescent Auko Exposure Preferences First Preference Second Prefer
178. ds cropping for example to remove structures such as the stage from the CT view follow step a to step c below If cropping is not needed proceed to step 4 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools To crop the data a Click the crop tool button H The crop tool appears and has six control points o Crops the data along the x axis Crops data along the y axis o Crops data along the z axis Figure 13 21 Crop data along the x y or z axis y Unat Laba kang Na arabia Phabin irio s Paskua Ciara Dra kal aha Ch Liep Tai Bary bp mraakimiiibabin ora Current crop inal baban NON No crop tool X axis crop tool Y axis crop tool Z axis crop tool b Click and hold a control point while you move the crop plane As you move the crop plane the slice views are updated Release the mouse button to crop the data c To reset the crop planes click the TA button When finished cropping press the Tab key to turn off the crop tool 4 Click the Manual Registration button JA The transformation tool appears Figure 13 22 The tool has three modes that enable you to translate scale or rotate the 3D volumetric data press the Tab key to change the tool mode The slice views are automatically updated when you use the tool 257 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools 258 Figure 13 22 Manual registration t
179. e Item Description Data Types 3D Volumetric Data Select this data type to measure the grayscale values of the source voxels of a 3D optical image Source Voxels Choose this option to measure the source intensity of the voxels of a 3D optical image Measurement Types 3D Volumetric Data Counts A measurement of a voxel value The scale is image specific and may not be consistent between images Absorption A measurement of the amount of X rays absorbed by the voxels Hounsfield A measurement of voxel grayscale value in Hounsfield units Note Absorption and Hounsfield units are only available for IVIS Spectrum CT data Source Voxels photons sec The radiance in each voxel summed or integrated over the 3D ROI cells Fluorescence yield for calibrated sources pmol M cm Fluorescence yield for uncalibrated sources pmol Fluorescence yield of calibrated sources Sequence Number The identifier of the active image data ROI Name of the 3D ROI Voxels The number of voxels within the 3D ROI 3D Volumetric Data Counts measurements 16 bit scale with values that change from image to image Total Counts the sum of all counts for all voxels inside the 3D ROI Average Counts Total Counts Number of voxels in the 3D ROI Min Counts The smallest number of counts in a voxel within the 3D ROI Max Counts The largest number of counts in a voxel within the 3D ROI 3D Volumet
180. e Bright and Contrast controls see Adjusting Image Appearance on page 43 Living Image 4 3 1 User s Manual IVIS Spectrum Appendix A IVIS Acquisition Control Panel 263 Table A 1 IVIS acquisition control panel continued Item Structure Description Choose this option to take a structured light image an image of parallel laser lines scanned across the subject when you click Acquire The structured light image is used to reconstruct the surface topography of the subject which is an input to the Diffuse Luminescence Imaging Tomography DLIT algorithm that computes the 3D location and brightness of luminescent sources When this option is chosen the f stop and exposure time are automatically set to defaults for the structured light image f 8 and 0 2 sec respectively The spatial resolution of the computed surface depends on the line spacing of the structured light lines The line spacing and binning are automatically set to the optimal values determined by the FOV stage position and are not user modifiable Overlay If this option is chosen the system automatically displays the overlay after acquisition is completed for example luminescent image on photograph Exposure time The length of time that the shutter is open during acquisition of an image The luminescent or fluorescent signal level is directly proportional to the exposure time The goal is to adjust the exposure time to produce a signal that is we
181. e average signal intensity in a Circle or square 105 user specified area of an optical 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 ina measurement ROI by subtracting an average background ROI from a measurement ROI BKG 1 7 81e 02 91 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data 92 Table 5 1 Types of ROls for optical images continued ROI Name Description Shape See Page Subject ROI for Identifies a subject animal in an optical image Square 105 optical data 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 Subject 1 Subject 2 The Living Image software records information about the ROIs you create during a session 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 Figure 5 1 Example measurement ROls on an optical image and the ROI measurements table E TUKAAN 005 Ures Hadang Pietra Demir Ovien eo 7 si imaga Pisni Ril magellan ta Coun Ang Count Sideshow
182. e flux of each highlighted voxel Host Organ The location of the selected source can be referenced to an organ atlas and the organ from the atlas that is closest to the source will be reported here This information is available if you select and register an organ atlas with the reconstruction For more details see page 223 Export Voxels Enables you to export the voxel measurements in their x y and z coordinates and source intensities csv file Center of mass Click to compute the center of mass for the source selected with the Measure Source ngo nfa tool For details using this tool see page 202 11 13 3D Tools Registration Mouse anatomy reference atlases are available for registration with 3D reconstructions A mouse anatomy reference atlas is used when volumetric data from another imaging modality is not available A reference atlas provides guidance for the bioluminescent or fluorescent source anatomical location Use the Registration tools to Display organs in the surface page 220 Manually adjust the location or scale of organs in the surface page 221 Check the organ fit page 222 Import an organ atlas page 223 You can check the organ fit in the 3D View window page 222 218 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Figure 11 32 3D registration tools and surface with fitted organs skin not displayed
183. e image data See page 54 for details Image acquisition begins and the upper area of the control panel changes to red color During acquisition the Acquire button in the control panel becomes a Stop button Click Stop to cancel acquisition The control panel returns to blue color when acquisition is finished and the image window appears Figure 3 21 See Table 3 2 on page 28 for details on the image window 40 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition Figure 3 21 Overlay fluorescent image on photograph in the image window E ALI20110615093703 001 bodka Too Palette B8 Units Display Overlay al Fluorescent on ES Photograph gt 7 Transillumination Location atnto nG i i Im ALI20110615093703_001 7 ner a gt Image Information a Wed Jun 15 2011 09 37 41 Group Level High Em 800 Ex 745 Trans illumination Bin 2 FOV 13 f2 0 5s pees Living Image Version 4 2 0 13627 Jun 9 2011 eee eee Camera 1S0702N4088 Andor iKon Commenti 50 ul xen10 injection 1e7 cfu Comment2 FLIT Time Point o n Animal Number 2 m gt ROI Tools r Trans fluorescence NTF Efficiency Color Scale Min 5 12e 1 Max 1 31 TIP See the tech note dentify Saturated Pixels in an Image for information on pixel measurements select Help Tech Notes on the menu bar 41 Living Image 4 3 1 User s Manual
184. e key while you drag the header left or right Release the mouse key to set the new position 58 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Table 4 1 Living Image Browser continued Item Description Load as Group Enables you to select particular images that you want to view as a sequence The images may be acquired during different sessions To select adjacent images in the browser press and hold the Shift key while you click the first and last file in the selection To select non adjacent images in the browser PC users Press and hold the Ctrl key while you click the images in the browser Macintosh users Press and hold the Cmd key apple key while you click the images in the browser Note The Load as Group option is only available when two or more images non kinetic are selected in the browser Tip See the tech note Loading Groups of Images for a quick guide select Help Tech Notes on the menu bar Load Opens the selected image or image sequence Remove Removes a user selected image sequence s from the browser Close Closes the Living Image Browser Opening Data from the Menu or Toolbar ei NOTE To open a recently viewed file select File Recent Files on the menu bar 1 Click the Open button G on the toolbar Alternatively select File Open on the menu bar 2 In the box that appears choose a file type fi
185. e left and right sliders to set the minimum and maximum colors Reverse Choose this option to apply the colors of the selected color table in reverse order to the source voxel scale For example the Red color table represents the source intensity from low to high using a color scale from transparent to red If Reverse is chosen the source intensity from low to high is represented using the color scale from red to transparent Log scale Choose this option to apply a logarithmic scale to the color table 217 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Table 11 11 3D Source tools continued Item Description Measured Sources Quantification DLIT For uncalibrated sources the total flux measured for the sources selected using the Measure Source tool For calibrated sources this unit will be in cell units For details on using this tool see page 202 Quantification FLIT For uncalibrated sources the fluorescence yield measured for the voxels selected using the Measure Source tool EE Fluorescence yield is expressed in units of pmol Mcm here for uncalibrated sources For calibrated sources this unit will be in either cells or pmol For details using this tool see page 202 Volume Volume of the selected source mm Center of Mass DLIT or FLIT The weighted average x y and z coordinates of the selected voxels where the weights are th
186. e located This information is available if organs are displayed with the reconstruction For more details on displaying organs see 3D Tools Registration page 218 Measuring Source Depth Follow the steps below after reconstruction is finished or results are loaded to measure source depth 1 If the surface includes voxels pasted from other results select a source from the drop down list 2 Confirm that Display Voxels is selected not Display Source Surface 3 Click the Measurement Cursor button The distance from the center of mass to the surface is measured in the three planes m Coronal and transaxial planes display the shortest distance from the center of mass to the surface m The sagittal plane displays the distance from the center of mass to the bottom of the subject 4 Click the ater button to display slice planes through the center of mass See page 204 for more information on planes Figure 11 19 Slice planes This example shows slice planes through a selected source center of mass and distance measurements from the source center of mass to the surface a Tool Palette 8 E7 11120050624145507 SEQ o amp 28 gt ROI Tools al a Sequence View L 3D View z lanar Spectral Imaging 3 pa ara Para gt Spectral Unmixing and DyCE R L BB JS pm 7 Ba oi Coronal z 19 5 Sagittal x 1 7 i Surface Topography gt 3D Multi
187. e selected color table Logarithmic Scale Choose this option to apply a log scale to the relationship between numerical data and the color range in the color table A log scale improves the visibility of dark areas in an image 69 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 70 Magnifying or Panning in the Image Window To incrementally zoom in or out on an image Click the E or ali button Alternatively right click the image and select Zoom In or Zoom Out on the shortcut menu To magnify a selected area in an image 1 Click the button Alternatively right click the image and select Area Zoom on the shortcut menu 2 When the pointer becomes a draw a rectangle around the area that you want to magnify The selected area is magnified when you release the mouse button To reset the magnification remove magnification Click the 4 button Alternatively right click the image and select Reset Zoom on the shortcut menu To pan the image window M NOTE Panning helps you view different areas of a magnified image If the image has not been magnified you cannot pan the image 1 Click the db button 2 When the pointer becomes a p click and hold the pointer while you move the mouse 4 6 Correcting Optical Image Data Use the Corrections Filtering tools to subtract background or apply corrections to the optical image data You can also apply smoothing and sof
188. e the currently selected row using the information from the control Add Adds a new row at the end of the sequence setup list Editing Image Parameters You can edit imaging parameters in the sequence table or in the control panel To edit a parameter in the sequence table 1 Double click the cell that you want to edit Figure 3 34 Figure 3 34 Control panel and sequence table j IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter PT Display Photographic Settings Subject Mouse Auto sec v v Medium vii v lock C Seq 1 O Mode Exposure Binning FStop Excitation Emission Structure FOY Height oe g Petr a 1 DES auto Medium 1 Block 560 Yes C 1 50 2 PE auto Medium 1 Block 580 No 1 50 3 DI auto Medium 1 Block 600 No 1 50 4 DI auto Block 620 No 1 50 s fia Auto i ENG 640 No 1 50 2 8 Imaging Wizard Subject height 1 50 cm Image Setup Focus use subject height v Temperature AN Locked C Number of Segments min Apply to All dg Update Insert Add Field of View System Status Acquire Sequence O MIS 2 Enter a new value in the cell or make a selection from the drop down list To apply the new value to all of the cells in the same column click Apply to all 3 Click outside the cell to lose focus To edit a parameter in the control panel 1 Select the ro
189. e the size of an ROI that was created using the auto ROI or free draw tool To resize an ROI using a handle 1 Select the ROI and put the mouse pointer over a handle W on the ROI 2 When the pointer becomes a arrow drag the handle Living Image 4 3 1 User s Manual IVIS Spectrum To resize an ROI using the ROI Properties box 1 Double click the ROI in the image Chapter 5 ROI Tools for Optical Data The ROI Properties box appears and displays the positions and dimensions of the selected ROI Figure 5 21 ROI Properties dialog box fe 7 ROI Properties mE ROI BKG 1 7 Label BKG 1 Shape Cirde Type Manual Background ROI Subject ROI Use as BKG for future ROIs in TLT20050624145507 005 only Entire sequence Lock Position Xc pix 117 00218 7 Yc pix 97 93839 7 Angle deg 0 0000 F Lock Size Dimensions of the ROI Width pix 2 06161 a selected in the image Height pix 20 43172 Enter a new width or height value in the ROI Properties box To lock the current ROI size choose the Lock Size option M NOTE The ROI size cannot be changed until the Lock Size option is cleared 114 Living Image 4 3 1 User s Manual IVIS Spectrum Editing the ROI Line 1 Double click the ROI that you want to edit The ROI Properties box appears Figure 5 22 Chapter 5 ROI Tools for Optical Data Figure 5 22 Editing ROI properties
190. e 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 Click to hide or show the x y z axis display in the 3D view window Click to hide or show coronal sagittal and transaxial planes through the surface in the 3D view window Click to show or hide a bounding box around the surface Click to show or hide a grid under the surface 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 7 ar Select this tool from the drop down list to display the perspective view E Click to show or hide measurement cursors in the coronal sagittal or transaxial views Click and drag the green handle I at either end of a measurement cursor to resize and reposition it If DLIT or FLIT results are loaded click a voxel in the 3D reconstruction then click this button to display measurements for the voxel in the 3D tools Source voxel measurements Enables you to save the 3D view to a graphic file for example jpg 180 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 10 Reconstructing a 3D Surface 181 Changing the View Perspective You can click and drag the surface to view it from different perspectives Alternatively do one of the following Select to change
191. e ves ter ests tere TETE IANG LENS ATIN SATA T NOT TITO IT AO DONT TATAE ert ot rs rere bets erry rae Crt Un te tstcstrtercrs rer ee terre INIT AAO SAAN Close Preview Label Set All 7 Add to Lit Brpwse View Default Configure Load as Group Load Remove Close Location KATHERINE PC Share Caliper LS Caliper Data Sample D ataf VIS200 data TraserBeadsPC TLT 20050624145507_SEQ TLT20050624145507_003 ClickInfo txt Images loaded in the browser as part of a sequence These images can also be selected for grouping into another sequence Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 89 2 In the browser select the images that you want to group together To select adjacent images in the browser press and hold the Shift key while you click the first and last file in the selection To select non adjacent images in the browser m PC users Press and hold the Ctrl key while you click the images of interest in the browser Macintosh users Press and hold the Cmd key apple key while you click the images of interest in the browser 3 Click Load as Group The image thumbnails are displayed together in an image window 4 Save the images as a sequence a Click the Save button el Alternatively select File 5 Save on the menu bar b In the dialog box that appears select a folder and click OK 5 ROI Tools for Optical D
192. eAF Mime x Sr 2 V AF680 l E zf 3 WI AF750 LNG ne Z Image Cube Viewer Z Overview Unmix Close 9 Click Unmix after you finish marking the probe locations and correct spectra for tissue autofluorescence The Unmixing window shows the analysis results unmixed images and a composite of the unmixed images Figure 8 19 See Spectral Unmixing Results page 154 for information about the results Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing Figure 8 19 Spectral unmixing results i TE Hx20120419114703 sEQ Cos E sequence View Unmixing V Show Labels V Individual Scale NG amp V Normalized Legend ee Tissue AF S Spectra plot See HG S page 154 for more Unmixed images details show the 0 50 extracted signal See page 157 for Min 0 00 p Mins 0 00 deta l Is on 004 a T Max 9 83e7 ere analyzing these Hanna avelenn cih hini AF750 Composite Images Spectrum List See Table 8 30n L A Xx i aaa detallson GG Composite of Select Name Color Pick the unmixed images See page 156 for more details 1 V TissueAF I ime vifr 2 V AF680 H iz 3 AF750 BB bie 4 Min 0 00 ax 6 4868 8 3 Correcting Spectra Spectra can be corrected for overlapping signal by subtracting one spectrum from another Click the niy button in the Unmix window 2 Choose the spectra to subtract in the d
193. ecessary change the threshold level to adjust the mask so that it matches the underlying subject photograph as closely as possible without including any area outside the subject image Figure 9 11 Auto unmix wizard KE RkG20110210131022 SEQ boltaje Sequence View Auto Unmix Select Image Mask Choose Components To Unmix Tips 10 time points selected Add the number of kinetics to analyze Kinectics Information Gx Click to add or remove components to unmIx Data Mask Options Photograph Threshold lel EG Draw Mask Rectangle Ellipse PCA Number of components to unmix 2 Ca 5 If you do not want to analyze the entire subject draw a data mask on a particular area using the data mask options a Select Draw Mask and choose the Rectangle or Ellipse option b Draw a mask over an area using the mouse If necessary click the mask to discard it and redraw the mask 6 Select a subject type from the drop down list 7 Click the paa button to add components to unmix i NOTE Two or three components are recommended for the initial automatic analysis The DyCE results obtained from the automatic analysis can be manually analyzed to identify possible additional components see page 169 for details on manual analysis 8 Click Finish The Unmixing window shows a time plot of the temporal spectra unmixed images and a composite of the unmixed images Figure 9 12 Living Image 4 3 1
194. ected by the software ot 7 TING SS ee er eas ey E 11120050624145507_SEQ Sequence View Units Radiance Photons v E Use Saved Colors Options z Info a A NG an ROI 3 4 J 1 433e 08 ROI 1 4 4 556e 07 4 ROI 4 4 J 2 517e 07 ROI 7 4 1 9798 09 Al a TA tH ROI 5 4 8 184e 08 a Min ee GETS RSL Ss Toot Patete Image Adjust ROI Tools ta KI e Measure ROIs Type Measuremer Auto All N Save ROIs Auto 1 ROI 2 KS parad ah Free Draw Delete Auto ROI Parameters Threshold B gt Spectral Unmixing and DyCE _ Surface Topography 3D Multi Modality Tools C gt DLIT 3D Reconstruction 3 Click the Measure button Wf Me ueROls in the ROI tools to show the ROI Measurements table 98 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data Figure 5 8 ROI Measurements table E ROI Measurements ROI Measurements Image Number TLT20050624145507 001 TLT20050624145507 002 TLT20050624145507 003 TLT20050624145507 003 TLT20050624145507 004 Image Layi Total Coun Overlay Overlay Overlay Overlay 6 536e 03 2 653e 04 6 290e 05 2 687e 05 1 621e 06 Avg Count 2 334e 02 6 982e 02 2 961e 04 4 405e 03 4 053e 04 Stdev Cour 4 395e 01 1 402e 02 5 80 7e 03 9 302e 02
195. ectrum Appendix B Preferences Table B 5 Image view preferences Item Description Color Palette Use these controls to select a color table for luminescent and fluorescent image data Choose the Reverse option to reverse the min max colors of the selected color table Use saved color palette while loading datasets If this option is chosen data are displayed using a user specified color palette For example after you load data specify a color table in the Image Adjust tools and save the data The user specified color table is automatically applied whenever the data are loaded Background amp Text Color Sets the color of the Background in the image window shown below a Text for the color bar To change a color click the button that opens the color palette BEA Options x Info NG KU ed TLT20050624145507_005 X j Display Overlay X Units Counts Luminescence 10000 8000 6000 4000 2000 Counts Color Scale Min 586 Max 10348 ROI Color Sets the colors for the ROI outline To change a color click the button that opens the color palette Luminescent Color of the ROI outline on a luminescent image Fluorescent Color of the ROI outline on a fluorescent image Restore Defaults Click to apply the default settings Figure B 7 3D view preferences CU Preferences Background Color
196. ectrum Chapter 2 Getting Started Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum continued continued Analyze Select Images Type Emission Methods Library Start Unmixing Living Image Tools and Functions See Page mage nrannaHon Bi mat EB units cm View information about an optical image for image example a pixel histogram or a 3D plot of pixel Binning 8 Width 12 6cm Height 12 6 cm intensities ma ee Make measurements in the image Image Data 9715 counts LB 00 0 00 Distance 0 00 yRolfols OOO O ROI Tools for Optical Data 90 O W Measureros X Specify a region of interest ROI in an optical E Apply to Sequence image and measure the signal intensity within the Type Measurement ROI v ROI Save ROIs Name ROI_1_KSA v Delete Load Save Auto ROI Parameters Threshold 26 a 24 E2 3D ROI Tools for Volumetric Data 124 EJ Fi Wf measure 3D ROIs pd Specify a 3 dimensional region of interest ROI in a CT volume and measure the signal intensity within the 3D ROI Save 3D ROIs Name 3D ROI 1 KSA oy Delete Load Spectral Unmixing 119 Use spectral unmixing to m Extract the signal of one or more fluorophores from the tissue autofluorescence s Analyze luminescent or fluorescent images when more than one reporter is used in the same animal model 14 Living Image 4 3 1 User s Manual IVIS Spectrum
197. ed Colors Options 4 Info 7 R A G a Min 34 Min 71 Min 68 f Min 68 5 Max 57497 Max 57148 Min 251 Max 2764 10 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 9 DyCE Imaging and Analysis Table 9 1 Sequence View window Item Description Units Select the measurement units for the image display from this drop down list The available units depend on the type of image data See the concept tech note Image Display and Measurement for more details select Help Tech Notes on the menu bar Use Saved Colors Choose this option to display the image data using the color table that was specified in the Preferences at the time of acquisition If this option is not selected image data are displayed using the color table currently specified in the Preferences Options Layout Choose a display option for the images in a sequence Default Dynamic or Film Strip For example here is Film Strip mode Sort by Options for ordering images in the sequence window This option only applies to images that were opened using the Load as Group function in the Living Image browser Default Order in which the images are stored in the folder TimeStamp Ascending order of the image acquisition time UserlD Ascending alphanumeric order of the user ID Display Choose the types of information to display with each image D mexani Cla D Sequence view
198. ee 10 Auto Exposure Feature 0 000 es 10 Overview of Living Image Tools and Functions 12 Managing User Accounts 6cacccag eed deed ot eS NG AGA ee ee eRe oe we 19 Adding USOS oheoeus new eenean ee ase AEEA A AA 19 Changing or Adding Passwords 0 00 cece eee eee eee 20 Deleting SEIS hana ama NANANG tee eesee gare seca AINA HANDA se ou 20 Locking User Accounts 0 00 eee es 21 Tracking System and User Activity 00 0 cece eee eee 22 Activity VWVINOOW 6 626604 5444060400558 48 RRA OTA RAE AG AG WA 22 Image Acquisition 5 06 vec eet dee Ee ee SS ee 23 Luminescent Imaging 00 cc ee 23 Quick Guide Acquire a Luminescent Image 24 Fluorescent Imaging With Epi Illumination aa 29 Quick Guide Acquire a Fluorescent Image With Epi Illumination 30 Fluorescent Imaging With Transillumination 0 0 00 cee ee 35 Acquire a Sequence Using the Imaging Wizard 2 0 0 cee ees 42 Set Up a Sequence a napana hanes eee Che ee ed Oe eo eee ee A 42 Acquire the Sequence 2 45 Acquire Multiple Sequences in Batch Mode 48 Manually Set Up a Sequence 2 tees 50 Editing Image Parameters aaa 52 Inserting Images in a Sequence 1 aa 52 Removing Images From a Sequence 0 eee ee ee 53 Manually Saving Image Data ua ana aaea 54 EXDOLLING IMAGES ccceeaxcaeee es aaa A AEREA ER E
199. ee page 46 for details 13 Enter information about the image in the Edit Image Labels box that appears optional Click OK M NOTE You can enter image label information at any time during or after acquisition If you do not want to enter image information click Cancel See page 58 for details on adding image information after acquisition 163 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 9 DyCE Imaging and Analysis 164 Figure 9 8 Enter information to include with the image optional 7 Edit Image Labels Ka UserID pry Living Image Universal Saved Labels LABELS _ 1 Ab 7 User v Baa 7 Information entered here appears in the O AA image label see Figure 9 9 on page 164 Male nu nu day 8 Mouse 4 7 Comment1 namaga Comment2 z Time Point v 7 Animal Number 4 5 T Animal Strain X T Animal Model v E Sex v View v Cell Line x C Reporter v E Treatment X E Luc Injection Time v T IACUC Number v Image acquisition begins During acquisition the Acquire button in the control panel becomes a Stop button Click Stop to cancel acquisition The image window appears when acquisition is completed Figure 9 9 See Table 3 2 on page 25 for more details on the image window Figure 9 9 DyCE sequence acquired using the Side Imager accessory _ C RkG20110210131022_SEQ bolo fes Sequence View Unmixing Units Counts 7 Use Sav
200. em Status Acquire Initializing C xFov 24 Service 12 8 naging Wizard 12 5 cm Imaging Wizard C SE Stage temperature controller pau Subject height 1 50 Sequence Setup Subject height 1 50 E Sequence Setup Focus use subject height Temperature NANG Locked Initialize Focus use subject height v Temperature NG Locked aabotin Save a batch sequence setup xsq 49 Living Image 4 3 1 User s Manual IVIS Spectrum 1 3 Living Image Help Chapter 1 Welcome There are several ways to obtain help on the software features and related information To view Do this A tooltip about a button function Put the mouse cursor over the button A brief description about an item in the Living Image user interface Click the k toolbar button then click the item The Living Image Software User s Manual Press F1 or select Help User Guide on the menu bar and select the manual specific for your imaging system Living Image technical notes see Table 1 2 on page 3 Select Help Tech Notes on the menu bar Note Please see the IVIS University download page for the most recent collection of technical notes Table 1 2 lists the tech notes that are available under the Help menu There are three types of tech notes m Tech Notes Quick guides for tasks using the Living Image software tools Biology Tech Notes Protocols and procedures
201. en the sequence view window displays images in the default sort order If the TimeStamp or UserID sort order is selected the images cannot be reordered 7 Click Close when you are finished editing the sequence The updated image sequence is displayed 4 13 Creating an Image Sequence from Individual Images This section explains how to create a sequence from images acquired during different sessions TIP Also see the tech note Loading Groups of Images for helpful information select Help Tech Notes on the menu bar 1 In the Living Image Browser browse for the images of interest See page 55 for more details on browsing M NOTE Browse for individual images which may or may not be part of a sequence not image sequences Figure 4 32 Living Image Browser ME Individual images that may or may not be part of a sequence can be selected 7 Living Image Browser koo x TLT20050624145507 003 Click Number EX Filter EM Filter Illumination Mode User ID User Group MB231D3H2LN luc Intracardiac EL MB231D3H2LN luc Intracardiac EL MB231D3H2LN Iluc i iIntracardiac i ME EL20100608105326 SEQ EL20100615094528 SEQ ME TLT20050624145507 ME TLT20050624145507 ME TLT20050624145507 bnan A STAND OTIN ANTO ire rete Ce TUN STS rose r DITTO TATOOT ATAT UTOT NC tree TOTO TATTO ANAN LUD TO ODI tre TATTOO ITS triste ree Tose terr
202. ence m If using the Imaging Wizard repeat step step 3 Each sequence is displayed in a separate tab If setting up the sequence manually click the button fp in the sequence table to add a new tab then proceed with manual setup in the new tab M NOTE Sequence tabs can be renamed Double click a tab name to edit it Alternatively right click the selected name to view a shortcut menu of edit commands for example Cut Copy Paste Figure 3 30 Multiple sequence tabs Three sequences are specified in this example Sequence tabs Removes the active tab and its sequence Click to open or save a batch sequence setup Subject xsq Exposure Binning FStop Excitation Emission FO Height Adds a new tab use with Auto Medium 1 Block 520 D 1 50 manual sequence setu sl p Auto Medium 1 Black 560 D 1 50 Auto Medium 1 Black 620 D 1 50 C Number of Segments Delay 0 0 min Apply ko All tf Update Insert Add 5 To remove a sequence click the sequence tab and then click the button 6 Click Acquire Sequence when you are ready to capture the sequences Image acquisition proceeds with no intervening time delay between sequences NOTE If the check mark is removed next to the Batch Sequences option in the control panel Figure 3 29 only the sequence in the active tab will be acquired To save the batch sequence setup 1 Click the Save button Gi 2 Enter a file name xsq and cho
203. ence Third Preference Target Counti Minimum Luminescent 3000 Exposure Time F Stop ka Epi fluorescent 6000 Trans Fluorescent 10000 Range Values Exp Time sec Binning F Stop Min 0 50 i i Max 60 Restore Defaults Table B 3 Auto exposure settings Item Description Luminescent Fluorescent Auto Exposure Preferences First Preference During auto exposure the software acquires a luminescent or fluorescent Second Preference image so that the brightest pixel is approximately equal to the user Third Preference specified Target Count Minimum If the target minimum count cannot be closely approximated by adjusting the first preference for example exposure time the software uses the first and second or first second and third preferences to attempt to reach the target max count during image acquisition Target Count Minimum A user specified intensity Range Values The minimum and maximum values define the range of values for Exp Time sec exposure time F Stop or binning that the software can use to attempt to Binning reach the target max count during image acquisition F Stop Restore Defaults Click to apply default settings Living Image 4 3 1 User s Manual IVIS Spectrum Appendix B Preferences 271 Figure B 5 Acquisition preferences Camera Settings C Freferences Acquisition Optical Properties Suto Exposure Camera Settings Default Image Exposure Default
204. ence for analysis with the Spectral Unmixing 138 Filter Scan tools to Extract the signal of one or more fluorophores from the tissue autofluorescence Determine the optimum excitation and emission filter for a probe DyCE Acquires a time series of optical images following a bolus injection of 159 radiotracer to enable detection of radiotracer distribution by tracking Cerenkov emission from charged decay products Note DyCE imaging and analysis requires a separate license FLIT Fluorescence Acquires an image sequence for analysis with the FLIT algorithm that 194 Imaging Tomography reconstructs the position geometry and strength of 3D fluorescent sources 5 Step through the rest of the wizard Each page of the wizard guides you with step by step instructions and descriptions When you finish the wizard it sets up the sequence to acquire Figure 3 25 Figure 3 25 Control panel and sequence setup Each row in the sequence table specifies the acquisition parameters for one image in the sequence See page 51 for details on the sequence table A IVIS Acquisition Control Panel Exposure Time Binning F Stop Excitation Filter Emission Filter 7 Display Photographic Settings Subject Mouse a eco Pose i CJ Seg 1 Mode Exposure Binning FStop Excitation Emission Structure FOY Height pao Sal al Estee o 1 DEZ auto Medium Block 560 Yes C 1 50 2 WG ato Medium 3 i m Auto Medium Block 580 No Le 1 50 Block 600
205. enu bar to open the Image Layout window Click the 2 button to paste the active image into the Image Layout window Drag a handle W at a corner of the image to resize the image Drag the image to reposition it in the window 85 Living Image 4 3 1 User s Manual IVIS Spectrum Figure 4 30 Image Layout window pam TC image Layout Window DO m z ee oO O le vy amp v X F7 LayoutStyle Freestyle v TE w a mo r Annotation TAN oe Table 4 14 Image Layout window Chapter 4 Working With Optical Image Data Item Description Clears the Image Layout window Note If you do not clear the layout click the button before you close the Image Layout window the same window contents are displayed the next time the window is opened Opens a dialog box that enables you to save the Image Layout window contents to a graphic file Pastes an image of the active data in the Image Layout window Copies the contents of the Image Layout window to the system clipboard Pastes the contents of the system clipboard to the Image Layout window Rectangle drawing tool Ellipse drawing tool Pointer tool MOMON Arrow and line drawing tool Yy Send backward WY send to back zE Bring forward 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 lis
206. er 11 3D Reconstruction of Sources 202 11 6 Measuring Sources This section presents a convenient way to measure the source voxels total flux or total florescence yield or if calibrated the abundance in cells or picomoles after the reconstruction is finished or results are loaded The volume center of mass and depth at the center of mass are also reported in the 3D Tools Source tab M NOTE If the surface contains voxels pasted from other reconstruction results choose a source in the 3D Source tools Figure 11 18 For more details on pasting voxels see page 205 Determining the Source Center of Mass Follow the steps in Figure 11 18 after reconstruction is finished or results are loaded to determine the source center of mass Alternatively use the 3D ROI tool for more precise measurements See page 124 for more details on 3D ROIs Figure 11 18 Select and measure source voxels in the 3D View window 1 Ifthe surface includes voxels pasted from other 3 Click the Measure Source button Por results select a source from the drop down list then draw a box around the source 2 Confirm that Display Voxels is selected not Display Source Surface Tool Palette 2 E TLT20050624145507 sEQ Sas 2 ROI Tools I Sequence View L 3D View _ Spectral Unmixing and DyCE ko _ Surface Topography _ 3D Multi Modality Tools Gronal z 15 6 Sagittal x 3 5 7 3D Optical Tools
207. er 4 _ Animal Strain Animal Model FE Sex View Cell Line 7 Reporter C Treatment E Luc Injection Time 4 4 4 4 4 4 4 4 4 4 4 T IACUC Number If this is the first image of the session you are prompted to enable the autosave function Figure 3 27 When Autosave is enabled all images acquired during the session are automatically saved to a user selected location A different location can be chosen at any time select Acquisition Auto Save on the menu bar Figure 3 27 Autosave prompt LC Living Image 4 3 64 bit Auto Save xs m Do you want to enable auto saving of acquired data for this session This can be changed anytime from the Acquisition menu Yes No 4 Click Yes in the prompt to enable autosave then choose a location in the dialog box that appears Alternatively click No in the prompt and manually save the image data See page 54 for details Image acquisition begins and the upper area of the control panel changes to red color During acquisition the Acquire button in the control panel becomes a Stop button Click Stop to cancel acquisition The image window displays the images as they are acquired The control panel returns to blue color when acquisition is finished and the Tool Palette appears Figure 3 28 45 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition Figure 3 28 Image window and Too
208. erse is chosen the source intensity photons sec from low to high is represented using the color scale from red to transparent Log Scale Applies a log scale to the color scale MIP When this option is chosen all maximum intensity voxels in the view are projected along the viewing direction into the viewing plane Copies the 3D View tab in the Longitudinal Study window to the system clipboard Opens a dialog box that enables you to export the 3D View tab to a graphic file for example png Enables you to select voxels for measurement Measurements are displayed in the Plots tab Measuring Intensity 1 Load 3D reconstruction results and click the button By default a selection box appears around each surface Figure 11 25 This means that measurements for the entire surface will be computed 2 To select a particular region of the surface for measurements draw a box by clicking and dragging the mouse around the area The same box is applied to the other surfaces in the Longitudinal Study window 3 To clear boxes click the button again 208 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 209 Figure 11 25 Selection boxes around each surface photons sec DUT Results Viewing Plots To view a graph make a selection from the Analysis Type and Plot drop down lists in the Plots tab Figure 11 26 The followi
209. et a stage z 0 for each field of view To focus at the top of the animal the stage moves down so that the top of the animal is at z 0 You can enter the height of the animal and select the use subject height option or use the manual focus option to determine the proper subject height for the area to be imaged See Appendix A on page 265 for manual focus instructions Figure 3 10 Choose a focus option in the control panel j IVIS Acquisition Control Panel Imaging Mode Exposure Time Binnin J FiStop al Excitation Filter Field of View C MIS Idle Service 2 8 cm System Status Emission Filter Imaging Wizard Subject height 1 50 3 cm Sequence Setup ocus use subject height w Temperature Locked 6 Select Overlay to view an overlay image registered photograph and fluorescent image after acquisition M NOTE If you want to check the subject inside the chamber before acquisition take a photograph uncheck the Fluorescent option choose the Photograph option and click Acquire Be sure to check the Fluorescent option after taking the photograph 7 Click Acquire when you are ready to capture the image NOTE If necessary click Image Setup in the control panel to operate in single image mode In single image mode the z Sequence Setup button appears in the control panel Use this button to set up sequence acquisition see page page 42 for more details
210. ets Rindiant Etfisieney Mire 1 2587 Max 1 0088 Pipes 7 LEEG Mina 6 Deg Max 1 0186 5 Min 1 they Hax 2 7 led Tool Palette Ca Image Adjust l A Ga Corrections j Filtering A gt ROH Tools a Spectral Unmixing and DyCE Sy De eit Select Images Tyra f x GC t Excitation wavelength Emission wavelengths of the sequence F E Lee ee ef we Mie teach Cap Select Guided 2 Click the Analyze tab of the Spectral Unmixing and DyCE tools By default all wavelengths are included in the analysis Remove the check mark next to wavelengths that you want to exclude from the analysis Select Guided from the Methods drop down list and click Start Unmixing The Unmixing window appears Figure 8 3 Figure 8 3 Unmixing window Image cube shows a pseudo color image composite of colorized sequence images I HXx20120419114703 seq ko laf LE sequence view Unmiing HO as V Normalized Legend a 5 2 H No data available Spectrum List See Table 8 3 on web x page 155 for more We details on the toolbar Background Name Color Pick buttons if if Mime P 2 AF Hired v Sr 3 AF Bi bie X a I Image Cube Viewer V Overview Next Cancel List of the spectral components to unmix If the Imaging wizard was used to set up the sequence the list includes the probes selected in the wizard The default list also includes a tissue autofl
211. etup xsq sequenceinfo txt or clickinfo txt file Displays a dialog box that enables you to save the information in the sequence table to a sequence setup file xsq Display Photographic Settings Choose this option to include the photograph exposure time binning and F Stop in the sequence table subject If a subject and probe are specified optional the software uses the information to automatically set parameters in the Surface Topography DLIT FLIT Spectral Unmixing and Planar Spectral Imaging tools If a subject or probe is not selected here the default parameters appear in the Tool Palette Number of Segments The sequence specified in the sequence table is called a segment Choose this option to set the number of segments to acquire and the time delay between segments This is useful for acquiring data for kinetic analysis Delay Specifies a time delay between each segment acquisition 51 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition Table 3 9 Sequence table continued Item Description Apply to Al Applies the selected cell value to all cells in the same column Se Remove Remove Selected Deletes the selected row from the sequence table Remove All Removes all rows from the sequence table tf iaei Updates the selected row in the sequence table with the acquisition parameters in the b control panel Inserts a row abov
212. f the Select Name Color Pick unmixed images 1 V Kidney Mime v Z bt 2 V Brain E wippr 3 V Bladder Er x F E l Min 0 00 Max 2 45e4 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 9 DyCE Imaging and Analysis 160 9 2 Acquire a DyCE Sequence A DyCE sequence includes images acquired with a user specified time delay between exposures An acquisition can include up to three different time intervals where each interval is defined by duration and the delay between exposures NOTE For optimum DyCE analysis results acquire images using the Side Imager accessory PN CLS135111 ei M NOTE The IVIS Spectrum should be initialized and the temperature locked before setting up the imaging parameters See page 7 for more details 1 Click Imaging Wizard in the control Panel Figure 9 2 2 If necessary click Restart in the Imaging Wizard to show the first page of the wizard Figure 9 2 Opening the Imaging Wizard E A MIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter Imaging Mode Bioluminescence Imaging Sadoc ti the option for magng biolumniraani cr chemtluminascenit reporters such as frefly luciferase ack boti Lidiferass roni or bacterial Loira Field of View System Status Acquire Mouse Imaging Shuttle Idle Pumescerce IMNO Ga 134 Jom Sub
213. face Description Topography Tools Name Name of the selected surface Delete Removes the selected surface from the system Load Opens the selected surface Save Saves a surface to the selected name Overwrite Saves the surface and overwrites the previous surface results Export or Import a Surface A surface can be shared with other users or viewed in other 3D viewer applications M NOTE Surface import capability is only available if Show Advanced Options is selected in the general preferences see page 235 Load a surface 2 Select File Export or Import 3D Surface on the menu bar 3 In the dialog box that appears select a folder enter a file name and select a file type see Table 10 2 M NOTE Importing a surface by this method is for viewing purposes only not for registration with optical reconstructions in Living Image software To import a surface or other organs for registration purposes import an organ atlas See page 191 for more details Table 10 2 Surface file types Export Option Description Export Import Surface mesh A native file format of the Living Image software that is used to yes yes xmh exchange 3D surface information between Living Image software and other third party analysis tools It is based on a basic indexed face set format which stores all of the vertex information first then stores the triangle information in terms of indexes into the vertex list
214. file of pixel intensities j TLT20050624145507 006 its Counts v Display Overlay Tool Palette 7 Lone el mo Luminescence Width 12 6 cm Height 12 6 cm Image X Y 4 417 11 14 cm Image Data 2 count 4 Line Profile Window Line Orientation Horizontal v Width 1 3 Position 138 L a amp Counts x Min lo e3 x Max 239 3 N Min 26 Y Max 117 ban Full Scale Logarithmic Scale Color Scale 4 TLT20050624145507_006 Overlay Min 1122 x10 Counts Max 19546 2 00 na Line Profile window 1 00 0 50 0 00 O pixels 2 To view the line profile at another location in the image put the mouse pointer over the line When the pointer becomes a drag the line over the image The blue part of the line indicates the pixel intensities that are plotted in the line profile graph The line profile 1s updated as you move the line move over the image Table 4 8 Line Profile window Item Description Line Orientation Choose Vertical Horizontal or Free Hand from the drop down list to set the orientation of the line in the image window The Free Hand orientation enables you to drag each line segment endpoint to a user selected position Width Sets the line width The Line Profile window displays the average of the pixel values included in the line width Position Line position pixels Enables you to choose the g
215. for noisy images with high intensity pixels where changing the Threshold value is not helpful You can also use this method to focus on particular sources to reconstruct and ignore others To change the Threshold for a selected image 1 Click Start in the Analyze tab Figure 11 7 The Data Preview window appears 2 Click an image in the Data Preview window M NOTE Changes to Threshold are applied to the selected image only To apply the change to all images choose the Select All option 3 Click Data Adjustment 4 In the window that appears enter a new Threshold value The new Threshold appears in the Analyze tab 5 To reset the Threshold to the default value for the selected images click Restore Threshold Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Figure 11 7 Adjusting the Threshold Tool Palette 83 DLIT 3D Reconstruction le Analyze Properties Results lo Sequence 71720050624145507 SEQ Tissue Mouse Tissue Source Firefy Select Filters Fiter Threshold NP 560 07 translates the Threshold to the minimum counts required for reconstruction Keep the minimum counts gt 200 NNLS v Start Data Preview window CHa Pers SEE an E TLI20050624145507 sEQ arm ai Sequence View A 3D view Data Preview V Image Label V Median Filter
216. g amp Width 12 6 cm Height 12 6 cm B 00 009 0 00 0 00 Distance 0 00 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Table 4 6 Image Information tools Item Description li Click this button to display a histogram of pixel intensity see page 74 Click this button to display a line profile see page 75 Click this button to display a 3D representation of signal intensity see page 77 Click this button to display the distance measurement cursor in the image window see page 78 Click this button to draw and measure a rectangle on an image see page 79 Click this button to display hide a scale on the x and y axis of the image window Click this button to display hide a grid the image window Units Choose the units cm or pixels for distance measurements in the image window Image Binning The binning applied to the image Note If soft binning is applied to the image data and the binning level is changed from 8 to 16 the new binning is indicated as 8x2 Width Height The FOV dimensions Note If Pixels is selected from the Units drop down list the dimensions are provided in terms of binned pixels Image X Y The x y pixel coordinates of the mouse pointer location in the image Image Data The intensity at the pixel location of the mouse pointer The intensity is represented in the un
217. ge or sequence The options available in the Tool Palette depend on the type of active image data A tool is only available if the data set includes the components that the tool requires to perform the analysis NOTE The 3D Multi Modality tools and DyCE tools require a separate license ell Figure 4 7 Tool palette See page 11 for an overview of the Living Image tools ea Tool Palette Tool Palette Click to expand a tool a a mA pla ETE aaa Photo Adjustment E Brightness B 100 Contrast la 1 5 saa Jio FE Color Scale Min f 6 60e6 Max fe 1 28e8 Color Scale Limits Auto gt Full 5 Manual Individual Color Table eon J a Reverse Logarithmic Scale Living Image 4 3 1 User s Manual IVIS Spectrum 4 3 Viewing Image Information At acquisition the software captures image information such as camera parameters and any image label information you entered at acquisition time Figure 4 8 Chapter 4 Working With Optical Image Data Figure 4 8 Image window displaying image information f Living Image 4 3 1 Click Info to display the image label and acquisition information DER a By i a amp R Units Radiant Efficiency C Apply to All l File Edit View Tools Acquisition Window Help j TLT20040217113205 f Units Radiant Efficiency Display Overlay info
218. ge window so that you can manually adjust the stage position For more details on manual focusing see page 265 Batch Sequences Choose this option if you want to specify multiple separate image sequences for batch acquisition multiple image sequences are automatically acquired one after another without user intervention See page 22 for more details Temperature L m E The temperature box color indicates the temperature and status of the system a White box System not initialized a Red box System initialized but the CCD temperature is out of range a Green box System is initialized and the CCD temperature is at or within acceptable range of the demand temperature and locked The system is ready for imaging Click the temperature box to display the actual and demand temperature of the CCD and stage See page viii for more details Acquire Click to acquire an image using the settings and options selected in the control panel or to acquire an image sequence specified in the Sequential Setup table Living Image 4 3 1 User s Manual IVIS Spectrum Appendix A IVIS Acquisition Control Panel 265 Table A 1 IVIS acquisition control panel continued Description Sequence Setup Click to display the sequence table so that you can specify and manage sequence acquisition parameters or open sequence acquisition parameters xsq See page 22 for more details on setting up an image sequence Imaging
219. ges of the light emission from the subject surface that is acquired at different filter bandpasses Table 11 1 FLIT The input data to the FLIT algorithm for 3D reconstruction of fluorescent light sources includes A surface topography of the subject generated from a structured light image m A sequence of images acquired at different transillumination excitation source positions using the same excitation and emission filter at each position Table 11 1 Table 11 1 IVIS Spectrum filters for luminescence or fluorescence tomography Filters Range Bandwidth 10 excitation filters 415 760 nm 30 nm 18 emission filters 490 850 nm 20 nm Quantification Database Optional If a quantification database is available it is possible to determine the number of cells in a DLIT source or the number of cells or dye molecules in a FLIT source The database is derived from an analysis of images of known serial dilutions of luminescent or fluorescent cells or dye molecules in a well plate See Chapter 12 on page 231 for more details on generating a database Using a quantification database is optional Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 186 Overview of Workflow for 3D Reconstruction of Sources Figure 11 1 Basic 3D reconstruction workflow KE naatasan nG m D pepee les Urti Radiance Pha ee Send Cola 1 T inky g al n Ci 1 Setup a DLI
220. ght click the selected table cells and choose Background Color on the shortcut menu b Choose a color from the color palette that appears Figure 12 4 Select the sample wells and enter the number of cells or molecules F Well Plate Quantification Window For Sequence EL20090414101005 SEQ Image EL20090414101005 001 r For Sequence EL20090414101005 SEQ Image L20090414101005_001 20090414101005 001 ma Fluorophore Type Fluorophore Type Alan ae poe ee Measurement Measurement Sample Wells 30 3A Sample Wells 3D 3A Background Wells Set Background Wells Set Well Volume uL Well Volume uL C Apply to Sequence E Apply to Sequence Well Plate Well Plate Set position and enter dilution values in nanoMolar units i Set position and enter dilution values in nanoMolar units For Image EL20090414101005 001 For Image EL20090414101005 001 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 12 Quantification Database 234 9 Enter the concentration values in the table cells in nanomolar units if calibrating fluorescent dyes Enter the cell values in dimensionless units if calibrating cells 10 To delete a concentration or cell value select the table cell and press the Delete key Alternatively right click a selected value to view a shortcut menu of edit commands for example cut copy paste 11 Enter the fluid volume microliters for the highlighted wells The h
221. graph option and click Acquire Be sure to select the Luminescent option after taking the photograph 6 Click Acquire when you are ready to capture the image NOTE If necessary click Image Setup in the control panel to operate in single image mode In single image mode the Sequence Setup button appears in the control panel Use this button to set up sequence acquisition see page 42 for more details on sequence setup 7 Enter information about the image in the Edit Image Labels box that appears optional Click OK M NOTE You can enter image label information at any time during or after acquisition If you do not want to enter image information click Cancel See page 65 for details on adding information to an image after acquisition Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition 27 Figure 3 4 Enter information to include with the image optional TC Edit Image Labels Ha UserID g xv Living Image Universal Saved Labels LABELS 1 AER lt User w aa 7 Information entered here appears in the am Saan image label see Figure 3 6 on page 28 Male nu nu day 8 Mouse 4 c ti Spectrum CT lt K S T Comment2 v Time Point v V Animal Number v Animal Strain v Animal Model v F Sex v View X Cell Line v Reporter v C Treatment v E Luc Injection Time v IACUC Number w Apply To Sequence If t
222. ground acquisition Acquisition Fluorescent Background Measure Fluorescent Background Starts a measurement of the instrument fluorescent background Acquisition Fluorescent Background Add or Replace Fluorescent Background Opens a dialog box that enables you to select an instrument fluorescent background measurement for the active image data If the Fluorescent Background Subtraction option is chosen in the Corrections Filtering tool palette the background measurement is subtracted from the image data Acquisition Fluorescent Background Measure and Replace Fluorescent Background Measures fluorescent background under the same conditions as the currently selected image When the measurement is complete the newly acquired background image will be included in the data set of the current image replacing any existing background image that may be present in the data set Acquisition Fluorescent Background View Available Fluorescent Background Opens a dialog box that displays the fluorescent background measurements for the system If a fluorescent background is selected the Fluorescent Background Subtraction option appears in the Corrections Filtering tool palette Choose the Fluorescent Background Subtraction option to subtract the user specified background measurement from the image data Acquisition Fluorescent Background Clear Available Fluorescent Backgr
223. he number of slices shown in the viewer Render Thick Slice This option is used to create a sequence of 3D or maximum intensity projection MIP renderings from the image stack When this option is selected Slice Spacing changes to Slice Thickness Increasing the slice thickness causes more slices to be extracted from the volume before creating the rendering Gradient Illumination Gradient Illumination is based on the idea that light is reflected at boundaries between different voxel intensities but is not affected when passing through homogeneous regions Choosing this option illuminates the voxels at boundaries more than voxels within a homogeneous region The boundaries are based on the gradient magnitude between heterogeneous regions or the change in intensities between neighboring voxels in heterogeneous regions Using this option enhances the variation in tissue properties and may be helpful for visualizing the boundaries of different tissues Maximum Intensity Projection MIP Projects all maximum intensity voxels in the view along the viewing direction into the viewing plane 247 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools Table 13 4 Volume Slice Viewer continued Item Description Min 15 00 mm p Max 15 00 mm Min The slice coordinate of the first slice being viewed Zero is defined as the center plane of the image Ma
224. hee eee ADA REEN AERES 246 Volume Information and Results 2 249 ManaGIMGURESUINS AA AA AA 249 Registering Optical and Volumetric Data 00 eee ees 250 Loading Data for Registration anaa aaaea 252 Registering Multi Modal Data 0 es 254 Volume Data Viewer 0 000 a 258 Viewing RAW Volumetric Data cc es 259 IVIS Acquisition Control Panel 02 002 ee eee eee 262 Control Panel 6 oa a ete ey ede ee oe ee a ee a AG al 262 Manually Setting the Focus 0 0 es 265 LALA AA ee a OR Ce ew ee ee oe 267 General Preferences cee eee eee eee eens 267 DODONG nama maTAPRGAGAADLI TANI eeeeauedaseeigeates ERI KAPANGANAKAN 269 ACOSO AA PA 270 WGCING 464665505545 BERS Reha he eh GEES PINADAAN Oe AA NA kA AG 271 Optical Properties 2445666445544 52 000 Se beeen esos BAAL DARE MGANDA NG 274 Menu Commands Toolbars and Shortcuts 20 275 Sn sey Sree da a vet oak Puy We et veh Vek BAN eae APA IA Pees av et se cere AG NA etm ret E ete ce et AA 279 5 1 Welcome About This Manual What s New in the Living Image 4 3 1 Software on page 2 Living Image Help on page 3 Caliper Technical Support on page 4 1 1 About This Manual NOTE This Living Image Software 4 3 1 User s Manual part no CLS135291 is only for use with the IVIS Spectrum instrument This user manual explains how to acquire optical and volumetric image data on the IVIS Spec
225. hen mark in the analysis See Table 11 4 for details on the tools the area to include in the reconstruction using the pencil tool gf In this example the red area marked with the pencil tool will be reconstructed Table 11 4 Region Selection Tools Item Description Draw Choose this option to display the pencil tool IS when the mouse pointer is over the data adjustment image Use this tool to apply markings that select regions to include in the reconstruction Erase Choose this option to display the eraser tool Use the eraser to remove pencil tool markings exclude pixels from the image Painting size Adjusts the width of the pencil tool mark or the eraser tool Segment Colors available for the pencil tool Opacity Adjusts the opacity of the pencil tool markings Reset Removes all pencil tool markings Living Image 4 3 1 User s Manual IVIS Spectrum 11 3 Reconstructing Fluorescent Sources Image Sequence Requirements Use the Imaging Wizard to set up the image sequence required for FLIT analysis For more details on the Imaging Wizard see page 42 If you plan to manually set up the sequence use transillumination on the IVIS Spectrum CT and the same excitation and emission filters from at least four source locations that form a rectangle Acquire the following Chapter 11 3D Reconstruction of Sources m Fluorescent image and photograph at the first transillumination locatio
226. hick Slice 2 Surface Topography lt 7 3D Multi Modality Tools Volume Process Slice Results Render Slice Using Slice Color Table Volume Color Table Color Table Red z aie Reverse Opacity Color Scale Min B 0 Max ij 33729 Slice Viewer E Choose a slice Orientation Coronal v Fil orientation Max 15 00 3mm Click the button to view the slices in a separate window See Figure 13 11 on page 247 for more details on the Volume Slice Viewer Use the slider to move through the selected slices 3D Optical Tools gt DLIT 3D Reconstruction 246 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools Figure 13 11 Volume Slice Viewer Multi View Single View CE Volume Slice Viewer Orientation Coronal bd Slice Spacing 1 50 Render Thick Slice Min 15 00 zE mm Total Slices 21 Q a Q nG terde Thick Sor Double click a slice to display the single view Table 13 4 Volume Slice Viewer Item Description Orientation Select a slice orientation from the drop down list Slice Spacing The distance between each slice in the Volume Slice Viewer Enter a smaller value to increase the number of slices in the viewer or a larger value to decrease the number of slices in the viewer Total Slices T
227. his is the first image of the session you are prompted to enable the autosave function Figure 3 5 When Autosave is enabled all images acquired during the session are automatically saved to a user selected location A different location can be chosen at any time select Acquisition Auto Save on the menu bar Figure 3 5 Autosave prompt Fa E Living Image 4 3 64 bit Auto Save xs r E Do you want to enable auto saving of acquired data for this session This can be changed anytime from the Acquisition menu 8 Click Yes in the prompt to enable autosave then choose a location in the dialog box that appears Alternatively click No in the prompt and manually save the image data See page 54 for details Image acquisition begins and the upper area of the control panel changes to red color During acquisition the Acquire button in the control panel becomes a Stop button Click Stop to cancel acquisition The control panel returns to blue color when acquisition is finished and the image window appears Figure 3 6 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition Figure 3 6 Overlay luminescent image on photograph in the image window FS ARS G AS 9Z414550 UL Image TLT20050624145507 001 Fri Jun 24 2005 07 55 59 Em Filter 560 Bin M 8 FOV 12 6 f1 1s Living Image Version 2 50 1 5 20 2005 Camera IVIS 200 Beta II SI620EEV int Ca
228. ht to adjust the gamma of an image displayed in overlay mode Alternatively enter a gamma value Gamma is related to image contrast Opacity Click and move the slider left or right to adjust the opacity of the pseudocolor luminescent data of an image displayed in overlay mode Alternatively enter an opacity value Color Scale Min The minimum pixel intensity associated with the color scale for an image Pixels less than the minimum value are not displayed Max The maximum pixel intensity associated with the color scale for an image Pixels greater than the maximum value are displayed in the maximum color Color Scale Limits Auto If this option is chosen the software sets the Min and Max values to optimize image display and suppress background noise The Min and Max settings can be manually adjusted to further optimize the image display for your needs Full Choose this option to set the Max and Min values to the maximum and minimum data values in the image Manual Choose this option to enter Max and Min values for the image display Individual Applies a separate color scale limits to each image in a sequence Note This option is only available when an image sequence is active Color Table ene KC Click the drop down arrow to select a color table for the image data For more details on color tables see the concept tech note Image Display and Measurement Reverse Choose this option to reverse th
229. ialog box that appears Figure 8 20 Click Apply to add the computed spectrum to the spectrum plot and list in the Unmixing window Alternatively select an existing spectrum from the Name drop down list and click Apply to overwrite the results 152 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing Figure 8 20 Choose spectra to subtract A x B C NG A Compute Pure Spectrum A Mixed Spectrum e g autofluorescence label V Normalized H 1 TissueAF Choose E 2 4F680 spectrum A E 54750 1 00 0 50 0 0 660 700 740 780 Emission Wavelength nm B Known Spectrum e g autofluorescence V Normalized Ej 1 TissueAF Choose BB 2 areso spectrum B Hl 54750 c Computed Spectrum e g label Result C A 1 3 B Computed spectrum C V Auto Scaling E Fit Offset Error Tolerance p 0 660 700 740 780 Emission Wavelength nm Normalized 8 x10 3 2 5 2 1 5 1 0 5 P 700 780 0 660 Computed spectrum C 740 Emission Wavelength nm Save to Spectrum Index Name Color New m uma Ill magenta Close Apply Table 8 1 Computed spectrum Item Description Normalized Choose this option to display spectra normalized on a scale from zero to one Result C A x B The subtraction performed by the software where x is a factor that ensures the residual signal is positive Auto
230. ide the selected range are not used in the color rendering process By default the entire color range is selected The wavelength range of the luminescent images in the sequence The two sliders determine the lower and upper end of the filter range Only the parts of the image that are within the selected wavelength range are colorized By default the entire filter range is selected Log Scale If this option is chosen the dynamic range of the brightness in the image is compressed using a log scale This improves the visibility of dark areas in the image Real Color If this option is chosen the colors are rendered using the wavelengths that directly correspond to the camera setup For example GFP appears green using real color rendering If this option is not chosen the original wavelength range of the image is modified to include the entire visible wavelength range of the camera setup This helps improve the color contrast Click this button to copy the colorize view to the system clipboard a Click this button to export the colorize view as a graphic file for example jpg Click this button to print the colorize view 4 11 Exporting or Printing Images The Image Layout window Figure 4 30 provides an alternative way to Annotate and export an image for example bmp m Print an image Copy an image to the system clipboard oS Select View gt Image Layout Window on the m
231. idual Images from the list to show the open Label single images in the User Label Name Set Living Image Universal ma g es d ro p d own ist luminescent image luminescent TIF photographic image photograph TIF Camera System Info readbiasonly image readbiasonly TIF Choose the Show All Sections option to display all categories of image information Key Acquisition Date Thursday October 27 2011 E Acquisition Seconds 3402592204 Bg Acquisition Time 13 30 04 Adapative FL corrected 0 Background Corrected 1 Binning Factor 4 Cosmic Drop down list of images in the selected sequence Or a list of single images if Individual Images is selected in the Sequences drop down list 64 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 4 To view particular information select a category in the upper box to show the associated information in the lower box For example select luminescent image in the upper box to show the luminescent image acquisition parameters Editing the Image Label You can edit image label information or add information to the label after acquisition To edit the image information 1 Open an image or sequence 2 Click Info to display the image label Figure 4 10 Image information EC TUT20050624145507 505 som x Units Counts ba Display Overlay v Options v 2n nG sj Im
232. iew select Help Tech Notes on the menu bar Table 3 1 Field of View FOV settings FOV Setting FOV cm A 4 B 6 5 C 13 D 22 5 19 5 E 22 5 26 Some IVIS Spectrum instruments may have the FOV in parentheses FOV 19 5 and 26 were replaced by FOV 22 5 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition 26 4 Select a focus option in the control panel Figure 3 3 The focal distance to the camera is set a stage z 0 for each field of view To focus at the top of the animal the stage moves down so that the top of the animal is at z 0 You can enter the height of the animal and select the use subject height option or use the manual focus option to determine the proper subject height for the area to be imaged See Appendix A on page 265 for manual focus instructions Figure 3 3 Choose a focus option in the control panel j IVIS Acquisition Control Panel Imaging Mode Exposure Time inning Excitation Filter Emission Filter Field of View System Status Acquire C MIS Idle 2 8 Imaging Wizard Subject height w Sequence Setup Focus use subject height Temperature AN Locked 5 Select Overlay to view an overlay image registered photograph and luminescent image after acquisition NOTE If you want to check the subject inside the chamber before acquisition take a photograph uncheck the Luminescent option choose the Photo
233. ighlighted well volumes must be equal 12 Choose the Apply to Sequence option 13 Choose the Background Wells option 14 In the well plate table select the background wells and click Set Clicking a row or column header selects the entire row or column To remove the background well designations click the button Figure 12 5 Set the background wells A Well Plate Quantification Window For Sequence EL20090414101005 SEQ Image EL20090414101005_001 v Fluorophore Type o Dye naleaes Cele Measurement f Sample Wells 3D 3A C Set V Background Wells 6D 6A Well Volume pL Apply to Sequence Well Plate Quantification Plots Results Set position and enter dilution values in nanoMolar units r For Image EL20090414101005 001 Quantify 15 Click Quantify The results are displayed Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 12 Quantification Database Figure 12 6 Example fluorescence quantification plot and results fa a a EEE a ae A Well Plate Quantification Window A Well Plate Quantification Window o Em For Sequence EL20090414101005_SEQ Click EL20090414101005_001 For Sequence EL20090414101005_SEQ Click EL20090414101005_001 Fluorophore Type Fluorophore Type H WellPlate Type EREEREER Well Plate
234. igure 13 1 shows how the histogram tool designed a color opacity map that shows both the skin and bone The histogram tool enables you to easily re design the color opacity map to show only the skin or only bone The 3D Multi Modality tools also enable you to classify the volumetric data by specifying color and opacity values for different intensity ranges so that you can easily view or hide certain parts of the data as needed A color opacity map can be saved Figure 13 1 Histogram tool specifies the opacity for different voxel intensities Histogram of voxel intensities Air tissue boundary Colors and opacities assigned to voxel intensities transparent Transparent Opaque to 60 Opaque Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools Specifying a Color Opacity Map m After the surface and volume data are loaded confirm that the Display Volume option is selected Figure 13 2 3D Multi Modality tools 63 Tool Palette E3 gt ROI Tools le gt Spectral Unmixing and DyCE E _ Surface Topography 3D Multi Modality Tools Volume i Process Slice Results esoscossesesessosoeossossossessessg tel Color Opacity Map Grays Logarithmic Histogram Select Counts Absorption option selected or Hounsfield units for the histogram display Autofit air noise bound
235. igure 3 17 Open the Transillumination Setup dialog box IVIS Acquisition Control Panel Excitation Filter Emission Filter F 2 w aw 8 j Transillumination Setup Single Location Mode C Move Motors To Selected Spot C Mask Grid Points To Subject Grid Type 15x23 C Raster Scan Seis z Field of view C ng System Status C MIS Idle Service 12 8 cm Subject height 1 50 cm Temperature Locked Focus use subject height Single location mode acquires one image at the location marked by a green square J Table 3 4 Transillumination Setup box Item Description Move Motors to Selected Transillumination motors will move the excitation light source to the grid Spot location selected in the Transillumination Setup dialog box Mask Grid points To When setting up a transillumination sequence choose this option to Subject automatically select only the grid locations within the subject boundaries Grid locations outside the subject are masked out The mask prevents the transillumination excitation source from selecting an uncovered hole Projecting light through an open hole would saturate the camera Raster Scan If this option is not selected the software generates one image per transillumination location per filter pair For example a sequence setup that includes 20 locations using two filters
236. image if you modify the Threshold move the slider or enter a new value the software automatically updates the ROls Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data Table 5 2 ROI tools for optical images continued Item Description Note The following Auto ROI parameters are only available if Show Advanced Options is selected in the general preferences For more details on setting Preferences see Appendix C page 168 Lower Limit Specifies a multiple 1 to 10 of the color scale minimum that sets the lower threshold for identifying an ROI For example if the lower limit 2 and the color scale minimum 1000 counts then the auto ROI tool will only draw an ROI on areas of 2000 counts or greater This helps create ROIs only within pixels visible on the image Minimum Size Sets the minimum size of an ROI measured in pixels For example if the minimum size is set at 50 then ROIs created on the image must be greater than 50 pixels in size Preview If this option is chosen the software draws the ROI each time a parameter is changed ROI parameters can be saved without drawing the ROI Use Bkg Offset Choose this option to measure background corrected signal This is typically used to remove natural animal background luminescence and should not be confused with the dark charge and read bias background corrections that are applied by default to the raw CCD
237. imation Na ly to All pply to All lap 3 tal Longitudinal Study Well Plate Quantification for ARW20050826124002 SEQ Image Overlay for AR W20050826124002_SEQ Colorize for ARW20050826124002 SEQ Image Math for ARW20050326124002 SEQ Ej ARW20050826124002 001 BAR TEC gt Image Adjust Units Radiant Efficiency v Display Overlay gt Corrections Filtering gt Image Information gt ROI Tools Radiant Efficiency t sec cm sh yWwicm Color Scale Min 9 38e7 Max 1 7369 Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum Chapter 2 Getting Started Apply corrections or filters to the raw data Field Correction Cosmic Correction Lens Distortion Correction Adaptive FL Background Subtraction Normalization Threshold Counts 100 secotine here 7 Living Image Tools and Functions See Page Image Adjust 68 l Tune the photograph brightness contrast or Brightness P t00 opacity et T i Change the minimum or maximum of the color an a i lied to the optical i dat scale applied to the optical image data Min 2140 Select a color table for image display Max fa 36446 z Color Scale Limits Auto Full Manual Individua Color Table Raww OO DN V Reverse E Logarithmic Scale AT area z Corrections Filtering Optical Data 70 13 Living Image 4 3 1 User s Manual IVIS Sp
238. ing the slider or enter a number in the box The organs are easier to view if you uncheck Skin in the Organs list To clear all organs from the surface click the Deselect All button 8 To hide a particular organ remove the check mark next to the organ name the Select All button To display a specific _ choose the organ name To display all organs on the surface click NOTE After fitting organs to the surface using the H or H tool if necessary you can click Reset button to restore the default fit F 220 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Manually Adjusting the Scale or Location of Organs 1 Load reconstruction results and confirm that the surface is in the perspective view click the toolbar button in the 3D View window or press the R key M NOTE It may be helpful to view the 3D image from different perspectives to check the organ position and size To turn and rotate the 3D image press and hold the left mouse key then drag the mouse when the hand appears In the 3D registration tools choose the Display Organs option and select an organ atlas The organs in the selected atlas appear on the surface In Figure 11 33 only Skin is selected Click the Transform tool button EB The transform tool appears Figure 11 34 explains the tool functions Figure 11 33 Displaying the transform tool T TLT20050624145507_SEQ o amp
239. ion 5US 00U 005 00U 0U5 NUN 005 PANUT 740 605 760 605 780 605 800 Index 2 Name AF680 Origin HX20120419114703 SEQ WavelengthPairs 605 660 605 680 605 700 605 720 605 740 605 760 505 720 505 800 Index 3 Name AF750 Origin HX20120419114703 5EG WavelengthPairs 605 560 605 520 505 700 505 720 505 740 605 760 605 730 605 300 show qualified only 0 0 660 FOO 740 Emission Wavelength rm Spectrum List Select 1 TissueArF AF680 Name Choose this option to show only spectrum libraries with excitation emission filters that match the data set being analyzed Ca Normalized U Legend Tall Color Use these controls to include or exclude spectra from the analysis rename a spectrum or change the spectrum plot color Figure 8 8 Spectral unmixing results E HX20120419114703 sEQ E sequence View Unmixing L V Show Labels V Individual Scale Co LO fata Normalized Legend a g x10 6 amp 5 Spectra plot See page 154 for more 3 details 2 1 0 660 700 740 780 Emission Wavelength nm Spectrum List See Table 8 3 on page 155 for details Note on this toolbar Select 1 2 V 3 M Name Color Pick TissueAF D ime zi M E AF680 z f AF750 ff bee ii Tissue AF Min 0 00 Max 9 13e7 AF 750 Min 0 00 x 2 3468 a GG Min 0 00 x 6 4666 Composite Unmixe
240. ion 64 bit Windows C Program Files Caliper Life Sciences Living Image NOTE All components of the IVIS Spectrum imaging system should be left on at all times due to the long cooling time required to reach operating demand temperature It is also important to leave the system on to enable automatic overnight electronic background measurements Periodically rebooting the computer is permissible and does not affect the camera operation el Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 2 Getting Started 6 To start the software 1 PC Users Double click the Living Image software icon F on the desktop Alternatively click the Windows Start button and select All Programs Caliper Life Sciences gt Living Image Macintosh Users Double click the Living Image icon Fg on the desktop or run the software from the application folder The main window appears Figure 2 1 Figure 2 1 Living Image main window at startup IC Select Add User ID Add New User 2 In the dialog box that appears select a user ID from the drop down list If the user ID is password protected enter the password and click OK Alternatively create a new user ID In the Select Add User ID box click the button Enter a user ID Enter and confirm a password This is optional Click Add and OK The control panel appears if the workstation controls the IVIS Spectrum Figure 2 2 For more
241. is obtained from a measurement ROI that is located in an area where no fluorophore signal is present The scale factor k accounts for different levels of tissue autofluorescence due to different excitation wavelengths and filter transmission characteristics After you acquire an image sequence that includes a primary and background image use the image math tool to subtract tissue autofluorescence For more details on acquiring an image sequence see Chapter 3 on page 42 135 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 7 Image Math 136 To subtract tissue autofluorescence 1 Load the image sequence that includes the primary and background fluorescent images Figure 7 3 Image sequence AASA a T3938T9 IC KSA20110305104918 SEQ So a Sequence View Units RadiantEfficiency F Use Saved Colors Info R 9 NG Radiant Efficiency pisecjcm3 UW cm Color Scale Min 1 75e6 Max 1 73e8 TLT20060510114512 0084 TLT20060510114512 0104 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 M NOTE You only need to draw the ROI on one of the images The software copies the ROI to the other image Figure 7 4 Draw measuremen
242. isplays the animal surface and the reconstructed sources m In the Tool Palette the Results tab displays the results data and the algorithm parameter values m The 3D Tools appear after a reconstruction is generated or loaded For more details on the 3D Tools see page 213 225 For details on managing results for example save load or delete see page 198 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 190 Figure 11 5 DLIT reconstruction results 3D View toolbar A TLT20050624145507_SEQ ka elle Tool Palette z3 Sequence View lZ 3D View gt ROI Tools fa gt Spectral Unmixing and DyCE gt _ Surface Topography A gt 3D Multi Modality Tools C gt 3D Optical Tools DLIT 3D Reconstruction Gronal z 15 6 Sagittal x 3 5 Analyze Properties Results DLIT Results Key Value Final voxel size mm 1 25 Number of voxels 37 Reduced Chi2 8 26e 02 Index Of Refraction 1 40 Damp Reduce Final 1 00e 02 Angle limit deg 70 Damping reduce 100 Data range Top view L_Curve 1 Mirror XOffset 37 ak Transaxial y 18 1 Photon Density Maps sk Export Results Save Results Name DLIT_3 ind Delete Load hotons sec Source Intensity Subject Height 26 2 mm ff Perspective Table 11 3 3D View tools Tool Description Image Tools A drop down list of to
243. it Space and Orientation button ET 2 In the dialog box that appears Figure 13 7 edit the pixel or slice spacing Figure 13 7 Volume Information dialog box p 7 Volume Information 7 xa Slice Information Pixel Spacing Row Column 0 1000 0 0016 mm Slice Spacing 0 0016 mm Subject Orientation Invert X Axis Invert Y Axis Invert Z Axis ox _ cance 13 4 Smoothing a Volume Smoothing can be applied to a volume to reduce noise in a CT MRI or PET image such as excessive variation in voxel grayscale values Smoothing computes the average grayscale value of a group of voxels for example a 3x3 group and applies the average value to the central voxel of the group To apply smoothing 1 Load the volumetric data 2 Choose the type of smoothing and group size in the Process tab of the 3D Multi Modality tools Figure 13 8 3 Click the 6 button to remove the smoothing Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools 245 Figure 13 8 3D Multi Modality tools Process tab 13 5 Rendering and Viewing Slices The Slice tab in the 3D MM tools contains rendering and viewing options for slices Rendering Slices Figure 13 9 Perspective view and slice views displayed using different color tables R Ela eo merene Render Slice Using Slice Color Table Volume Color Table Color Table Reverse Opacity
244. its currently selected for the image Crop Distance The x y pixel coordinates at the upper left corner of the crop tool panira OR kalng The x y pixel coordinates at the A end of the distance The x y pixel coordinates at the lower right corner of the crop tool i pi OR PAA The x y pixel coordinates at the B end of the distance The width and height of the image crop tool OR Ax Ay from the A to B end of the distance measurement cursor For more details see page 78 and 79 73 Living Image 4 3 1 User s Manual IVIS Spectrum Viewing X Y Coordinates and Intensity Data 1 Open an image and the Image Information tools choose Cm or Pixels from the Units drop down list 2 Put the mouse pointer over a location in the image The x y coordinates and intensity data are displayed in the Tool Palette Chapter 4 Working With Optical Image Data Figure 4 18 x y coordinates and intensity data at the mouse pointer location 11120050624145507_006 Units Counts Display Overlay v DER orons v emre yn Luminescence Counts Color Scale Min 1122 Max 19546 Tool Palette gt Image Adjust 4 gt Corrections Filtering a Image Information paan l gl w DY TB units cm Image Binning amp Width 12 6 cm Height 12 6 cm Image x Y 6 171 7224 cm If Image Data 7555 counts Crop Distance Ps i iB i 2 00 8 00
245. izard Subject height 15 cm Image Setup Focus use subject height Temperature fg Locked C Number of Segments F Delay 0 0 min Apply to All Ad Update Insert Add 3 Choose a subject and probe from the drop down lists Figure 3 32 Figure 3 32 Choose a subject and probe Ei C Display Photographic Settings Subject Mouse Bioluminescent Probes Unselect Probes Mode Exposure Binning FStop Excitation Emission Structure FO Height Fluorescent Probes at i Bacteria l 1 DEE auto Medium 1 Block 560 Yes C 1 50 Unselect Probes CBGreen 2 ES ato Medium 1 Block 580 No C 1 50 CBRed 3 KJ awto Medium 1 Block 600 No c 1 50 4 old 4 WI ato Medium 1 Block 620 No c 150 ta pa s DBA Auto Medium 1 Block 640 No C 1 50 YPM 2 LED 2n5 C Number of Segments DEJ Delay min Apply to All A Update Insert Add Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition 4 Specify the imaging settings for the first image in the sequence See Appendix A on page 262 for details on the imaging parameters in the control panel M NOTE f you selected Photograph and the photograph Reuse option in the control panel Figure 3 33 the IVIS Spectrum acquires only one photograph for the entire sequence If this option is not chosen the system acquires a photograph for each image in the sequence
246. ject height 1 50 cm B Focus use subject height v Temperature Locked cancel Newt If this screen does not appear when the wizard starts click Restart Wizard on the wizard screen to restart the wizard 3 Double click Bioluminescence ff Mor Fluorescence i imaging in the wizard Bamryarin M NOTE Choose Fluorescence imaging if using near infrared probes Choose Bioluminescence imaging if using a Cerenkov radioactive probe 4 Choose the DyCE option Figure 9 3 and click Next Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 9 DyCE Imaging and Analysis Figure 9 3 Choose the DyCE option Imaging Wizard Bioluminescence options TE imago wer soore ita Imaging Wizard Fluorescence options ej Imaging Wizard Fluorescence bale Bioluminescence DyCE DyCE in the luminescent mode is an optical imaging approach that can provide detection of radiotracer distribution in preclinical animal models by tracking the Cerenkov emission from the charged decay products While the charged particles travel faster than the speed of light in the medium such as tissue photons in the visible band are emitted via the Cerenkov effect and these photons can be detected by optical imaging Open Filter This technique utilizes a time series of optical images acquired following a small bolus injection of a radiotracer As the radiotracer circulates through the body each o
247. just the mask so that it matches the underlying subject photograph as closely as possible without including any area outside the subject image Draw Mask Choose this option to manually draw a data mask on an area of the photograph Rectangle Specifies a rectangular shape for the manual data mask Ellipse Specifies an elliptical shape for the manual data mask 103 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data Figure 5 14 Mirror ROls on a fluorescent image acquired with the Side Imager I EL20120411170231 Col fee Units Radiant Efficiency v Display Overlay v Options Info G ij Epi Fluorescence Radiant Efficiency t secicm sn ywiicrn Color Scale Min 4 41e9 Max 7 89e9 Tool Palette Image Adjust _ Corrections Filtering _ Image Information 7 ROI Tools O OF amp W Measure ROIs x Apply TO sequel Type Mirror ROI v V Photo Mask Save ROIs Name ROI 2 KSA v el NOTE The ROls are numbered in the order they are created You may want to arrange the ROls in a known order for easier comparison between images To renumber the ROls ascending order from right to left right click the image and select Sort ROIs on the shortcut menu If the Apply to Sequence option is selected in the ROI tools choose Sort ROIs in Sequence to sort a
248. k to apply the default settings 273 Living Image 4 3 1 User s Manual IVIS Spectrum B 5 Optical Properties Figure B 8 Set the default optical properties preferences left for the Properties tab in the Planar Spectral Imaging DLIT or FLIT tools Tool Palette _ ROI Tools gt Spectral Unmixing and DyCE _ Surface Topography 3D Multi Modality Tools gt 3D Optical Tools DLIT 3D Reconstruction Analyze Properties Results gt La JL IG A Preferences General Options Acquisition Theme Optical Properties Preview Tissue Properties Mouse Tissue Bioluminescent Source Spectrum Firefly Fluorescent AF680 Source Spectrum Plot Tissue Properties v Restore Defaults Tissue Properties Mouse Tissue z Source Spectrum Firefly v Plot Tissue Properties z Luminescent Calibration Database not found cm eff Wavelength nr Wavelength nm Table B 7 Tissue properties preferences Appendix B Preferences Item Tissue Properties Description Choose a default tissue type that is most representative of the area of interest This tissue type will be used if a Subject Type is not selected in the Imaging Wizard and saved during acquisition Source Spectrum Choose the default lu
249. l IVIS Spectrum Chapter 11 3D Reconstruction of Sources Table 11 8 3D Scene Exporter dialog box continued Item Description Slice Orientation Choose transaxial coronal or sagittal slices for the export Export voxels using original resolution Choose this option to export source voxels without any smoothing or binning The original resolution of the source voxels is the resolution obtained after DLIT or FLIT reconstruction approximately 1mm resolution Slice Resolution Sets the number of slices required to accommodate the slice orientation with good slice sampling spacing Total Slices Slice spacing Pixel spacing Parameters that determine the number and resolution of the slices to export Solid mesh If this option is chosen voxels generated inside the hollow mesh are assigned an intensity so that they are displayed as tissue when loaded into visualization software If no intensity is associated with the voxels they are considered noise or air and appear hollow Hollow mesh Viewing the DICOM Data The intensity of pixels inside the surface is set to zero so that the exported surface appears as a hollow empty structure The 3D scenes exported to DICOM can be viewed in the Living Image 3D Browser 1 Select File Browse 3D Volumetric Data on the menu bar 2 In the dialog box that appears select the DICOM data dcm or dc3 and click Open The 3D Browser wind
250. l Palette x Sequence View Units Counts gt E Use Saved Colors Options Info A Pa aa Min 2140 Max 36446 Sequence View Tool Palette fe ese TootPatette B Lo Image Adjust gt Corrections Filtering lle Lo Image Information ROI Tools ARA ASA O TLI20050624145507 006 Units Counts z Display Overlay ba Image TLT20050624145507 006 Fri Jun 24 2005 07 57 18 Em Filter Open Bin M 8 FOV 12 6 f4 1s Living Image Version 2 50 1 5 20 2005 Camera IVIS 200 Beta II SI620EEV Click Info to show the Image Label information Cortes Series Male Nn nu Experiment DOB 03 21 05 Label kidney Comment dorsal Luminescence Counts Color Scale Min 1122 Max 19546 Check the image min and max in the color scale to determine whether the signal of interest is above the noise level and below CCD saturation The Image window may include multiple tabs depending on the type of acquisition m Sequence View Displays the image sequence m 3D View Displays the 3D volume if the acquisition included CT mode TIP See the tech note Saturated Pixels In an Image for information on pixel measurements Table 3 8 Sequence View window Item Description Units Select the measurement units fo
251. label information at any time during or after acquisition If you do not want to enter image information click Cancel See page 65 for details on adding information to an image after acquisition Figure 3 19 Enter information to include with the image optional Comment2 v Time Point V Animal Number 4 _ Animal Strain Sex View Cell Line Reporter X C Treatment F Luc Injection Time v T IACUC Number C7 Edit Image Labels UserID B 7 Living Image Universal Saved Labels LABELS 1 Ab 7 User M T cao Information entered here appears in the SP image label see Figure 3 21 on page 41 Male nu nu day 8 Mouse 4 7 ti Spectrum CT Hi If this is the first image of the session you are prompted to enable the autosave function Figure 3 20 When Autosave is enabled all images acquired during the session are automatically saved to a user selected location A different location can be chosen at any time select Acquisition gt Auto Save on the menu bar Figure 3 20 Autosave prompt LC Living Image 4 3 64 bit Auto Save xs Do you want to enable auto saving of acquired data for this session This can be changed anytime from the Acquisition menu Yes No 11 Click Yes in the prompt to enable autosave then choose a location in the dialog box that appears Alternatively click No in the prompt and manually save th
252. led to the imaging chamber The initialization procedure is started from the control panel Figure 2 3 NOTE The control panel is only available on the PC workstation that controls the IVIS Lumina Il The items available in the control panel depend on the imaging mode selected and the type of acquisition Image Setup or Sequence Setup Initialization 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 IVIS Spectrum Hardware Manual part no 133577_Rev A Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 2 Getting Started 8 Initializing the IVIS Spectrum 1 Start the Living Image software double click the amp 7 icon on the desktop 2 In the control panel that appears click Initialize Figure 2 3 After several seconds you will hear the instrument motors move Figure 2 3 IVIS Acquisition Control Panel during initialization j IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter Red color appears in the control panel during initialization The color turns blue when initialization is finished System Status Acquire Initializing 12 8 Imaging Wizard
253. les you to save the voxel information from the active data File Export 3D Scene as DICOM Opens a dialog box that enables you to save a 3D reconstruction and or surface in DICOM format The Multi Frame DICOM option supports 3D CT reconstruction in third party software File Print Displays the Print box File Print Preview a Displays the Print Preview box that shows what will be printed Living Image 4 3 1 User s Manual IVIS Spectrum Appendix C Menu Commands Toolbars and Shortcuts Table C 1 Menu bar commands and toolbar buttons continued Menu Bar Command Toolbar Description Button File Recent Files Shows recently opened data sets Note The number of files displayed can be set in the Preferences box select Edit Preferences and click the General tab File Logout Opens the Select Add User ID dialog box so that another user can logon or a new user ID can be added to the system File gt Exit Closes the Living Image software Edit Copy Copies the active image window to the system clipboard Edit Image Labels Opens the Edit Image Labels dialog box that enables you to edit the label set information for the active data see page Figure 4 8 on page 64 Edit Preferences Opens the Preferences box see page 267 View Tool Bar Choose this option to display the toolbar View Status Bar
254. ll 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 sec to 10 minutes Integration times less than 0 5 seconds are not recommended due to the finite time required to open and close the lens shutter Binning Controls the pixel size on the CCD camera Increasing the binning increases the pixel size and the sensitivity but reduces spatial resolution Binning a luminescent image can significantly improve the signal to noise ratio The loss of spatial resolution at high binning is often acceptable for in vivo images where light emission is diffuse For more details on binning see the reference article Detection Sensitivity select Help gt References on the menu bar Recommended binning 1 4 for imaging of cells or tissue sections 4 8 for in vivo imaging of subjects and 8 16 for in vivo imaging of subjects with very dim sources F stop Sets the size of the camera lens aperture he aperture size controls the amount of light detected and the depth of field
255. ll of the ROIs in the sequence The sort options are only available if the ROIs have not been sorted 5 Adjust the ROI position a Place the mouse pointer over the ROI Click the ROI when the pointer becomes a b Drag ROI s M NOTE To move multiple ROIs at the same time press and hold the Shift key while you click the ROls and then drag them to a new location Contour ROIs cannot be moved using this method 6 Adjust the ROI dimensions a Place the mouse pointer over the ROI Click the ROI when the pointer becomes a b Place the mouse pointer over an ROI handle so that it becomes a Drag the handle to resize the ROI M NOTE You can also change the ROI position or size using the adjustment controls in the ROI Properties box see Moving an ROI page 112 and Editing ROI Dimensions page 113 7 Click the Measure button W MeasureRols The ROI table appears For more details on the table see Managing the ROI Measurements Table page 119 104 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data 105 5 6 Measuring Background Corrected Signal If a subject has significant autoluminescence or autofluorescence you can obtain a background corrected ROI measurement by subtracting an average background ROI from a measurement ROI The software computes Background corrected intensity signal Signal in the measurement ROI Average signal in the average backg
256. ls pixe HX20070420121444 001 ROI1 Overlay 1 425e 06 4 167e 02 6 726e 02 8 462e 00 2 545e 03 3420 2 189e 05 4 141e 02 1 020e 03 2 87 HX20070420121444 001 ROI2 Overlay 1 489e 06 3 309e 02 6 061e 02 8 642e 00 2 545e 03 4500 2 880e 05 4 168e 02 9 001e 02 2 822 HX20070420121444 001 ROI3 Overlay 1 676e 06 3 637e 02 5 988e 02 1 056e 01 2 545e 03 4608 2 949e 05 4 168e 02 1 152e 03 2 84 HX20070420121444 002 ROI1 Overlay 7 261e 06 2 123e 03 3 445e 03 2 181e 01 1 031e 04 3420 2 189e 05 4 141e 02 1 020e 03 2 87 HX20070420121444 002 ROI2 Overlay 7 572e 06 1 683e 03 3 107e 03 2 181e 01 1 031e 04 4500 2 880e 05 4 168e 02 9 001e 02 2 825 nla 1 Customized Selections Measurements Types Image Attributes ROI Dimensions Copy Select All Counts v All Possible Values v Pixels Configure Export Table 5 7 ROI Measurements table Item Description Measurement Types Make a selection from the this drop down list to select the type of image unit for the ROI measurements in the table None Excludes ROI measurements from the table 119 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data Table 5 7 ROI Measurements table continued Item Description Counts luminescence 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
257. lter from the drop down list Figure 4 4 Figure 4 4 Opening data from the toolbar or menu bar A Choose a file to open Organize v New folder Jr Favorites E Desktop Mm Downloads Recent Places GJ Libraries Documents d Music t Pictures E Videos 2 Homegroup J Computer amp Local Disk C Ga Network File name Gori CaliperLS Caliper Data CerenkovDyCE RKG20110210131022 SEQ gt v by Search RKG20110210131022 S m A Name Date modified Type Size di RKG20110210131022_001 5 2 2012 3 42 PM File folder Ji RKG20110210131022 002 5 2 2012 3 42 PM File folder Ji RKG20110210131022 003 5 2 2012 3 42 PM File folder i RKG20110210131022_004 5 2 2012 3 42 PM File folder Ji RKG20110210131022 005 5 2 2012 3 42 PM File folder Ji RKG20110210131022 006 5 2 2012 3 42 PN File folder Ji RKG20110210131022 007 5 2 2012 3 42 PM File folder JN RKG20110210131022 008 5 2 2012 3 42 PM File folder Ji RKG20110210131022 009 5 2 2012 3 43 PM File folder J RKG20110210131022 010 5 2 2012 3 43 PM File folder Ji XML 5 1 2012 8 56 AN File folder _ Sequencelnfo 4 16 2012 10 03 AM Text Document WW w Living Image Files Click txt Sequence txt dcm TIFF Image Files tif tiff All Files Select the file type s 59 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 60 Table 4 2 File filter
258. lyzing Images 2 4 steca texaghededataeeeaeesebiend eee enas ex 157 Managing Spectral Unmixing Results 0 0 00 ee 157 Chapter 9 DyCE Imaging and Analysis 20002 022s nnen 159 About DyCE Dynamic Contrast Enhancement 0 000 eee eee 159 Acquire a DyCE Sequence 2 ccc eee ee eens 160 DYCE YEN PAA eae ae a 166 Automatic DyCE Analysis 0 0 ee ee ees 166 Manual DyCE Analysis 0 0 cc ees 169 EC HESU swa bee at dee eg aed po ge eg ah ode ae ee ea eo 172 Viewing Unmixed Images ccc ee 172 Viewing the Composite Image 0 00 cece es 173 Chapter 10 Reconstructing a 3D Surface 0 00 0 177 Generating a Surface 7 65n656 s00e55 bi ebidcd betes st NANANA ewan bi ehessn 178 Managing Bo Ngo e lt ecucteue ces eee APA AA AA e 182 Export or Import a Surface 22x 4400 bs BA Ok he Re Ee eh 183 Chapter 11 3D Reconstruction of Sources 02002 eee es 184 Overview of Reconstructing Sources 00 cece ee 184 Overview of Workflow for 3D Reconstruction of Sources 186 Reconstructing Luminescent Sources 000 eee eee eee ee ee 187 General Considerations a eee eee aaa 187 3 Living Image 4 3 1 User s Manual IVIS Spectrum Contents Chapter 12 Chapter 13 DLIT Image Sequence Requirements 2 00 ee eee ee eee 187 Including or Excluding Data for 3D Reconstruction 191 Reconstructing Fluorescent Sources
259. mage Appearance on page 68 Correcting Optical Image Data on page 70 Viewing Intensity Data and Making Measurements on page 72 Creating a Transillumination Overview on page 80 Overlaying Multiple Images on page 81 Rendering Intensity Data in Color on page 84 Exporting or Printing Images on page 85 Editing an Image Sequence on page 87 Creating an Image Sequence from Individual Images on page 88 4 1 Loading Optical Image Data You can load open optical images from the m Living Image Browser see below m Toolbar or menu bar page 59 Multiple data sets can be open at the same time M NOTE Select File Recent Files on the menu bar to view recently opened files Loading Optical Images From the Living Image Browser The Living Image Browser provides a convenient way to browse and preview optical data view information about the data and load the data To start the browser 1 Click the Browse button By Alternatively select File gt Browse on the menu bar 2 In the dialog box that appears select the folder of interest and click OK The Living Image Browser appears Figure 4 1 It displays all Living Image data located in the folder and its subfolders along with the user ID label information and camera configuration information M NOTE The next time you start the Living Image software and open the Browse For Folder box the software automatically returns to the last folder visited Living Image 4 3 1 U
260. mage window click Display Result For Measuring If necessary use the Color Scale Min and Max sliders in the Image Adjust tools to adjust the image display 8 To save the new image a Click the Save button fg Alternatively select File Save on the menu bar b Select a directory in the dialog box that appears and click Save A folder of data is saved to the selected location AnalyzedClickInfo txt ClickInfo txt luminescent and photographic TIF images 9 To export the new image to a graphic file a Click the Export button g b Select a directory in the dialog box that appears enter a file name and select the file type from the Save as type drop down list c Click Save 137 3 Spectral Unmixing About Spectral Unmixing Spectral Unmixing Methods on page 139 Correcting Spectra on page 152 Spectral Unmixing Results on page 154 8 1 About Spectral Unmixing The Living Image software applies spectral unmixing to distinguish the spectral signatures of different fluorescent or luminescent reporters and calculate the respective contribution of each on every pixel of an image Use spectral unmixing to m Extract the signal of one or more fluorophores from the tissue autofluorescence Images are acquired using epi illumination excitation light above the stage or transillumination excitation light below the stage a Analyze luminescent or fluorescent images when more than one reporter is used in the
261. mation Setup Time Scale Key Frame 3 Key Frame 4 Key Frame 5 Keyframe 1 Keyframe 2 Play Frames Per Second 10 Total Duration secs 5 Load Save The L box shows the key frames in the current animation setup Click a key frame in this box to display the associated 3D view and time stamp position in the time scale 0 100 at which the frame occurs in the animation Click to view the Rey icles Keyframe 4 animation composed of the key frames Keyframe 5 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Table 11 13 3D animation tools Item Description Time Scale 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 2 5 seconds after the start of animation Key frame 3 Time stamp 50 frame occurs 5 0 seconds after the start of animation Key frame 4 Time stamp 75 frame occurs 7 5 seconds after the start of animation Key frame 5 Time stamp 100 last frame of the animation Presets A drop down list of predefined animation setups Key frame A 3D view The software interpolates the key frames to create intermediate frames in real time then generates an animated sequence from all
262. measurement and an average bkg ROI that are included in the same subject ROI Using this type of ROI is optional Mirror ROI Measures the signal intensity in an area of an image acquired using the Side Imager taking mirror reflection effects into account Save ROls Creates a file that includes the ROI parameters for example the X Y coordinates type of ROI color shape width height ROls that have been saved to file can be recalled and applied for another image at any time Name The name of the selected ROI set or the default name for a new ROI set Delete Deletes the selected ROI set from the system Note This permanently removes the ROI from the system Load Applies the ROI set selected from the Name drop down list to the active image Save Saves the ROI set in the active image Note This is a global save the ROI is saved to the system and the ROI set can be loaded onto any image If you use the File Save commands to save an image that includes an ROI the ROI is saved with the image only not a global save and is not available for loading onto other images For more details see Save Load or Delete ROIs page 116 Auto ROI Parameters that specify how the auto ROI tool draws an ROI Parameters Threshold If the Auto All or Auto 1 method is selected the Threshold specifies the minimum percent of peak pixel intensity that a pixel must have to be included in an ROI identified by the software After ROIs are drawn on an
263. ment Show Advanced Options Counts Tatal Count bd Radiance Photons Tatal Flux Efficiency Total Efficiency Kok cancel Apply Table B 1 General preferences Item Description Start Up Defaults Dock Tool Palette Choose this option to set the position of the Tool Palette in the application window Choose left or right Note To undock the Tool Palette click on the palette title bar and drag ita distance greater than its width Window Size Specifies the dimensions of the main application window Width Height Sets the dimensions of the image window Restore Defaults Click to apply the default settings Living Image 4 3 1 User s Manual IVIS Spectrum Appendix B Preferences Table B 1 General preferences continued Item Description Apply Individual Color Scale for Sequences Choose this option to apply a separate color scale to each thumbnail of a sequence If this option is not chosen all of the thumbnails are displayed using the same color scale Show Transillumination Locations Choose this option to display a cross hair at each transillumination location when you load transillumination data When you mouse over a cross hair a tool tip displays the transillumination coordinates If this option is not chosen you can choose the Transillumination Location option in the sequence view window to display the transillumination loca
264. min sec per user ID from logon until switching users or system shut down The software creates a separate record for each month for example LILUSAGE_ lt MONTH gt _2011 csv located at C Program Files Caliper Life Sciences Living Image Usage Figure 2 13 Activity window File EGR View Toos Acquisition Window Help GAB B8 AS NM a4 Description Living Inage 43 0 15210 64 bit RC Dec 13 2011 12 12 19 Po K3 Logged IN lt lt a a MS confeguration file found and loaded Act IVI ty win d OW Reading user preferences 3 Image Acquisition Luminescent Imaging Fluorescent Imaging With Epi Illumination on page 29 Fluorescent Imaging With Transillumination on page 35 Acquire a Sequence Using the Imaging Wizard on page 42 Acquire Multiple Sequences in Batch Mode on page 48 Manually Set Up a Sequence on page 50 Manually Saving Image Data on page 54 Exporting Images on page 54 3 1 Luminescent Imaging Luminescent imaging captures signals from luminescent molecular reporters This section explains how to acquire a single luminescent optical image a Quick guide See Figure 3 1 on page 24 m Detailed instructions See page 25 See page 42 for information on acquiring a luminescent sequence Living Image 4 3 1 User s Manual IVIS Spectrum Quick Guide Acquire a Luminescent Image Chapter 3 Image Acquisition Figure 3 1 Quick Guide Acquire a luminescent image 1 art the Living Im
265. minescent source spectrum This Source Spectrum will be used if a Subject Type is not selected in the Imaging Wizard and saved during acquisition for DLIT sequences Plot Tissue Properties Choose this option to display a graph of the absorption coefficient u effective attenuation coefficient u and reduced scattering coefficient u or usp Source Spectrum Choose this option to display the source spectrum for DLIT reconstructions Bioluminescent Spectrum Choose this option to display the spectrum of the bioluminescent source available for DLIT reconstructions only Fluorescent Spectrum Choose this option to display the spectrum of the fluorescent source available for FLIT reconstructions only Restore Defaults Click to restore the defaults in the Optical Properties tab 274 Appendix C Menu Commands Toolbars and Shortcuts Figure C 1 Living Image toolbar Xx gt A amp WP unts Counts Apply to Al Table C 1 Menu bar commands and toolbar buttons Menu Bar Command Toolbar Description Button File Open Displays the Open box so that you can select and open an image data file Double click a Sequencelnfo txt file or ClickInfo txt file to open the image data file see page 59 File Browse 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
266. ms lay ET Click Info to show the Image Label information Tool Palette Series Male Nn nu Experiment DOB 03 21 0 Label kidney Comment dorsal Luminescence Counts Color Scale Min 220 Max 3432 TIP See the tech note Determine Saturation for information on pixel measurements select Help Tech Notes on the menu bar Table 3 2 Image window Item Description Units Select the measurement units for the image display from this drop down list The available units depend on the type of image data See the concept tech note Image Display and Measurement for more details select Help gt Tech Notes on the menu bar Display A list of image types available for display for example overlay For more details on the different types of image displays see Table 2 1 on page 11 Info Click to display or hide the image label The image label includes information you enter in the Edit Image Labels dialog box Figure 3 6 and other image information automatically recorded by the software a Opens a dialog box that enables you to export the active view as a graphic file 28 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition Table 3 2 Image window Item Description wj Creates a preview picture snapshot of the image or thumbnails that the Living Image Browser displays when the data are selected in the browser For more details on the
267. n The entire image cube is calibrated and visualized on the same scale To view a particular image remove the check mark next to the Overview option and move the slider or enter an image number M NOTE Mark a region of tissue autofluorescence only on the image cube for the Tissue AF component The spectra of components that you mark on the image cube are raw spectra from the data when using the manual method 5 Move the mouse pointer over the image cube to see the spectrum at a particular location The spectrum at the pointer location is updated as you move the pointer 6 To specify a probe location for unmixing a Click the button for a spectrum b Using the mouse draw a mark on an area of the image cube which represents the probe location The software plots a normalized spectrum of the signal Figure 8 18 c If necessary right click the image cube to erase the mark 7 Repeat step step 5 to specify other probe locations Manually subtract autofluorescence background See Correcting Spectra page 152for instructions Figure 8 18 Mark the probe locations for unmixing on the image cube Kpa C HX20120419114703_SEQ bol Sequence View Unmixing a GG J Normalized Legend S ImageCube 1 00 Raw spectra at the probe locations marked on the image cube HA 0 0 660 700 740 730 Emission Wavelength nm Spectrum List U btr x Note Select Name Color Pick 1 V Tissu
268. n m Fluorescent image at the remaining transillumination locations a A structured ligh image Figure 11 9 shows an example image sequence Figure 11 9 Example sequence setup for FLIT eo I E Display Photographic Settings Subject Mouse Probes x Exposure Binning FStop Excitation Emission Lamp Level FOY Height Transillumination Auto Auto Auto Auto Auto Auto Auto Mediun Medium Medium Medium Medium Medium EC Number of Segments 1 Delay 0 0 bf bf b15 b15 HTI 615 675 Tall T20 Tall dal lal T20 T20 High High High High High High min Apply to All bd Remove C 1 50 1 50 1 50 1 50 1 50 1 50 1 50 9x19 4 6 gx19 4 T 9 19 46 9 19 45 9 19 14 4 9 19 43 9 19 14 2 mo Insert Add A Update EE Steps to Reconstruct Fluorescent Sources 1 2 Generate or load a surface in the Surface Topography tools For details on generating the surface 3 Load a FLIT image sequence see Chapter 10 on page 177 In the Tool Palette choose FLIT 3D Reconstruction The Analyze tab shows the images that the algorithm automatically selects for the reconstruction based on an appropriate signal level Figure 11 2 For more details about the Threshold see page 191 194 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 195 Figure 11 10 FLIT 3D Reconstructio
269. n the menu bar Mathematically combine add multiply subtract or divide two user selected images For example subtract a blue shifted background filter image from the primary excitation filter image to remove tissue autofluorescence signal 1 Select Edit User settings on the menu bar 2 Click the Add User tab in the dialog box that appears Figure 2 8 User Settings Add User si A User Settings amp Add User 1 p change Password 2 Delete User fe Security P 83 Add New User User ID 7 Password Confirm Password Deletes all entries 19 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 2 Getting Started 3 Enter a user ID 4 Optional enter and confirm a password 5 Click Add Changing or Adding Passwords 1 Select Edit User settings on the menu bar 2 Click the Change Password tab in the dialog box that appears Figure 2 9 User Settings Change Password y 7 User Settings es 8 Add user amp Change Password amp Delete User searity Change Password User ID HLA x New Password Confirm Password Deletes all entries 3 Select a User ID enter and confirm a new password and click Submit Deleting Users M NOTE User accounts can be locked If this security is applied a master password is required to delete users from the system See page 21 f
270. n tools Analyze tab EWL EmWL Threshold e Images selected for reconstruction Type of image used in the reconstruction If no NTF data are available only Radiance is available SEEEEEEEE 2882888288 n A gt Optimization algorithm 4 Select the type of image used in the reconstruction Radiance or NTF Efficiency Figure 11 10 NTF Efficiency data is the default because it affords higher sensitivity to the embedded fluorescence sources 5 Inthe Properties tab make a selection from the Tissue Properties and Source Spectrum drop down lists Figure 11 3 Figure 11 11 FLIT 3D Reconstruction tools Properties tab Tool Palette Fluorescent Quantification WPQUANT_1 X The selected plot type is displayed below Wavelength nm 6 To view the tissue properties u Plot drop down a Hep HI for the tissue you selected make a selection from the Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 196 7 To include the number of fluorescent molecules source in the results select a fluorescent quantification database For details on generating a fluorescent quantification database see page 231 8 In the Analyze tab click Start 9 The Data Preview window appears and display
271. ndow in the list to make it the active window indicated by a check mark Window Other Windows 5 lt window name gt Lists other windows that are open For example If the Living Image Browser is open use these commands to make the browser the active window and display it on top of all other open windows Help User Guide Displays the Living Image User Manual Help Tech Notes Displays a folder of technical notes Note For the most recent collection of technical notes please see the IVIS University download page Help License information Displays the license information Help Plug in Information Displays a list of tool plug ins and Tool Palette plug ins Help IVIS Reagents Opens the Caliper LS web page for In Vivo Imaging Reagents Help About Living Image Displays information about the Living Image software and Caliper technical support contact information Table C 2 Keyboard shortcuts Click this button then click an item in the user interface to display information about the item Keys Shortcut Description Ctri B Opens the Living Image Browser Ctrl C Copies the active image to the system clipboard Ctrl D Arranges open windows in a cascade Ctrl O Displays a dialog box that enables you to open data Ctrl P Open the Print dialog box Ctrl S Saves the active file or window Ctrl T Tiles the open wi
272. ndows Ctri W Closes the active window Shift F1 Changes the mouse pointer to the What s This tool Ng Click this button then click an item in the user interface to display information about the item M NOTE Macintosh users use the Cmd key apple key instead of the Ctrl key 278 Index Symbols 3D Multi Modality tools adjusting image resolution 242 classifying 3D volumetric data 239 color opacity map 240 control points 240 fiducial registration 250 gradient illumination 244 loading data 252 254 manual registration 255 258 maximum intensity projection 244 requirements 238 238 239 volume display options 242 3D quantification database 231 236 create 232 235 manage results 236 samples 231 3D reconstruction fluorescent sources 194 197 luminescent sources 187 193 reconstruct particular regions 192 3D reconstruction results DLIT or FLIT 197 199 3D ROI draw 125 128 Measurements table 130 132 properties 128 129 3D signal intensity 77 3D tools 213 230 Animate tab 225 230 Registration 218 224 Source tab 216 218 Surface tab 214 215 3D Volumetric Browser 252 3Dsurface generate 178 179 A acquire a sequence batch mode 48 49 acquire a sequence manual 50 53 acquire a sequence 42 48 animation 225 230 custom 227 229 edit an animation setup 229 230 preset 227 animation tools 225 230 autofluorescence 105 See tissue autofluorescence autoluminescence 105 automatically draw ROIs 98 99
273. ng graphs are available in the Plots tab Plot Type Description Quantification Profile Plots the measured intensity within the user selected area on the surface If no box was drawn on the surface measures the total intensity for the entire surface Reduced Chi Squared A measure of the difference between the computed and measured photon Profile density maps at the optimum solution A smaller y value indicates a better quality of fit Voxel Size Plots the voxel size at the start of the 3D reconstruction and at the end of the 3D reconstruction Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Figure 11 26 Example Quantification plot 3D View Plots Analysis Type Plot Quantification FLIT x10 9 12 Quantification photomwsec 1 Results 0 First surface 1 Second surface 2 Third surface and so on 11 9 Exporting a 3D Scene as DICOM The items in the perspective 3D View are called a 3D scene For example the 3D scene in Figure 11 27 includes a surface and voxels The 3D scene can be exported to DICOM format and viewed in the Living Image DICOM Viewer or third party software 210 Living Image 4 3 1 User s Manual IVIS Spectrum Figure 11 27 3D scene E TUT2nosoe2a14s507 seQ CI Sequence View PU D ve To export the 3D scene 1 Load the results that you want to export Chapter 11 3
274. nmixing and DyCE y gt Surface Topography o gt 3D Multi Modality Tools gt 3D Optical Tools C Ck20080407145405 SEQ o oO Ka Sequence View lA 30 View Coronal 2 4 3 Analyze Properties Results FLIT Results FLIT_45405 Loaded Key Value Final voxel size mm 3 1 25 E Number of voxels 203 Reduced Chi2 2 02e 03 Starting vsize 5 00 Nsurf best 36 Total surf samples 1296 Threshold angle 70 00 Damping Reduction 100 00 Median Filter TRUE Uniform Surface Sam TRUE x Transayial 420 5 Photon Density Maps Export Results Save Results Name FLIT 45405 v Delete Load Subject Height 19 6 mm f Perspective 11 4 3D Reconstruction Results The Results tab displays information about the photon density voxels and algorithm parameters DLIT or FLIT Results M NOTE For more details on DLIT see the see the reference article DLIT and FLIT Reconstruction of Sources select Help References on the menu bar Sometimes adjusting the DLIT algorithm parameters improves the fit of the simulated photon density to the measured photon density data Figure 11 14 Example DLIT 3D reconstruction results Tool Palette 3 _ ROI Tools al gt Spectral Unmixing and DyCE 3 gt Surface Topography a Cs 3D Multi Modality Tools 2 3D Optical Tools DLIT 3D Reconstruction
275. of the frames Each successive key frame in a sequence should differ slightly from the preceding one so that motion is smoothly depicted when the frames are shown at a proper frame rate frames second The Living Image software provides preset key frames or you can specify the 3D views for the key frames Preset Key Frame Factor Determines how many key frames are used to generate one revolution in a spinning animation No of frames 4 x Key Frame Factor 1 Increasing the key frame factor reduces the time period between key frames and creates the appearance of finer movement Decreasing the key frame factor increases the time period between key frames and creates the appearance of coarser movement Frames displayed per second in the animation sequence Creates a new key frame from the current 3D view Updates the selected key frame to the current 3D view Deletes a selected or all key frames from the key frame box Moves a selected key frame up in the key frame box Moves the selected key frame down in the key frame box The total time of the animation sequence Play Click to view the animation sequence defined by the current key frames and animation parameters Record Displays a dialog box that enables you to save the current animation to a movie mov mp4 or avi mpg Animation Setup Load Displays a dialog box that enables you to open an animation setup xml Save
276. ols for viewing and working with the surface or DLIT results h fy or Rotates or spins the surface in the x y or z axis direction a h Moves the surface in the x or y axis direction gn eg Zooms in or out on the image To zoom in right click Cmd key apple key click for Macintosh users and drag the H toward the bottom of the window To zoom out right click and drag the isa toward the top of the window Displays the x y z axis display in the 3D view window Displays coronal sagittal and transaxial cross sections through the subject in the 3D view window Displays a bounding box around the subject Displays a grid under the subject 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 11 36 page 223 es fe Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 191 Table 11 3 3D View tools continued Tool Description fe Select this tool from the drop down list to display the perspective view kE al Rotates the 3D reconstruction results in the 3D view window 3D scene Click the or ga key to increase or decrease the rotation speed To stop the rotation click the 3D scene or the m button via Displays measurement cursors in the coronal sagittal or transaxial views Click this button then select a so
277. om right to left right click the image and select Sort ROIs on the shortcut menu If the Apply to Sequence option is selected in the ROI tools choose Sort ROIs in Sequence to sort all of the ROIs in the sequence The sort options are only available if the ROIs have not been sorted Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data Figure 5 3 ROI intensity measurements TE TLT20050624145507 seQ Sequence View Units Radiance Photons M E Use Saved Colors Gasan Gansa 2m 9 Ng m ROI 1 50 2 278e 06 ROI 2 50 J 1 017e 07 i 4 na a ROI 7 50 4 664e 08 ROI 8 50 2 455e 08 r by ROI 3 50 J 2 853e 08 LO a ROI 4 50 9 246e 07 ROI 9 50 2 552e 09 j ROI 10 50 1 361e 09 f f i 5 Use the Threshold slider or a arrows to adjust the ROI boundaries parameters 2 C HE G W Measure ROIs Type Measurement RO save ROIs Name ROI 1 KSA Delete Auto ROI Parameters hreshold Yar PA M ig ig ag ge gt gt ig ig ig igh ig tg gt gt ig AH YE Ng gt ig ag ig YA Nyt Ng gt ag agi gt gt gt Ng ig ag ig gt gt gt gt ig ag ig gt gt Ng ig ag ig gt gt gt gt ig ag gt gt gt Ng ig ag ig gt gt gt gt ig ag gr gt gi Ng ig ag agi gt gt gt gt ig ag gt gt gi Ng ig gi igure 5 4 Auto K Threshold Specifies the minimum per cent of peak pixel intensity that a pixel must have to be included in an ROI identifie
278. omatic from the Methods drop down list and click Start Unmixing The Auto Unmix window appears The purple data mask shows the data that will be included in the analysis the entire subject is included by default 145 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing 146 Figure 8 10 Auto Unmix window a TE Hx20120419114703_SEQ OI Sequence View Auto Unmix Select Image Mask Choose Components To Unmix Tips 8 wavelength pairs selected Choose the number of components to unmix Pick the significant background signals first then add the probe information to the table if it is not listed If you are undear about the probe or it is not in the library choose Unknown Imaging Subject Mouse Data mask Background Signals V Tissue Autofluorescence Food Signal Probe Information Data Mask Options Photograph Threshold Bi 23 5 Draw Mask Rectangle O Ellipse PCA Number of components to unmix 3 Ensh J cod 4 If you do not want to analyze the entire subject draw a mask on a particular area Figure 8 11 Figure 8 11 Drawing a data mask See Table 8 2 on page 147 for more details on the data mask options Select Image Mask Select Image Mask Data Mask Options Data Mask Options D Photograph Threshold 3 H Photograph Threshold 23 Draw Mask Rectangle Ellipse Draw Mask Rectangle Ellipse Draw
279. on 000 0c eee 220 Importing an Organ Atlas lt 4 ganda wa ce wb Ca eG ee KPA MP os 223 JOANA O sokcaseeaxaacny ead toe eo en ee ee eee oS eae 225 Viewing a Preset Animation 0 00 a 227 DLIT FLIT Troubleshooting 20 0000 eee 230 Quantification Database 0 0002 eee eee es 231 Preparing and Imaging the Samples 0 cee ees 231 Creating a Quantification Database 0 es 232 Managing Quantification Results 0 000 cc eee 236 Exporting Quantification Results 0 aaa 237 3D Multi Modality Tools am es 238 About the 3D Multi Modality Tools 00 eee 238 3D Multi Modality Tool Requirements nannaa nnana aaa 238 Classifying 3D Volumetric Data aaa 239 Specifying a Color Opacity Map cc ee 240 Volume Display OptiOnS 22s cue ese ae oe ae ae oe Mee ee ee 242 Adjusting the Image Quality 0 0 0 0 es 242 Adjusting Volume Opacity cee eee ee eee 243 Maximum Intensity Projection MIP 2 a 244 4 Living Image 4 3 1 User s Manual IVIS Spectrum Contents Appendix A Appendix B Appendix C Index Gradient Illumination 0 0 ees 244 Modifying Volume Resolution aaa 244 Smoothing a Volume 2 008640444524404 00 80 eRN RE owe ee DOG AA 244 Rendering and Viewing Slices 0 aaa 245 Rendering Slices 02 aa 245 Viewing SlICES 6 EERE AG PEBAG MAGNG BLAME hee
280. on Two registration methods are available a Automatic fiducial registration For experiments in which the optical data are acquired on the IVIS Spectrum and the CT data are acquired on the Quantum FX uCT instrument The subject must be contained in the Mouse Imaging Shuttle during both optical and CT imaging and the CT data must be exported to DICOM format See page 254 for more details Manual registration Use the 3D Multi Modality tools to register a 3D surface reconstruction with 3D volumetric data acquired on a third party instrument See page 255 for more details Figure 13 13 shows an overview of the steps to register these types of multi modal data After registration classify the 3D volumetric data to help identify and separate objects see page 239 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools Figure 13 13 Steps to register multi modal data 1 Load the optical data s Bioluminescence or fluorescence image sequence and structured light surface m 3D source reconstruction DLIT or FLIT results page 187 or page 194 2 Load 3D volumetric data CT or MRI page 252 3 Register the 3D source reconstruction and the 3D volumetric data by performing either a Automatic fiducial registration Available for data acquired on the Quantum FX uCT instrument using the Mouse Imaging Shuttle page 250 or m Manual registration Match animal surface representations u
281. on Filter Field of View System Status HU C MIS Subject height 5o S cm Sequence Setup Focus use subject height v Temperature AN Locked tie CT 2 Select an excitation and emission filter from the drop down lists The instrument has 18 narrow band excitation filters that span 490 850nm with a 20nm bandwidth enabling spectral scanning over the blue to NIR wavelength region Figure 3 9 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition 32 Figure 3 9 IVIS Spectrum excitation and emission filters Excitation Filters Transmission 400 440 480 520 560 600 640 680 720 760 800 Wavelength nm Emission Filters 550 600 750 800 Wavelength nm Transmission 3 Put a check mark next to Photograph 4 Select a Field of View size of the stage area to be imaged O TIP See the concept tech note Detection Sensitivity for more information about the Field of View select Help Tech Notes on the menu bar Table 3 3 Field of View FOV settings FOV Setting FOV cm A 4 B 6 5 E 13 D 22 5 19 5 E 22 5 26 Some IVIS Spectrum instruments may have the FOV in parentheses FOV 19 5 and 26 were replaced by FOV 22 5 Living Image 4 3 1 User s Manual IVIS Spectrum 5 Select a focus option Figure 3 10 Chapter 3 Image Acquisition The focal distance to the camera is s
282. on sequence setup 8 Enter information about the image in the Edit Image Labels box that appears optional Click OK M NOTE You can enter image label information at any time during or after acquisition If you do not want to enter image information click Cancel See page 65 for details on adding information to an image after acquisition 33 Living Image 4 3 1 User s Manual IVIS Spectrum Figure 3 11 Enter information to include with the image optional TE Edit Image Labels Ba UserID B v Living Image Universal Saved Labels LABELS 1 AER V User v V Group V Experiment 87 MG uc2 Intracranial Implantation v Male nu nu day 8 Mouse 4 V Commenti S v E Comment2 v Time Point v V Animal Number 4 x E Animal Strain x Animal Model v Sex X View v Cell Line C Reporter C Treatment E Luc Injection Time v T IACUC Number Information entered here appears in the image label see Figure 3 13 on page 35 Chapter 3 Image Acquisition If this is the first image of the session you are prompted to enable the autosave function Figure 3 12 When Autosave is enabled all images acquired during the session are automatically saved to a user selected location A different location can be chosen at any time select Acquisition Auto Save on the menu bar Figure 3 12 Autosave prompt Fa E Living Image 4 3 64 bit Auto Save
283. ool transformation modes ive Tab here Bi crate beet eran AT Use Tab hey ko rtdh babaman trarakarmabon toot Use Tab hey bo sakt betmeen transformation tools Use X Yor 2 lungs to restrict scaling to only oon asis Scag ON KYT Translate Moves the volume in Scale Increases or decreases scale Rotate To rotate the volume on the x y or z axis Drag the tool to the size of the volume drag a red the x y or z axis click the blue adjust the position of the volume cube at a corner of the volume To green or red circle and drag the restrict scaling to a particular axis mouse arrow in the direction of press the X Y or Z key then drag a interest red cube M NOTE Make sure that you click the transformation tool so that it is highlighted before you use it Otherwise the dragging operation is applied to the optical data structured light surface 5 To return the 3D volumetric data to the default position and size click the Reset Registration button i 6 Save the registration information see page 254 M NOTE Registration information is saved with the results for the volumetric data and is specific for a particular optical data set 13 8 Volume Data Viewer The Living Image software provides a viewer for volumetric data The 3D Multi Modality tools are not required to view DICOM or TIFF data 1 Select View gt Volume Data Viewer on the menu bar The Volume Data Viewer appears 2 Select volume da
284. or more details on locking user accounts 1 Select Edit User settings on the menu bar 2 Click the Delete User tab in the dialog box that appears Figure 2 10 User Settings Delete User In this example user accounts are locked ra 7 User Settings 2 2 add User 2 change Password 8 Delete User fae Security Delete User User ID HLA x Enter master password Enter Master Password Clear Deletes all entries 20 Living Image 4 3 1 User s Manual IVIS Spectrum 3 Select a User ID Chapter 2 Getting Started 4 If the accounts are locked enter the master password 5 Click Delete and Close Locking User Accounts If user accounts are locked a master password is required to change user passwords delete users or unlock user accounts To lock user accounts 1 Select Edit User settings on the menu bar 2 Click the Security tab in the dialog box that appears 3 Click Lock User Accounts Figure 2 11 User Settings Security 2 Add user 2 Change Password 2 Delete user Security Currently User Accounts are Unlocked pi A User Settings 2 add User Change Password amp deleteuser Security E vod ver Accounts Currently User Accounts are Unlocked Enter Master Password Enter a Master Password to Lock the User Account settings Master Password
285. ormation and results 249 volumetric data classify 239 240 color opacity map 240 display options 242 information and results 249 rendering slices 245 246 smoothing 244 viewing slices 246 248 VSIZe starting 198
286. ose a location for the file in the dialog box that appears 49 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition 50 3 6 Manually Set Up a Sequence This section explains how to set up an image sequence if you do not use the Imaging Wizard The sequence parameters in the sequence table can be saved as a Living Image Sequence Setup file xsq For details on image acquisition see Acquire the Sequence on page 45 TIP It may be convenient to create an image sequence by editing a sequence setup generated with the Imaging Wizard or an existing sequence setup xsq Save the modified sequence setup to a new name 1 Click Sequence Setup in the control panel Figure 4 30 The sequence table appears 2 If necessary click the Remove button and select All to clear the sequence table Figure 3 31 Opening the sequence table IVIS Acquisition Control Panel Imaging Mode Exposure Time p Excitation Filter Emission Filter Field of view C v System Status O ms Idle 12 8 cm Imaging Wizard Sequence Setup Subject height 1 50 3 cm Focus use subject height w inning Excitation Filter Emission Filter C Display Photographic Settings Subject None v hao Be Bfearn E ope fee Seat O Field of View C v System Status 7 Acquire Sequence O MIS Idle 12 8 cm Imaging W
287. ound Opens a dialog box that enables you to remove the fluorescent background measurements from the system Acquisition Auto Save If Auto Save is selected all images are automatically saved to a user selected folder Acquisition CT gt Generate Alignment data Acquires images of the Rotation Stage Alignment tool that are used to generate alignment data for the IVIS Spectrum CT Acquisition CT Acquire Reference images Acquires dark and bright reference images that are used to determine corrections that are applied to the raw projection images during the CT reconstruction process Acquisition Auto Save To Opens a dialog box that enables you to select a folder where images will be saved to automatically Window Close Closes the active image window Window Close All Closes all image windows Window Cascade Organizes the open image windows in a cascade arrangement see page 60 Window Tile Organizes the open image windows in a tiled arrangement see page 60 277 Living Image 4 3 1 User s Manual IVIS Spectrum Appendix C Menu Commands Toolbars and Shortcuts Table C 1 Menu bar commands and toolbar buttons continued Menu Bar Command Toolbar Description Button Window 1 lt Image or Sequence name gt Window 2 cImage or Sequence name gt Window etc A list of the open image windows Click a wi
288. ow appears Figure 11 29 Living Image 3D Browser Browse 3D Volumetric Data Lo JO Jea Add to list Browse Folder Path AAAH AA AASA AAAH AA AA ar Slices Dimension Modality Pixel Spacing Slice Spacing Data Type Type Start Index 1 M Move the slider to select a particular slice for viewing or click an image dcm 20110306103654 094 dcm dcm 20110306103654 095 dcm dcm 20110306103654 096 dcm dcm 20110306103654 097 dcm dcm 20110306103654 098 dcm dcm 20110306103654 099 dcm dcm 20110306103654 100 dcm dcm 20110306103654 101 dcm dcm 20110306103654 102 dcm dcm 20110306103654 103 dcm dem 20110306103654 104 dcm x Load data in new window Load 100 Data will be loaded in TLT20050624145507_SEQ bI EndIndex 256 2 Table 11 9 Living Image 3D Browser DICOM viewing controls Item Description Start Index Specifies the first image slice for viewing 212 Living Image 4 3 1 User s Manual IVIS Spectrum Table 11 9 Living Image 3D Browser DICOM viewing controls Chapter 11 3D Reconstruction of Sources Item Description Auto Preview Select this option to automatically play back the images End Index Specifies the last image slice for viewing Load Opens the DICOM data in a 3D View window window click Load 11 10 3D Tools Overview After you reconstruct or load a surface or 3D sources the Tool Palette includes the 3D
289. p button and select Delete Current 11 15 DLIT FLIT Troubleshooting Issue Solution No sources in This can occur in DLIT or FLIT if the surface is not correct For example if a surface is solution imported into the 3D View from another source other than a Surface Topography analysis Surface has spikes The most common source of spiky surfaces are folds in the animal skin or fur which corrupt the desired smooth lines projected on the animal from the laser galvanometer Choose the Fur Mouse option for Subject Smoothing the surface by using the Smooth feature in the Surface Topography tools can help improve the surface Tool Palette Optical Surface Reconstruction orentaton Dorsal gt sie Surface Smoothing Save Results Name Dorsal surface 3D ti ty Tools gt 3D Optical Tools gt DLIT 3D Reconstruction Bad Photon The optical properties or source spectrum may have been incorrectly chosen For Density or NTF example Mouse Tissue optical property is appropriate or mice but XPM 2 XFM 2 is Efficiency fit only appropriate for the mouse phantom 1 2 Quantification Database Preparing and Imaging the Samples Creating a Quantification Database on page 232 Managing Quantification Results on page 236 It is possible to determine the number of cells in a DLIT source or the number of dye molecules or cells in a FLIT source if a quantification database is available The database is de
290. ptical image sequence Table 11 2 Recommended DLIT optical image sequence for manual sequence setup Image Type Emission Filter Options 560 580 600 620 640 660 Photograph J Select the Reuse option in the control panel Luminescent V V V V V V M NOTE It is recommended that the binning level be the same for all of the luminescent images Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 188 Steps to Reconstruct Luminescent Sources Using DLIT Load a DLIT image sequence 2 Generate or load a surface using the Surface Topography tools For details on generating the surface see Chapter 10 on page 177 3 Inthe Tool Palette choose DLIT 3D Reconstruction The Analyze tab shows the data that the algorithm automatically selects for the reconstruction Figure 11 2 For more details about the Threshold see page 191 Figure 11 2 Analyze tab Threshold 10 6 8 1 4 Inthe Properties tab make a selection from the Tissue Properties and Source Spectrum drop down lists Figure 11 3 Figure 11 3 Properties tab Database not found The selected plot type is displayed below Normalized Amplitude 8 Wavelength nm Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sou
291. quisition Control Panel 264 Table A 1 IVIS acquisition control panel continued Item Description Lamp Level Sets the illumination intensity level of the excitation lamp used in fluorescent imaging Off Low High and Inspect The Low setting is approximately 18 of the High setting Inspect turns on the illumination lamp so that you can manually inspect the excitation lamp Note Make sure that the filters of interest are selected in the filter drop down lists before you select Inspect The Inspect operation automatically positions the selected filters in the system before turning on the lamp Subsequent changes to the filter popup menus will have no effect until another Inspect operation is performed Lights Turns on the lights located at the top of the imaging chamber Alignment Grid Choose this option to illuminate an alignment grid on the stage when the imaging chamber door is opened The alignment grid shows the sizes and positions of the possible fields of view If subject alignment is not completed in two minutes place a check mark next to Alignment Grid to turn on the grid Field of View Sets the size of the stage area to be imaged by adjusting the position of the stage and lens The FOV is the width of the square area cm to be imaged A smaller FOV gives a higher sensitivity measurement so it is best to set the FOV no larger than necessary to accommodate the subject or area of interest The FOV also affects
292. r 3 Select a directory in the dialog box that appears and click OK M NOTE The software automatically includes the user ID and a data and time stamp with the data 3 8 Exporting Images The active image view can be saved in different file formats for example bmp dcm 1 Open an image or sequence 2 Click the Export Graphics button Figure 3 36 Figure 3 36 Exporting an image to a graphic file IG TUT20050824145507 006 ex NG n TENG A NAS v Otsptay Over v T go Ir p Favorites bg Organize a Orsktop AJ Libraries Downloads e3 Homegroup 872 byt Computer nts Counts e sg Fe Favorites th Network File name vE ais ci C Bitmap Image bmp IPEG Image jpa l Postscript Enhanced Metafile eps Tagged Image File Format t Windows Metafile Format nf Enhanced Metafile Format Pani 8 DICOM dem Hide Folders Select a directory in the dialog box that appears and enter a file name 4 Click Save M NOTE To export a sequence to DICOM dcm format select Export gt Image Sequence as DICOM on the menu bar This creates a directory that contains the dcm files and a Sequencelnfo txt 54 4 Working With Optical Image Data Loading Optical Image Data About the Image Window and Tool Palette on page 61 Viewing Image Information on page 64 Adding Comments or Tags to an Image on page 66 Adjusting I
293. r Color Scale Min 2 35e7 Max 4 16e8 Select an ROI or all ROIs from the drop down list LP Histogram Wind pS Full min Bin 2 35e7 MaxBin 4 16e8 2 Bins 512 lplot Roas Eg S TLT20050624145507 005 Overlay 3 000 2 000 j 0 000 1 00 2 00 3 00 4 00 Bins 108 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data 109 5 8 Managing ROI Properties In the ROI Properties box you can view information about an ROI change the position of the ROI on the image and edit the ROI label or line characteristics Viewing ROI Properties 1 To view ROI properties do one of the following a Double click an ROI in the image m Right click the ROI and select Properties from shortcut menu that appears m Select the ROI then select View Properties on the menu bar The ROI Properties box appears for more details see Figure 5 19 2 To view properties for another ROI click the ROI in the image Alternatively select an ROI from the ROI drop down list in the ROI Properties dialog box Figure 5 17 Figure 5 17 Opening the ROI Properties dialog box lt i units ahas Photons Joss ey Cee Cc Luminescence Subject 1 Background ROI Image Number TLT 2005062414550 BKG 1 3 495e 05 ROI ROI 7 BKG 1 50 4 663e 08 Lock Position Xc cm 5 93250 Ye am 7 23844 Angle deg
294. r the image display from this drop down list The available units depend on the type of image See the concept tech note Image Display and Measurement for more details on measurement units Use Saved Choose this option to display the image data using the color table that was specified in the Colors Preferences at the time of acquisition If this option is not selected image data are displayed using the color table currently specified in the Preferences 46 Living Image 4 3 1 User s Manual IVIS Spectrum Table 3 8 Sequence View window continued Chapter 3 Image Acquisition Item Description Options Layout Choose a display option for the images in a sequence Default Dynamic or Film Strip For example here is Film Strip mode TE TLT20050624145507 sEQ Sequence View Spectra Units Counts x E Use Saved Colors Options 7 mo PA Mm 4 a Layout gt Default SortBy b Dynamic Display gt Y Film Strip Labels gt Sort by Options for ordering images in the sequence window This option only applies to images that were opened using the Load as Group function in the Living Image browser Default Order in which the images are stored in the folder TimeStamp Ascending order of the image acquisition time UserID Ascending alphanumeric order of the user ID Display Choose the types of information to display with each image a gemme
295. ral Unmixing and DyCE gt Surface Topography 3D Multi Modality Tools 3D Optical Tools Surface Source Registration V Display Subject Surface A O Opacity lel V Display Photon Density Map Apply Simulated Wavelengths 560 4 07e 5 E c Ss F D Intensity Color Table Rainbow V Reverse Log Scale Table 11 10 3D Surface tools Item Description Display Subject Surface Choose this option to display the surface in the 3D View window Drawing styles for the surface Point cloud Paki Wire frame Fi Surface face Wire frame 4 surface face 214 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Table 11 10 3D Surface tools continued Item Description Ga ecu Shading styles for the surface Q Smooth surface qy Reflect surface face 1 face Reflect smooth bagi Surface face surface face m Click to open the color palette from which you can select a display color for the surface and the cross section views Opacity Adjusts the surface opacity Display Photon Density or NTF Efficiency Map Choose this option to display the photon density or NTF Efficiency on the surface Apply Choose measured or simulated photon density or NTF Efficiency maps for display Wavelengths DL
296. rces 189 5 To view the tissue properties U Hepp H for the tissue and source you selected make a selection from the Plot drop down 6 Select a luminescent quantification database to compute the number of cells per source optional For details on generating a luminescent quantification database see page 231 7 In the Analyze tab click Start The Data Preview window appears and displays the image data that will be included in the reconstruction Usually no data adjustment is required However it is possible to exclude or include user selected pixel data from the analysis For more details see Including or Excluding Data for 3D Reconstruction page 191 Figure 11 4 Data Preview window SS z SL Tool Palette E TUT20050624145507 sEQ aa lt _ NG PLCLAHPHMJU Sequence View LA 3D View Data Preview Analyze Properties Results Sequence 71720050624145507 SEQ Tissue Mouse Tissue Source Firefly Filter e Threshold 7 Image Label V Median Filter Restore Threshold Data Adjustment T Select All Cancel Reconstruct Data Preview window 8 In the Data Preview window click Reconstruct The reconstruction normally requires less than one minute depending on the reconstruction volume parameter settings and computer performance When the analysis is finished m The 3D View window d
297. rease or decrease the size x y Or z axis click the blue green or red circle and drag the mouse arrow in the direction of interest 4 Press the Tab key to switch between the transform tools The position of the organ s is updated in the slice windowpanes coronal sagittal and transaxial views after each adjustment 5 Turn off the transform tool when you are done adjusting the position of the organ s click the button To check the organ fit 1 Check the fit in the coronal sagittal and transaxial windowpanes 2 Click the Change view toolbar button The Top view is displayed Figure 11 35 Skin pink fitted to surface gray A TLT20050624145507_SEQ Sequence View b 3D View Gronal z 16 2 Sagittal x 3 0 Transaxial y 17 6 Subject Height 26 3 mm k Eas mee 3 Press the V key or the ia button to display alternative views of the surface 222 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources 223 Figure 11 36 Alternate views of the surface In this example skin is selected from the organ atlas pink surface The mouse surface is gray Top Bottom Front Back Left Right Importing an Organ Atlas An organ atlas iv dxf or stl one organ per file consisting of segmented organ surfaces derived from an MRI or CT scan can be imported into the Living Image software for registration with
298. reconstruction of the animal surface 9 EG topography derived from the CT image siect _ NideMese 7 AA i A surface is a required input for a DLIT diffuse light t hy analysis which ae rT iffuse light tomography analysis whic generates a 3D reconstruction of luminescent Name SURFACE 4 WMIC_Right_Mouse sources PASTE FLIT fluorescence imaging tomography analysis which generate a 3D reconstruction of fluorescent sources 3D Multi Modality Tools 238 15 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 2 Getting Started 16 Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum continued continued Living Image Tools and Functions See Page eb Optical Tool eae Surface Surface tools Adjust the appearance of the W Display Subject Surface reconstructed animal surface and the photon A density maps aaa 100 E Source tools Adjust the appearance of reconstructed sources make source measurements export voxel measurements Display Photon Density Map Apply Wavelengths Registration tools Display organs on the kanal Looe EF reconstructed surface adjust the location or scale Gala Tabie of organs on the surface import an organ atlas Log Scale DLIT 3D Reconstruction 187 Diffuse light tomography DLIT analysis provides ee ERA a complete 3D reconstruction of the luminescent source distribution within the subject The 3D Threshold reconstruction i
299. reporters M NOTE The FLIT results selected for display in the Longitudinal Study window must have the same type of units The DLIT results selected for display in the Longitudinal Study window must have the same type of units Viewing Results in the Longitudinal Study Window M NOTE The Longitudinal Study window can display FLIT results or DLIT results but not both at the same time Only 3D reconstruction results with the same type of units can be loaded 1 Load the DLIT or FLIT sequences with the results that you want to display Select Tools gt Longitudinal Study on the menu bar The Longitudinal Study window appears Figure 11 23 Longitudinal Study window 7 Longitudinal Study Window o pg g Angle Of View Se Select a Sequence 3D View Plots Loaded DLIT or FLIT EE DLIT EL20100601160926 sEQ sequences GS DLT EL20100608105326 sEQ Wavelength EEE DLIT EL20100615094528_SEQ DLIT TLT20050624145507_SEQ Results saved with the Select Analysis Result sequence selected in ie the upper box Select the results to display from this list Unit DLIT photons sec cells Display Voxels DLIT Color Scale v Display Surface Opacity P 50 v Display Photon Density Map NOTE After the Longitudinal Study window is open more sequences can be added to the window by clicking the Open button Cg and selecting sequenceinfo txt files found in the sequence
300. rescence Choose background signal s Food Signal Probe Information X ses z Probe list shows the probes to AET z unmix initially based on probes selected in the Imaging Wizard during sequence set Data Mask Options E u p Photograph Threshold la 73 PE J Draw Mask Rectangle Ellipse PCA Number of components to unmix 3 Number of components to unmix no of probes suggested by the software plus background signal s ss 6 Click the PCA button The Principle Component Analysis window shows the amount of signal explained by the suggested components Figure 8 13 The three components in this example tissue autofluorescence probe AF680 and probe AF750 explain more than 99 5 of the signal The small residual is due to noise If the explained variance is low add more components probes to unmix using the fu button 147 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing Figure 8 13 Principle component analysis E Principle Component Analysis Suggested of components 3 Explained variance 26 ofComponents Explained Vamance 6 1 58 7555 92 3562 99 5914 99 8463 99 9128 99 9572 99 9855 7 Click Finish The Unmixing window shows the analysis results which include unmixed spectra unmixed images and a composite of the unmixed images Figure 8 8 See Spectral Unmixing Results page 154 for information about the results
301. rescence sequence acquired in transillumination mode into a single image All of the individual fluorescent signals are stacked over one photograph and the intensity is summed One overview is created per filter pair If two filter pairs were used during acquisition then two overview images will be created All transillumination locations are displayed simultaneously a tool tip displays the transillumination position when you mouse over a transillumination point An overview image is displayed by default in radiant efficiency and if transmission images are available in normalized transmission fluorescence efficiency Transillumination overview images can be analyzed using the tools in the Tool Palette M NOTE If you choose the Raster Scan option in the Transillumination Setup box the overview image is automatically generated see Figure 3 22 on page 45 1 Load a sequence that was acquired in fluorescence transillumination mode 2 Click the button Alternatively select Tools gt Transillumination Overview for lt name gt _SEQ on the menu bar The overview appears Figure 4 24 Transillumination overview NG conos oe e f 7 a eee ieee Linita Panduit Didan Ut Sree Beker V Tainan Locaia Geman Dobari T ES Bo XU 4 1 E oversen e Unite Radiant Dfa 7 Dimis Over 7 Pusrescent om E hanga 7 ov Tranclumnasonlocion Opiom inde cA wj Trane Th eecence Living Image
302. rgan corresponds to characteristic pharmacokinetics depending on whether the isotope is accumulating washing through or being metabolized Spectral unmixing kt Dow sgnal relative to first time point gt w Filter Pair Spectral Unmixing Filter Scan Fluorescence DyCE DyCE in the fluorescent mode is an imaging approach that can provide coregistered anatomical information by exploiting in vivo pharmacokinetics in small animals DyCE can be applied singly or in combination with functionalized marker probes DyCE utilizes a time series of optical images acquired following a small bolus injection of dye or contrast agent As the dye circulates through the body each organ corresponds to a characteristic pharmacokinetics depending on whether the dye is accumulating washing through or being metabolized ow sgnal o first time point Ss MN relateve For fluorescence DyCE only select a probe from the Name drop down list For bioluminescence DyCE go to step 6 If your fluorescent probe is not in the list select Input and enter the fluorescence excitation and emission peak wavelengths Click Next Figure 9 4 Select the probe fluorescence DyCE only ic E imaging Wizard Fluorescence DyCE Probes Add a QD705 500 707 Remove Filter Config Options Y 4 HI Excitation Filters 400 500 600 700 800 90
303. ric Data Absorption Measurements Fixed 32 bit scale with values that are consistent between images Note These measurements are only available for IVIS Spectrum CT data Total Value The sum of the absorption measurements of all voxels in the 3D ROI Average Value Total Value Number of voxels in the 3D ROI Stdev Value Standard deviation of the absorption values for all voxels inside the ROI Min Value The smallest absorption value for any single voxel in the 3D ROI Max Value The largest absorption value for any single voxel in the 3D ROI 3D Volumetric Data Hounsfield measurements Calibrated CT scale Fixed from image to image Note hese measurements are only available for IVIS Spectrum CT data Total Hounsfield The sum of the Hounsfield unit values for all of the voxels in the 3D ROI Average Hounsfield Total Hounsfield unit value Number of voxels in the 3D ROI Stdev Hounsfield Standard deviation of the Hounsfield unit values for all voxels inside the ROI Min Hounsfield The minimum Hounsfield unit value for any single voxel in the 3D ROI Max Hounsfield The maximum Hounsfield unit value for any single voxel in the 3D ROI Living Image 4 3 Software User s Manual Chapter 6 3D ROI Tools for Volumetric Data Table 6 1 3D ROI Measurements table continued Item Description Source Voxels photons sec measurements Total Flux ph s The flux in
304. rid line pattern to display in the line profile window Exports the line profile data to a csv or txt file Copies the line profile graph to the system clipboard Opens the Print dialog box X Min X Max Displays the minimum and maximum value of the x axis Use the bi arrows to change the x axis min or max If a calibrated unit such as radiance is selected in the image window the x axis units cm If counts is selected in the image window the x axis units pixels To display the range available for the Min or Max place the mouse pointer over the Min or Max edit box Y Min Y Max FA a Displays the minimum and maximum value of the y axis Use the arrows to change the y axis min or max To display the range available for the Y Min or Y Max place the mouse pointer over the Min or Max edit box 76 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Table 4 8 Line Profile window Item Description Click to reset the X and Y Min and Max values to the defaults Full Scale Select this option to display the full X and Y axis scales Logarithmic Scale Select this option to apply a log scale to the y axis Viewing 3D Signal Intensity 1 Open an image and then click the Plot 3D button in the Image Information tools A 3D representation of all signals in the image is displayed in the 3D Plot window Figure 4 21 Fig
305. rived from an analysis of images of known serial dilutions of luminescent cells or fluorescent cells or dye molecules 12 1 Preparing and Imaging the Samples 1 Prepare a well plate 4 x 6 6 x 4 8 x 12 or 12 x 8 well format that contains a dilution series of luminescent cells or fluorescent dye at four or more concentrations Include at least four background wells that contain diluent only Place the well plate on the IVIS stage positioning it so that it is centered and square in the field of view M NOTE All of the wells must be within view in the image For wells containing fluorophores FOV D is recommended to reduce shadows from well walls and ensure more uniform excitation of the wells Acquire the images m Bioluminescent samples Acquire one Open filter image of the well plate m Fluorescent samples Acquire reflectance illumination Filter Scan images using the appropriate excitation and emission bandpass filters The well plate in Figure 14 1 contains a dilution series of a sample at four concentrations The image sequence is a filter scan set of images with the excitation filter centered at 465 nm for all the images and emission filter images centered at 520 nm 540 nm 560 nm and 580 nm Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 12 Quantification Database Figure 12 1 Well plate data Ee EL2060a14 10005 Sto LI Sequence Yen Eperira nets Radiant Efe 7 Use Saved tok
306. round ROI m NOTE This is an optional background correction that is applied in addition to the electronic dark charge and read bias corrections that are applied to the raw CCD data The Image Adjust tools and zoom feature are helpful for selecting an appropriate area for an ROI By setting the image minimum close to zero and zooming in on a background area in the image you can determine where naturally occurring background luminescence or autofluorescence is present For more details on the Image Adjust tools and the zoom feature see Adjusting Image Appearance page 68 and Magnifying or Panning in the Image Window page 70 Subject ROIs A subject ROI identifies a subject animal in an image It provides a convenient way to automatically associate link a measurement and average background ROI for background corrected ROI measurements when there is significant autoluminescence or autofluorescence Using a subject ROI is optional To draw a subject ROI using the auto ROI feature 1 Select Subject ROI from the Type drop down list 2 Click the O button 3 Select Auto All To manually draw a subject ROI M NOTE If the image was acquired using the Side Imager draw three subject ROIs one for each view Select Subject ROI from the Type drop down list 2 Click the O button and select 1 3 Position the subject ROI so that it includes the measurement ROI s and the associated average background ROI Living Image 4 3
307. row s Deletes the selected row s from the sequence table Replace Row s Replaces the row s selected in the sequence table with the rows in the system clipboard Note The Replace function is only available when the number of rows in the system clipboard is the same as the number of rows selected in the sequence table Paste Row s Adds copied rows to end of the sequence Removing Images From a Sequence Method 1 1 Select the row s that you want to delete 2 Click and choose Selected from the drop down list Method 2 Select the row s of interest and right click the sequence table to view a shortcut menu of edit commands Figure 3 35 53 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition 3 7 Manually Saving Image Data When you acquire the first image s of a session you are prompted to enable the autosave feature If autosave is enabled all images acquired during the session are automatically saved to a user selected location You can choose a different location at any time select Acquisition gt Auto Save on the menu bar This section explains how to manually save data 1f you do not want to use the autosave feature 1 Turn off the autosave feature select Acquisition on the menu bar and remove the check mark next to Auto Save After image or sequence acquisition click the Save button Gr Alternatively select File gt Save on the menu ba
308. s File Type Filter Shows Living Image files 8 Click txt an image Living Image file format Sequence txt an image sequence Living Image file format dcm kinetic data or an image that was exported to a DICOM file TIFF Image Files Graphic files tif tiff All Files All file types 3 Navigate to the file and click double click it Alternatively select the data and click Open Organizing Images When multiple image windows are open you can organize them in a cascade or tile arrangement Choose Window Cascade or Window gt Tile on the menu bar Figure 4 5 Image windows cascade top or tiled bottom fie bdi View Took Wirdow Hrip SPA eee Ts Fic Ed Vor Teh Wen Hd coo eS Bw ice a kay baa TUR ah bg erata BP niregan Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 4 2 About the Image Window and Tool Palette Image Window An image or image sequence is displayed in an image window Multiple image windows can be open at the same time Figure 4 6 Image windows sequence view and single image The options available in the image window depend on the type of active image data he ILI 4vusvuetitoous OCW a Sequence View a Haran ree Units E Use Saved Colors gt Image Adjust l i o
309. s enable real time pharmacokinetic spatio temporal biodistribution studies of probe or dye signal Improved Spectral Unmixing Tools 138 Choose from four methods of spectral unmixing depending on your knowledge of the probe 139 spectral response and the probe location If a user created spectrum library reference spectral data from known probes at khown locations is available it can be selected in the Imaging Wizard during sequence setup This provides a convenient way to select filters Ability to subtract compute pure spectra by subtracting unmixed spectra which overlap 152 Export unmixing results as an image or sequence which can be analyzed using image 157 analysis tools ROIs Mirror ROIs for optical data support measurements on the left and right views of images 102 acquired using the Side Imager accessory View a histogram of measurement ROI pixel intensities 108 Improved Acquisition Features The color of the upper control panel is an indicator of instrument activity Red color means the instrument is initializing or acquiring images blue color means the instrument is idle IVIS Acquisition Control Panel IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter g F Stop Excitation Filter Emission Filter 8 1 x Imaging Mode Exposure Time md Fluorescent Field of View C System Status Field of view D v Syst
310. s of the Living Image software require a separate license DyCE imaging and analysis is intended for biodistribution studies DyCE imaging captures a time series of optical images immediately following a bolus injection of a probe or dye The Living Image software temporally unmixes the data on a pixel by pixel basis for each image of the time series and determines real time spatio temporal distribution of the probe or dye signal The Living Image software presents the spatio temporal information as Temporal spectra Line plots of signal intensity as a function of time Each line plot represents the signal time course within a particular anatomical region a An unmixed image An image that represents the peak signal time point for a particular temporal spectrum A composite image An overlay of the unmixed images Figure 9 1 Example DyCE results Images were obtained using the Mouse Side Imaging Kit i RKG20110210131022_SEQ Sequence View Unmixing V Show Labels X Individual Scale Normalized E Legend a o h A P PAN Unmixed images Each Temporal spectra Wa 3 image is a representation show signal time J meee a of a temporal spectrum at course of different aai West 7 a the peak signal time point anatomical regions 0 40 Min 0 00 f Min 0 00 00 1200 Max 2 90e4 Max 1 5564 400 8 Time Point s Bladder Spectrum List wo py Xx Note Composite o
311. s presented as volume elements 3 4 called voxels 2 9 06 If a luminescent calibration database is available 620 0 5 the number of cells per source can be determined cso 06 in addition to source intensity photons sec FLIT 3D Reconstruction 194 Fluorescent imaging tomography FLIT analysis provides a complete 3D reconstruction of the fluorescent source distribution within the subject EmWL Threshold The 3D reconstruction is presented as volume 31 elements called voxels 31 2 51 If a fluorescent calibration database is available 5 0 the number of fluorophore molecules or cells per 51 source can be determined in addition to the total 51 fluorescence yield 51 51 Source Unknown ISS SSS S288 i G aka kakak 138828888888 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 2 Getting Started Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum continued continued V Apply to Sequence WellPlate Quantification Plots Results Click E120090414101005 001 a an a ROI vs well plate population 6 Total Efficiency cm Linear Fit 4 m 5 0 x10 0 0 1 0 2 0 3 0 40 5 0 well plate population Living Image Tools and Functions See Page 3D Animation Tools 225 E 3DAnimation Select Tools 3D Animation on the menu bar Preset Animations Presets
312. s the image data that will be included in the reconstruction Usually no data adjustment is required However it is possible to exclude or include user selected pixel data from the analysis For more details see page 191 You can also include or exclude image data by adding or removing the check mark next to the images listed in the Analyze tab Figure 11 10 Figure 11 12 Data Preview window G ALUMANA NU el TE ck20080407145405_SEQ V Image Label V Median Filter RestoreThreshold Data Adjustment Select All Cancel Reconstruct 10 Click Reconstruct The reconstruction normally requires less than one minute depending on the reconstruction volume parameter settings and computer performance When the analysis 1s finished a The 3D View window displays the surface and the reconstructed sources a In the Tool Palette the Results tab displays the results data and the algorithm parameter values Figure 11 14 m The 3D Tools appear in the Tool Palette For more details on the 3D Tools see page 213 225 For details on managing results for example save load or delete see page 198 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 11 3D Reconstruction of Sources Figure 11 13 FLIT results 3D View window and Results tab For details on the 3D View toolbar see Table 11 3 page 190 3D View toolbar Tool Palette 8 ROI Tools lol gt Spectral U
313. same animal model Image Requirements Use the Imaging Wizard to set up an image sequence for spectral unmixing See page 33 for more details on the wizard If you generated a spectrum library you can select it in the Imaging Wizard Figure 8 1 Figure 8 1 Select a spectrum library in the Imaging Wizard Imaging Wizard Fluorescence Spec Unmix Filter Scan Excitation Peak Emission Peak AF750 v 752 N a o Open the Filter Section Configuration dialog box Filter Config Filter Scan Type O Excitation an 9 Emission Scan C Bath Filter Selection Configuration Excitation Filters 1 00 Excitation Filters E Block 430 465 500 535 570 605 640 675 AE i NDS Block ND3 430 ND3 465 ND3 500 ND3 535 ND3 570 ND3 605 ND3 640 ND3 675 ND3 710 ND3 745 ND3 s GFP Block GFP 430 GFP 465 GFP 5 DsRed Block DsRed 430 DsRed 465 DsRed 500 DsRed 535 DsRed ae i Cy5 5 Block Cys 5 430 Cy5 5 465 Cy5 5 500 Cy5 5 535 Cy5 5 570 Cy5 5 605 Cy5 5 640 Cy5 5 npa n ICG Block ICG 430 1CG 465 1CG 5OD ICG 535 ICG 570 ICG 605 ICG 640 ICG 675 ICG 710 ICG 745 ICG Open Block Open 430 Open 465 Open 500 Open 535 Open 570 Open 605 Open 640 Open 675 Open 710 Open 745 Open M m _ PP ES Select by Spectrum Library Click Select by Spectrum Library and choose a spectrum library in the dialog box th
314. scaling Choose this option to display computed results on a normalized scale starting a zero Fit Offset If this option is chosen the software computes and removes an intensity baseline from the spectra Error Tolerance The software computes a default error tolerance the factor x for A x B such that signal B is maximally removed from signal A with no negative result Moving the slider adjusts the error tolerance and automatically updates the computed spectrum Choose New to save computed spectrum with the specified name and color Click Apply to add the computed spectrum to the spectrum plot and list in the Unmixing window Choose a spectrum number from the drop down list to overwrite that spectrum with the computed spectrum when you click Apply 153 Living Image 4 3 1 User s Manual IVIS Spectrum 8 4 Spectral Unmixing Results Chapter 8 Spectral Unmixing The results include a signal distribution map of each unmixed result and a composite image of all signals each displayed in a different color Figure 8 21 Spectral unmixing results Information about the analysis method and analysis inputs pr 7 HX20120419114703_SEQ Sequence View Unmixing V Show Labels V Individual Scale mana Tool Palette 8 Spectral Unmixing and DyCE o a q E Analyze Results 2 B g amp pectral Unmixing Re V Normalized Legend a
315. se A O Sequence View LA 3D View Rv EJ 9 BR e s Be Transaxial y 47 7 i nnna n ee ee na i Subject Height 19 4mm Perspective 0 00 Counts v 4 Logarithmic Histogram Maximum Intensity Projection MIP Gradient Illumination gt 3D Optical Tools E DLIT 3D Reconstruction Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools 244 Maximum Intensity Projection MIP MIP projects all maximum intensity voxels in the view along the viewing direction into the viewing plane Gradient Illumination Gradient Illumination is based on the idea that light is reflected at boundaries between different voxel intensities but is not affected when passing through homogeneous regions Choosing this option illuminates the voxels at boundaries more than voxels within a homogeneous region The boundaries are based on the gradient magnitude between heterogeneous regions or the change in intensities between neighboring voxels in heterogeneous regions Using this option enhances the variation in tissue properties and may be helpful for visualizing the boundaries of different tissues Modifying Volume Resolution Changing the pixel or slice spacing modifies the volume resolution Increasing the pixel or slice spacing reduces resolution while reducing either increases resolution 1 In the Volume tab click the Ed
316. ser s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 56 Figure 4 1 Opening the Living Image Browser ki ie lab View Tools Window Help BeOS RSW Armitage 4 J Caliper Ls 4 Caliper Data gt J ALI20110120171831 SEQ bal AutoFluorBkgCorr gt Ji BkgSub 1 Ceeio DE gt J RKG20110210131022 SEQ gt Ni CoReg Demo J dem 20110306103654 m Gose previen tabe set Al J ad to tist C ronse _ view Location KATHERINE PC Share Caliper LS Caliper Data CerenkovDyCE RKG20110210131022_SEQ Sequencelnfo txt Living Image Browser ME Image HED Image sequence DEM Image exported as DICOM file ove DyCE image sequence Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data 57 Figure 4 2 Living Image Browser To expand a sequence click the Click a column header to sort the To view data properties gt arrow next to sep browser contents in ascending right click a row and select alpha numeric order Click the Properties on the column header again to sort in shortcut menu descending alpha numeric order Group Experiment Commenti a iXFM SN1007 irod7 srcA 745 800 ALIAGA iXFM SN1007 irod7 srcA 745 800 eeesssiessseessssssssessssossecasssseiessosseessessssessosssessesssdesssssessssssesesssssossessssseesssssosssssssseessssssse iXFM SN1007 irod7 src A ___ EXFilter EM Filter Illumination Mode
317. sing the Manual Registration tool page 255 4 Classify the 3D volumetric data to help identify and separate objects page 239 Save the color opacity map optional 5 Save the registered 3D multi modality results page 249 251 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools 252 Loading Data for Registration 1 Loada DLIT or FLIT image sequence and the 3D reconstruction results M NOTE The 3D Multi Modality tools appear in the Tool Palette after you load optical image data If the 3D Multi Modality tools do not appear in the Tool Palette confirm that the 3D Multi Modality Tools license is installed and that the workstation graphics card meets the specifications in Table 13 1 on page 238 2 Select the DICOM or TIFF volumetric data a Select File Browse 3D Volumetric Data on the menu bar b Select a data folder in the Browse For Folder box that appears and click OK The Living Image 3D Volumetric Browser appears Figure 13 14 NOTE Only DICOM or TIFF data can be added to the 3D Volumetric browser For details on loading other data types raw or vox files see page 258 Figure 13 14 Opening the 3D Volumetric Browser Fae kat Yaw Tacit Magasin Window Hep SAW a BAS E PASAN Fer elds nang mage Huirt Pokie ai Caliper LS a Ji Caliper Data JK ANTE NG Ji AutePicebieg Con E BkgSub ub Cofteg Demo AL dem Sones Ji DUT
318. single image This enables you to see both intensity and spectral information in a single view The tool provides a useful way to visualize multiple probes or scale probe signals that are not in the visible range 18 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 2 Getting Started Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum continued continued Living Image Tools and Functions See Page Transillumination Overview 80 Select Tools Transillumination Overview for lt sequence name gt on the menu bar The transillumination overview tool combines the images of a FLIT sequence a fluorescence sequence acquired in transillumination mode into a single image All of the individual fluorescent signals are stacked over one photograph and the intensity is summed One overview is created per filter pair If two filter pairs were used during acquisition then two overview images will be created IE Wrage Math Winders Slag boae EAL FIO TE AG A TUDO LO ASL OA TORSO LL SLD OLGA E NI AOCSER ILIAN DLA PAS TAB TIAE ADS DIGA 18 ka 8 Pal kik PAN Pl Camie Uk BE F mi Pho bom a Fa Ma 2 5 Managing User Accounts Adding Users New users can be created in the m Main window at startup see page 6 m User Settings dialog box Figure 2 8 Image Math Window 133 Select Tools Image Math for lt sequence name gt o
319. sk Setup box that enables you to set the photo mask for adaptive fluorescent background subtraction Tip See the tech note Adaptive Fluorescence Background Subtraction select Help Tech Notes on the menu bar Read Bias Subtraction Dark Charge Subtraction Select this check box to subtract dark background from the image data If a dark charge image is available for the imaging conditions the dark background image including read bias noise will be subtracted Otherwise only read bias noise will be subtracted Note In Radiance Photons mode dark background or read bias subtraction is a mandatory default In counts mode the check box can be cleared Tip See the tech note Luminescent Background Sources and Corrections select Help Tech Notes on the menu bar Flat Field Correction Select this check box to apply flat field correction to the image data 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 See the tech note mage Data Display and Measurement for more about cosmic correction select Help Tech Notes on the menu bar 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
320. t 86 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Table 4 14 Image Layout window continued Item Description Ng Deletes the selected image A drop down list of formatting options for the Image Layout window For example the 2x2 layout style provides 4 separate layout areas in the window A different image can be pasted into each layout area Layout Style Layout ene kag fmatahon To apply notes to an image enter text in the annotation box and press Enter Drag the IMA text to the location of interest in the image 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 4 12 Editing an Image Sequence You can add or remove individual images from a sequence Only individual images not an image sequence can be added to a sequence 1 Open the image sequence that you want to edit 2 If you plan to add images to the sequence browse for the images that you want to add in the Living Image browser See page 55 for more details on browsing 3 Click the Edit button K in the image window Figure 4 31 Figure 4 31 Opening the Edit Sequence dialog box Single images in the Living Image Browser that can be added to the sequence
321. t ROI on an area that represents background signal Radiant Efficiency secicm shy uwicm Color Scale Min 2 62e6 Max 1 7388 3 Select Tools Image Math for lt name gt _SEQ on the menu bar Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 7 Image Math 4 Inthe Image Math window that appears select the primary image in box A Select the background image in box B For more details on items in the Image Math window see Table 7 1 page 134 5 Select the math function A B k in the Result drop down list Figure 7 5 Select a math function and view the mathematical result P ye 7 Image Math Window Co e is E TLT M20060510114512 008 l x Sequence KSA20110305104918 SEQ Units Counts Display TLT20060510114512_008A TLT20060510114512 010A B TLT20060510114512 008A TLT20060510114512 010A Counts Color Scale Limits for A and B Full 5 Auto Result Color Scale Limits 3 2 Counts Full Auto E Min 0 Result A B k x k 0 787938 Compute k from ROI 7 with Photo from A X Display Result For Measuring Epi fluorescence 6000 2000 Counts Color Scale Min 1773 Max 6087 6 Click and select the ROI created in step 2 from the drop down list The background corrected signal is displayed 7 To view the mathematical result overlay mode in a separate i
322. t binning to the image data TIP See these technical notes for helpful information select Help gt Tech Notes on the menu bar m Detection Sensitivity includes information about binning and smoothing m Luminescent Background Sources and Corrections a Fluorescent Imaging for more about fluorescent background Figure 4 15 Tool Palette Corrections Filtering tools Tool Palette Fx Corrections Filtering le Read Bias Subtraction Note Read Bias Subtraction and Flat Field Correction Flat Field Correction are default V Cosmic Correction mandatory corrections in Radiance E Adaptive FL Background Subtraction units mode These corrections can be cleared in counts mode Binning 8 gt 4 Smoothing None lo Image Information ROI Tools Spectral Unmixing and DyCE Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Table 4 5 Corrections Filtering tools Item Description Lens Distortion Correction Select this option to correct for distortion at the perimeter of an image due to curvature of the CCD lens Lens distortion correction is available for data acquired by Living Image software version 4 3 and higher The correction is particularly important for IVIS Spectrum CT data acquired for DLIT or FLIT Adaptive FL Background Subtraction Opens the Photo Ma
323. t the new location Figure 5 23 Figure 5 23 Move or edit the ROI label EF 1L120050624145507 006 oO 8 Units Radiance Photons v Display Overlay v Options Y Info NG igj Luminescence Contour 2 5 Auto 25 Background ROI Subject ROL Image Number 20 TLT20050624145507 006 v ROI BKG 1 v BKG 1 1 278e 05 V Lock Position ROI 1 BKG 1 25 4 825e 09 ROI 2 BKG C Radiance Height am 0 89250 p sec cm2 sr Color Scale Line Size 3 Min 1 46e8 Max 2 54e9 Line Color AN saa Done To edit the ROI label 1 Double click the ROI of interest Alternatively right click the ROI Ctrl click for Macintosh users and select Properties on the shortcut menu 2 In the ROI Properties box that appears edit the name in the ROI Label box and click Done Figure 5 23 Save Load or Delete ROls The software automatically saves ROIs with an image The ROI measurements are saved in the AnalyzedClickInfo txt file associated with the image ROIs are saved per user and can be applied to other sequences Additionally ROI parameters can be saved per user and applied to other sequences To save ROls to the system 1 In the Name drop down list confirm the default name or enter a new name for the ROI s 116 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data 117 Aa a eee ee a a ee eee ee ee ee Jl Jl a ig
324. t to unmix a Click the mm button A new name appears in the spectrum list Figure 9 16 b Specify the region by using the mouse to draw a mark on the image cube If necessary click the button next to the spectrum name to select a different line thickness from the drop down list c If necessary right click the image cube to erase the mark 5 Repeat step 4 to specify additional temporal components M NOTE A maximum of 10 components can be unmixed 170 Living Image 4 3 1 User s Manual IVIS Spectrum Figure 9 16 Mark the area of a temporal component on the image cube i 4 Normalized 7 RKG20110210131022 SEQ Hola Sequence View Unmixing Spectrum List Mark a region aso py Xx Note Select Name i Color Pick 1 V Kidney Mime X ar 2 V Brain I red X fir 3 V Bladder l DG X ar 4 V umx4 Wanta 7TF Thin Medium Thick Image Cube Viewer Overview Unmix Close Double click to edit a name B g amp Legend a ImageCube 400 800 1200 Time Point s Change the line thickness optional 6 Click Unmix after you finish marking the regions The software generates unmixed images for the new temporal spectra and updates the composite image with these components Table 9 2 Spectrum list toolbar Chapter 9 DyCE Imaging and Analysis item Description Enables you to view and save the unmixed images as a seq
325. ta by doing either of the following m Drag the data file DICOM TIFF from Windows Explorer to the Volume Data Viewer window OT In the Volume Data Viewer click the Open button G F and in the dialog box that appears select a DICOM or TIFF file and click Open 3 To clear the Volume Data Viewer click the ET button Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools Figure 13 23 Drag volume data from Windows Explorer to the Volume Data Viewer Organize b BkgSub gt wi DLIT Datasets gt Kidney b di Lung b Ni ALI20110120171831 SEQ gt Ss AutoFluorBkgCorr So dem 20110306103654 p di I20110114162356 SEQ d Katherine Quantum gt Ni KSA20110305104918_SEQ b gt LognitudinalData di MAT20090302190433 b Ji Multi Modality Datasets Share with Go Ie TIFF mouse 3_3 20100528 140051 FOV60m Bs Open Slide show I kn BAG Search mouse 3 Z a2 Print E mail Burn Paa a SO mouse 3 3 20100528 140051 in J RAW TIF File mouse 3 3 20100528 140051 _ State BR Shared Date taken Specify date taken EE Volume Data Viewer mouse 3 3 20100528 140051 To view a particular slice move the slider or enter a slice number 3 MING GIO AN1 E FAT aR 1 f mi AG LAAL Sya Pp Hi Starts playback of the DICOM files Ta
326. ter 6 3D ROI Tools for Volumetric Data M NOTE Preset table configurations cannot be deleted Copying or Exporting the ROI Measurements Table To export the table 1 In the ROI Measurements table click Export 2 In the dialog box that appears a Select a folder and file type txt or csv b Enter a name file and click Save To copy the table to the system clipboard m Copy selected rows Select the rows of interest and click Copy Alternatively select the rows then right click the table and choose Copy on the shortcut menu a Copy all rows Click Select All and click Copy Alternatively press Ctrl A then right click the table and choose Copy on the shortcut menu Figure 6 11 3D ROI table shortcut menu z C ROI Measurements Cola xs ROIMeasurements 3D ROI Measurements Data Types 3D Volumetric Data Z Measurement Types Refresh B120111027132749_SEQ ROI 1 54015 2640219 o Copy Ctrl C Select All Ctrl A Copy Select all Configure Export Close 132 7 Image Math Creating a New Image Using Image Math Subtracting Tissue Autofluorescence on page 135 The Image Math tool is used to mathematically combine two images to create a new image Image math is primarily for subtracting tissue autofluorescence background from signal To perform image math open an image sequence or a group of images For more details
327. ter 7 Image Math Figure 7 2 Image Math window and new image Click to export the image to a graphic file r A Image Math Window A TLT20060510114512_001 TLT20060510114512_002 i a TLT20060510114512_003 TLT20060510114512_004 TLT20060510114512_005 TLT20060510114512 006 TITINNEANEIMI1AE17 ANT TLT20060510114512 001 TLT20060510114512_002 TLT20060510114512_003 TLT20060510114512 004 TLT20060510114512_005 TLT20060510114512_ 006 TITINNANGIN114517 ANT Color Scale Limits for A and B Full Auto Result Color Scale Limits Full Auto E Min z0 Result A B k M k 1 00 Compute K from ROI v V with Photo from A M Display Result For Measuring Sequence TLT20060510114512 SEQ Radiant Efficiency Radiant Efficiency o amp 83 E7 TLT M20060510114512 005 Units Radiant Efficiency Display Options vY si TLT20060510114512 005 TLT20060510114512 001 1 00 p Epi fluorescence Radiant Efficiency t pisecicm sn WWwicm2 Color 5cale Min 3 98e7 Max 7 77e8 M NOTE For more details on items in the Image Math window see Table 7 1 page 134 2 a gt To save the new image Select a mathematical function from the Result drop down list To include a scaling factor k in the function enter a value for k To view the new image click Display Result for Meas
328. the drop down list In this example the Contour shape was selected for the free draw method The ROI shapes that are available depend on the type of ROI selected If you selected DJ or O Use the pointer to draw the ROI Use the pointer to click around the area of interest and draw line segments that define the ROI Right click when the last point is near the first point in the ROI KOO OO OO TOOT OO OOTD QO TOO MM MM aM MM MM I aM MM MMM TOO igure 5 11 Drawing an ROI using the free draw method Ko Ta a DT DD a DD aaa DD DT eae a m o ge e a aa a DD DT aaa DT ae a ai ets Mp PAA k a a a a a a Pa AD EL ELLE a a a a a Gi Gi Gi i Gil Gi Ni Mi Mi Gi il il Nail Ni il Mil Ni Mil Mi Ni Ni Nil Ni Mi Mi il Mi Nil Gi ai Nil Mi Mi Mil Mil Mil Gi Mil il Nil il Mil Nil Nil Nil Mil Nil Mil a a Mai E7 1 120050624145507 006 Co ss fl Units Counts v Display Overlay x Options v Info NG isj Pat M a a ea Sie Tool Palette gt Image Adjust gt Corrections Filtering gt Image Information 7 ROI Tools O O Yf Measure ROIs x F Apply to Seque Auto All ype Measuremer Save ROIs Name ROI 3 KS C gt REg Luminescence Auto 1 Free Draw Delete Load Save Auto ROI Parameters Thresnot 15000 Lower Limit 10000 5000 Counts Color Scale Min 1122 Max 19546 4 Click the Measure but
329. the time series exceeds 100 If necessary adjust the duration or delay between images of one or more intervals to reduce the number of images d Click Next The specified sequence appears in the sequence table Figure 9 6 Example DyCE sequence Mode Exposure Binning FStop Excitation Emission Lamp Level FOV Height DyCE Duration DyCE Delay 1 Pia Auto 2 640 TOU High C150 4min Ssec 2 Pa auto 2 640 700 High Co 150 Simin A5 sec 3 Dee Auto 2 640 700 High C150 2min S sec Number of Segments 1 Delay 0 0 5 min Apply ko All x Remover Cf Update Insert Add c aga Subject Mouse Probes x 11 Click Acquire when you are ready to capture the image If this is the first acquisition of the session you are prompted to enable the autosave function Figure 9 7 When Autosave is enabled all images acquired during the session are automatically saved to a user selected folder A different folder can be chosen at any time select Acquisition Auto Save on the menu bar Figure 9 7 Autosave prompt A Living Image 4 3 64 bit Auto Save x Do you want to enable auto saving of acquired data for this session This can be changed anytime from the Acquisition menu a Yes No 12 Click Yes in the prompt to enable autosave then choose a folder in the dialog box that appears Alternatively click No in the prompt and manually save the image data S
330. the view Figure 10 6 m Click the surface in the 3D View window then press the V key to cycle through the different views of the surface a Figure 10 7 shows examples of the available views You can view the surface from different perspectives by doing one of the following Figure 10 6 Surface perspective view E TLT20050624145507 sEQ oko Sequence View 4 3D View Gronal z 13 0 Sagittal x 2 0 Transaxialfy 13 0 Subject Height 26 2 mm eo View name Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 10 Reconstructing a 3D Surface Figure 10 7 Alternate views of a surface Click the surface then press the V key to change the view Top Bottom Back Left Right 10 2 Managing Surfaces After the surface is saved it can be shared by the DLIT or FLIT tools Figure 10 8 Tool palette Surface topography tools Tool Palette ROI Tools Spectral Unmixing and DyCE Surface Topography Optical Surface Reconstruction Orientation Dorsal bd Surface Smoothing Level Low bd D Save Results Name SURFACE 1 Surface name Delete Load gt 3D Multi Modality Tools 3D Optical Tools gt DLIT 3D Reconstruction 182 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 10 Reconstructing a 3D Surface 183 Item in the Sur
331. tion ig pa Shading styles for the source surface see Display Source Surface m Click to open the color palette from which you can select a display color for the source surface Opacity Adjusts the source surface opacity Display Voxels Choose this option to display the sources reconstructed using DLIT or FLIT Maximum Intensity Choose this option to project all maximum intensity voxels in the view along the Projection viewing direction into the viewing plane Threshold Choose this option to apply a minimum threshold intensity to the voxel display DLIT FLIT Gradation Use this slider to set a threshold for the percentage voxel intensity above which voxels DLIT FLIT are opaque and below which voxels will gradually face to transparent The percentage voxel intensity is the percentage relative to the maximum intensity Voxel size The 3D grid spacing size for interpolation of the reconstructed source Smoothing The smoothing box filter size Display voxels as The voxel display mode cubes spheres points or texture Color Scale Color Scale Min la 2 10e3 5 Color Table tet jam TC Reverse E Log Scale Min Use the slider or up down arrows to set the minimum value of the source color scale Voxels with intensities less than the color scale minimum are not displayed in the reconstruction Color Table Color scheme for voxel display Use th
332. tion flipped or reversed along the x y or z axis As a result the slice views transaxial coronal sagittal may be flipped or rotated so that the actual view that is displayed does not match the 3D View windowpane name for example the Sagittal windowpane does not display a sagittal slice or the data appears flipped with respect to the surface derived from the IVIS Spectrum In such cases you can Invert the data along the x y or z axis a Manually rotate the data using the Transformation tool for more details see page 258 260 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools 261 To invert the subject orientation 1 Click the Edit Spacing amp Orientation button lm 2 In the dialog box that appears choose a Subject Orientation option and click OK Figure 13 27 Volume Information dialog box E Volume Information Slice Information Pixel Spacing Row Column 0 1000 0 0015 mm Slice Spacing 0 0016 mm Subject Orientation E Invert X Axis E Invert Y Axis E Invert Z Axis Appendix A IVIS Acquisition Control Panel Control Panel Manually Setting the Focus on page 265 A 1 Control Panel The control panel provides the image acquisition functions Figure A 1 Figure A 1 IVIS acquisition control panel auto exposure selected To acquire an image using auto exposure click the arrow and select Auto Ex C mis
333. tions Show Advanced Options If this option is selected advanced features are available in the menu bar and Tool Palette including a Additional ROI functionality for Auto ROI parameters Additional export and import option for 3D surfaces and voxels m Planar Spectral Imaging tools in the Tool Palette Show Activity Window on A drop down list of options for when to display the activity log Figure B 2 Save Settings Save float corrected image Saves an image after all corrections are applied read bias subtraction flat field correction cosmic correction Color Selections Applies the color settings of the active image data to subsequently opened image data Folder Locations Sets the default folder path to the current folder path setting Click the Export button ey in the image window to view the current folder path setting Figure B 2 Window Size amp Position Applies the active image window size and position settings to subsequently opened image data Most Recently Used Dataset History Defines the number of recently opened data sets to remember and display when you select File gt Recent Files 5 Menu Display ROI Label As Measurement Sets the type of measurement in counts radiance photons or efficiency to show in the ROI label Some of the general preferences specify how the main application window is organized To undock the Tool Palette click on the palette title bar and
334. ton Wf Measure rat The ROI measurements and table appear For more details on the table see Managing the ROI Measurements Table page 119 For information on how to save ROIs see page page 116 101 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 5 ROI Tools for Optical Data 102 5 5 Mirror ROIs Use a mirror ROI to measure bioluminescence or fluorescence in the right or left mirror reflected view of images acquired using the Side Imager Measure signals in the center view using a measurement ROI See page 97 for more details on drawing a measurement ROI m NOTE Do not apply mirror ROIs on the center view or measurement ROIs on the left or right mirror reflected views Placing an ROI on the wrong view will result in incorrect ROI measurements 1 Open an image or image sequence acquired with the Side Imager M NOTE Fluorescent image data acquired in reflectance epi illumination mode must include a photograph 2 Select Mirror ROI from the Type drop down list in the ROI tools If analyzing a fluorescent image choose the Photo Mask option Figure 5 12 ROI tools Tool Palette I Corrections Filtering gt Image Information ROI Tools LI YW Measure ROls x Apply to Sequence ypei Mirror ROL Photo Mask Save ROIs 3 Select the ROI shape a Click the Circle O or Square GJ button b Select the number of ROIs to add to the image on the drop down
335. trum and analyze the data using the Living Image software The manual provides detailed instructions and screenshots Sometimes the screenshots in the manual may not exactly match those displayed on your screen When analyzing data acquired on a different type of IVIS instrument say for example the IVIS Spectrum CT please see the Living Image Software User s Manual specific for the VIS Spectrum CT Table 1 1 Living Image 4 3 1 software manuals Please see the IVIS Spectrum Hardware Manual part no 121450 Rev00 for information on the IVIS Spectrum instrument Living Image Software Manual for the Part No IVIS Lumina II CLS135289 IVIS Lumina XR CL5135290 IVIS Kinetic CLS135288 IVIS 200 CLS135287 IVIS Spectrum CLS135291 IVIS Spectrum CT CLS135292 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 1 Welcome 1 2 What s New in the Living Image 4 3 1 Software The Living Image 4 3 1 software controls optical image acquisition on the IVIS Spectrum instrument and provides tools for optimizing image display and analyzing images The major new or improved features are listed below New or Improved Features See Page New DyCE Dynamic Contrast Enhancement Acquisition and Analysis Tools 159 Note DyCE acquisition and analysis features require a separate license DyCE acquisition supports Cerenkov radioactive luminescent or fluorescent imaging DyCE acquisition and analysis tool
336. ts Item Description Delete Removes the active quantification sequence Sequence Name PC3M_GFP x Save Saves the quantification results with overwrites previous results Load Opens quantification results from the sequence path Overwrite Saves the results with the selected image sequence and results from the image the selected image sequence Database Name WPQUANT_1 v available for DLIT or FLIT reconstruction overwrites previous results Delete Deletes the database from the system Load Opens quantification results from the system path Save Saves the quantification results to a system database that is Overwrite Saves the results to the selected database name and Living Image 4 3 1 User s Manual IVIS Spectrum Exporting Quantification Results Right click the results table to view copy and export options Copy Copies the selected rows to the system clipboard m Select All Selects all rows in the results table Chapter 12 Quantification Database a Export Results Opens a dialog box that enables you to export the selected results to a text file Figure 12 8 Well plate quantification results For Sequence EL20090414101005 SEQ Click EL20090414101005 001 v Fluorophore Type E Well Plate Type i Dye molecules Cells Measurement Sample Wells 3D 3A C Set 7 Background Wells 1D 1A Apply to Sequence
337. uction results will be displayed in calibrated units for cell numbers or molecule quantities in picomole units Figure 12 7 Save the quantification results 7 Well Plate Quantification Window Col Ha Tool Palette 3 For Sequence EL20090414101005_SEQ Click EL20090414101005 001 LC Image Adjust a Fluorophore Type Corrections Filtering gt H Well Plate Type Dye molecules Cells Z d Measurement gt ROI Tools Sample Wells 3D 3A C Set Pd Surface Topography J Background Wells 6D 6A _ set 6 FLIT 3D Reconstruction 7 Apply to Sequence Analyze Properties Results Well Plate IF Quantification Plots J Results Tissue Properties Mouse Tissue Well Plate Quantification Results Unsaved Fluorescent Quantification Excitation Emission Extinction Coeff Cross Section WPQU Il nm nm Qe M cm 1000 Qoa mm ANTA N E pa Tissue Properties Plot 1465 520 2 919e 07 1 115e 08 2 465 540 1 262e 07 4 821e 09 n pa 3 465 560 5 894e 06 2 251e 09 29 em ueff 4 465 580 2 230e 06 8 518e 10 5 Wavelength nm Sequence Database Name WPQUANT 1 w Name WPQUANT 1 v ete Save Delete ad Save _ 3D Multi Modality Tools a Saves the results with Saves the results to a database that is the image sequence available for DLIT or FLIT analyses Table 12 2 Managing quantification resul
338. uence data set The image adjust corrections filtering image information or ROI tools are available for the images Enables you to subtract one spectrum from another see page 175 Adds a component to the spectrum list A Deletes the last spectrum in the spectrum list 171 Living Image 4 3 1 User s Manual IVIS Spectrum 9 4 DyCE Results Chapter 9 DyCE Imaging and Analysis The Unmixing window shows the DyCE results The example in Figure 9 17 shows three temporal spectra signal as a function of time Figure 9 17 DyCE results showing three temporal spectra RKG20110210131022 SEQ h O Sequence View Unmixing Show Labels V Individual Scale Normalized E Legend 1 00 Temporal spectra 080 show signal time course of different 0 60 anatomical regions 0 40 D 400 800 1200 Time Point s Spectrum List by Xx Note Select Name Color Pick 1 V Kidney Hime vifir 2 V Brain mr 3 V Bladder be ze Unmixed N Kidney 1 ee 7 Min 0 00 Max 2 90e4 Bladder Min 0 00 Max 2 4564 Composite Unmixed images Each image is a representation of a temporal spectrum at the peak signal time point Mins 0 00 Max 1 58e4 Composite of the temporal spectra Viewing Unmixed Images An unmixed image shows the maximum signal for a temporal spectrum a Double click an unmixed image to view it in an image window
339. umber of voxels Stdev pmol Standard deviation of the picomole values in the 3D ROI Min pmol Smallest picomole value in the 3D ROI Max pmol Largest picomole value in the 3D ROI Refresh Updates the ROI Measurements table for example after you draw new ROls move an ROI and close or open image data Copy Copies the selected row s in the table to the system clipboard Select All Copies all rows in the table to the system clipboard Configure Displays the Configure Measurements box that enables you to specify and organize the data categories column headers for the table See page 130 for more details Export Opens a dialog box that enables you to export the ROI measurements txt or cSV Close Closes the ROI Measurements table ROI Properties You can view information about the location and dimensions of a 3D ROI Click the 3D ROI Transform button Fi and select an ROI from the drop down list 2 Double click the 3D ROI The 3D ROI Properties dialog box appears 3 Enter new values or use the arrows in the dialog box to modify the location and or dimensions of the 3D ROI 128 Living Image 4 3 Software User s Manual Chapter 6 3D ROI Tools for Volumetric Data 129 Figure 6 6 3D ROI Properties EE 3D ROI Properties felts For Sequence BI20111027132749_SEQ r Ea 8 Mm tee ss GR ROIName ROT 1 Use Tab key to switch between transformation t Su
340. uorescence background component Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 8 Spectral Unmixing 141 The image cube represents a stack of the sequence images sorted according to the spectral axis When the Overview option is selected the image cube shows a pseudo color image that is a composite of the stack images which have been colorized to encode spectral information The entire image cube is calibrated and visualized on the same scale To view a particular image remove the check mark next to the Overview option and move the slider or enter an image number NOTE In the Guided method the Tissue AF component is preset as background After you define the Tissue AF component mark a region of tissue autofluorescence only on the image cube the spectra of the other components that you mark on the image cube will be background subtracted not raw spectra from the data ell 4 Move the mouse pointer over the image cube to see the spectrum at a particular location The raw spectrum at the pointer location is updated as you move the pointer 5 To specify a probe location for unmixing a Click the button for a spectrum b Using the mouse draw a mark on an area of the image cube which represents the probe signal The software plots a background subtracted spectrum of the signal Figure 8 4 6 If necessary right click the image cube to erase the mark Repeat step step 5 to specify other pro
341. urce or a point in a source to obtain source gee measurements total flux volume center of mass host organ in the 3D tools Source tab For more details see page 202 Copies or pastes voxels or a source surface so that DLIT and FLIT reconstructions can be displayed on one surface For more details see page 205 Enables you to save the 3D view to a graphic file for example jpg Including or Excluding Data for 3D Reconstruction The Data Preview window shows the image data that are automatically selected for reconstruction Figure 11 7 In special cases you may want to include or exclude particular data from this default selection There are two ways to do this m Change the Threshold value see below Applying a Threshold value excludes or includes some pixels from the reconstruction The software computes the minimum and maximum pixel values of an image based on an histogram of pixel intensities If Threshold 0 5 then pixels with intensity less than 0 5 of the maximum intensity value are excluded from the reconstruction The Threshold can be edited for individual images The Data Preview window is updated when you change the Threshold value Min Counts translates the Threshold to the minimum counts required for reconstruction Keep the minimum counts gt 200 m Region selection see page 192 Use the pencil tool to mark particular regions to include in the reconstruction This may be useful
342. ure 4 21 3D intensity signal F 7 3D Plot Window Lolo a For Click TLT20050624145507 005 Plot Full Image zZ Max 240 2 69 Ba 6 2 Refresh Perspective 2 To change the display make a selection from the Plot drop down list and click the Refresh button 2 Refresh Table 4 9 3D Plot window Item Description Plot Full Image Displays all signals in the image ROI lt ROI number or name gt Displays the signal within the selected ROI All ROIs Displays the signal within all ROIs in the image Z Max Height of the z axis Use the up down arrows to change the height of the z axis Click to reset the z axis to the default setting 3 Copies the 3D window to the system clipboard 77 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 4 Working With Optical Image Data Table 4 9 3D Plot window continued Item Description ES Opens a Print dialog box that enables you to print the 3D window Making Measurements To measure distance with the measurement cursor 1 Open an image and click the Distance Measurement Cursor button in the Image Information tools A measurement cursor s4 5 appears on the image Figure 4 22 The Tool Palette shows the position and length of the cursor Figure 4 22 Measurement cursor The Tool Palette displays the measurement cursor position and length j T1T2005062414
343. ure 5 24 Name and save the ROIs to the system Ca a e E Apply to Sequence Type Measurement ROI bd Save ROIs Mame ROI 1 KSA Delete Load Auto ROI Parameters Threshold 36 25 K 2 Click Save The ROI s from the image are saved to the system and can be selected from the Name drop down list To load ROIs on an image 1 Open an image 2 In the ROI tools make a selection from the Name drop down list and click Load NOTE If you load ROI s onto an image then draw additional ROIs the Save button changes to Overwrite If you want to save this collection of ROIs using the existing name click Overwrite To delete ROIs from an image M NOTE This does not delete ROIs saved to the system global save m Select the ROI and press the Delete key OR a Click the XJ button in the ROI tools and select a delete command from the drop down list Living Image 4 3 1 User s Manual IVIS Spectrum Figure 5 25 Delete ROIs from an image Tool Palette ST O a EE HE o YW Measure ROIs Apply to Sequence save ROIs Name ROI 2 KSA Il Measurements Delete 5 Il Autos Auto ROT Parameters Il BEG Threshold Si Il Subjects Il Mirrors ROI1 To permanently delete ROIs from the system Chapter 5 ROI Tools for Optical Data 1 Select the ROI s that you want to delete from the drop down list of saved ROIs 2 Click Delete Figure 5 26
344. uring a Click the Save button fg Alternatively select File Save on the menu bar b In the dialog box that appears select a directory and click Save A folder of data is saved to the selected location AnalyzedClickInfo txt ClickInfo txt luminescent and photographic TIF images 8 To export the image to a graphic file a Click the Export button g Figure 7 2 b Select a directory in the dialog box that appears enter a file name and select the file type from the Save as type drop down list c Click Save Table 7 1 Image Math window Item Description Color Ranges for Aand B Full Choose this option to set the Max and Min values to the maximum and minimum data values in the image Auto When this option is chosen the software sets the Min and Max values to optimize image display and suppress background noise The Min and Max settings can be manually adjusted to further optimize the image display for your needs Note The color scale does not affect the image math result 134 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 7 Image Math Table 7 1 Image Math window continued Item Description Color Ranges for Result Full See above Image Auto See above Min 0 Choose this option to set the minimum data value to zero Results Drop down list of mathematical functions that can be used to generate the new image including A B k A B k A B k
345. use a click and drag operation to move the tag then click the mouse to set the tag location 3 A line between the pixel and the tag identifies the location associated with the tag 4 5 Adjusting Image Appearance Use the Image Adjust tools to adjust the appearance of an image Figure 5 14 NOTE Not all tools are available for all image display modes Some tools are available for single images but not image sequence and vice versa For example the Correction Filtering and Image Information tools are available for an image but not for an image sequence el Figure 4 14 Tool Palette Image Adjust tools j 11120040217113205 Sele Tool Palette Units Radiant Efficiency K Display Overlay info NG isj i LES Photo Adjustment Image TLT20040217113205 Series Tue Feb 17 2004 03 33 20 Dey mio Tan Experiment 2 Female Nuj Nu mic Level High Em Cy5 5 Ex Cy5 5 Epi illumination Bin Labal 1 S200 Brightness M38 FOV 12 6 F2 1s Living Image Version 2 50 2 12 2004 restructured Comment Contrast E Camera IVI52 51620EEY Fluorescent Background Tests Opacity Color Scale Epi Fluorescence Min J Max Color Scale Limits Auto O Full Manual Individual Color Table J m C Reverse C Logarithmic Scale Radiant Efficiency secicm T yim Color Scale Min 3 04e8 Max 9 75e8 Color scale Min and Max Table 4 4
346. use subject height option or use the manual focus option to determine the proper subject height for the area to be imaged See Appendix A on page 265 for manual focus instructions Figure 3 18 Choose a focus option in the control panel 4 IVIS Acquisition Control Panel Imaging Mode Exposure Time inning Field of View Set System Status Excitation Filter Emission Filter MIS Subject height 1 Focus use subject height w Temperature PSS Locked tam Imaging Wizard Sequence Setup 8 Select Overlay to view an overlay image registered photograph and fluorescent image after acquisition M NOTE If you want to check the subjects inside the chamber before image acquisition take a photograph uncheck the Luminescent option choose the Photograph and Auto options and click Acquire 9 Click Acquire when you are ready to capture the image o 7 l NOTE If necessary click Image Setup single image mode the Sequence Setup in the control panel to operate in single image mode In button appears in the control panel Use this button to set up sequence acquisition see page 42 for more details on sequence setup 39 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition 10 Enter information about the image in the Edit Image Labels box that appears optional Figure 3 19 Click OK l NOTE You can enter image
347. use with the library method of spectral unmixing See page 142 for more details on the library method Bal Enables you to view and save the unmixed images as a sequence data set which can be analyzed using the tool palette Opens a dialog box that enables you to correct a spectrum for overlapping signal by subtracting one spectrum from another see page 152 Adds a component to the spectrum list A Deletes the last spectrum in the spectrum list Adding Spectra to the Plot To Add Do This A spectrum library Click the button and select a spectrum library in the dialog box that appears Note A spectrum library is a user created set of reference spectra generated by analyzing probes with known spectra and known locations defined region A spectrum from a user Add a new spectrum to the list in the Unmix window and identify the region by drawing a mark on the image cube See page 150 for more details 155 Living Image 4 3 1 User s Manual IVIS Spectrum Composite Image Chapter 8 Spectral Unmixing The composite image includes all of the signals each displayed in a different color Double click the composite image to view it in a separate window Figure 8 23 Figure 8 23 Composite window C HX20120419114703_SEQ Sequence View Unmixing V Show Labels V Individual Scale V Normalized C Legend 1 00 0 0 Emission Wavelength nm
348. using the Mouse Imaging Shuttle see the Mouse Imaging Shuttle Instructions Caliper part no 127820 RevA To perform automatic fiducial registration 1 2 Load the data that you want to register see page 252 Click the Fiducial Registration button IB The multi modal data are automatically registered and cropped Figure 13 17 To undo the registration click the Reset Registration button th To save the registration information a Confirm the default name or enter a name for the results in the Results tab b Click Save 254 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 13 3D Multi Modality Tools Figure 13 17 Registered 3D optical and 3D volumetric data A EL20101119144617_SEQ Sequence View L 30 View Spectra Coronal z 6 3 vai vV amp gt Af w Ng e 0 Transaxialfy 8 0 Subject Height 15 0 mm Perspective Figure 13 18 3D Multi Modality tools Results 3D Multi Modality Tools Key StudyDate NameOfVolume PatientsName Modality Manufacturer BitsAllocated SamplesPerPixel Rows Columns PixelSpacing SliceThickness Volume Information MULTIMODALITY_6 Loaded NumberOfFram ImagePositionP Results Value 20101119 Mouse3_3_Day 28_145522_00 Mouse3 CT Rigaku 16 1 256 256 512 0 23610 236 0 236 30 208000 30 208000 60 4160 ImageOrientati 1 0 0 0 1 0
349. w that you want to modify in the sequence table 2 Set new parameter values and or imaging mode in the control panel 3 Click in the sequence table Inserting Images in a Sequence Method 1 1 Select the sequence table row that is below where you want to insert a new image row 2 Set the imaging mode and parameters in the control panel 3 Click to insert the new image above the selected row 52 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition Method 2 1 Select the row s of interest and right click the sequence table to view a shortcut menu of edit commands Figure 4 34 on page 57 Figure 3 35 Sequence table edit commands in the shortcut menu 3 Display Photographic Settings a Subject Mouse Probes 7 B Seq 1 Seg 2 Seg 3 Seg 4 Seg 5 Mode Exposure Binning rag Excitation Emission FOV Height la DI Auto 8 Block 1 50 Ca 3 WG Auto 8 1 Block 600 C 1 50 Copy rows KI BET 1 Block 520 C 150 Select All 5M ato 8 1 Block 640 C 150 Delete rows Replace Row s Paste Rows Number of Segments 1 Delay 0 0 min Apply to All x Remaver A Update Insert Add Table 3 10 Sequence table shortcut menu edit commands Command Description Copy row s Copies the selected row s to the system clipboard Select All Selects all rows in the sequence table Delete
350. x Remove lt Column headers in the active ROI table mar Delete Move Up Move Down 2 Select a configuration from the User Lists drop down list and click Customize 3 To add column header to the ROI table make a selection from the Available Item list and click Add 4 To remove column header from the ROI table select the item that you want to remove in the Selected Items list and click Remove 5 To reorder an item in the Selected Items list select the item and click Move Up or Move Down The columns in the ROI Measurements table are updated 6 Enter a name for the custom configuration in the Name box and click Save 7 Select the custom configuration from the Measurements Unit drop down list Figure 6 10 Select a custom configuration for the 3D ROI Measurements table ROIMeasurements 3D ROI Measurements Data Types 3D VolumetricData v Measurements Unit My Custom Configuration Counts amp Refresh Absorption Sequence Number ROI Voxels Total Counts A Hounsfield 5 My Custom Configuration BI20111027132749 SEQ ROI1 8000 8987e 07 1 123e 04 3 369e 03 s Min Counts Max Counts 3 084e 03 3 341e 04 6 ROI Depth mm To delete a custom table configuration Select the configuration from the User Lists drop down list and click Delete Living Image 4 3 Software User s Manual Chap
351. x The slice coordinate of the last slice being viewed Specify the position range to include in the viewer using the Min and Max sliders or enter values Slice position Click to show the single view of the active slice in the multi view Alternatively double click a slice in the multi view to show the single view Click to show the multi view If the single view has been magnified click this button to zoom out incrementally Magnifies the single view Resets the single view to the default magnification DP OR Click to export the slice view as a graphic file for example bmp 248 Living Image 4 3 1 User s Manual IVIS Spectrum 13 6 Volume Information and Results Chapter 13 3D Multi Modality Tools The Results tab displays information about the loaded data taken from the DICOM file header Figure 1 ele Figure 13 12 Volume information L gt ROI Tools Spectral Unmixing and DyCE gt Surface Topography J 3D Multi Modality Tools Key StudyDate NameOfVolume PatientsName Modality Manufacturer BitsAllocated SamplesPerPixel Rows Columns NumberOfFram PixelSpacing ImagePositionP ImageOrientati volume Process slice Results Volume Information Unsaved Value 20111027 BI20111027132749 dcm DevpPhase OT Caliper Life Sciences 16 1 100 400 400 0 30000010 300000 6 000010 84001 6 00
352. ype Epi Illumination Trans Illumination This option selects the best excitation and emission filters for a specific fluorescent probe The fluorescent signal is then detected on the surface of the subject Table 3 6 Imaging Wizard bioluminescence imaging options Spectral Unmixing Option Description See Page Open Filter Acquires a luminescent image at maximum sensitivity Acquires an image sequence for analysis using the Spectral Unmixing 138 tools to analyze luminescent or fluorescent images when more than one reporter is used in the same animal model 43 Living Image 4 3 1 User s Manual IVIS Spectrum Chapter 3 Image Acquisition Table 3 6 Imaging Wizard bioluminescence imaging options continued Option Description See Page DyCE Acquires a time series of optical images following a bolus injection of 159 probe radiotracer bioluminescent or fluorescent to track probe biodistribution Note DyCE imaging and analysis requires a separate license DLIT Diffuse Light Acquires an image sequence for analysis with the DLIT algorithm that 187 Imaging Tomography reconstructs the position geometry and strength of 3D luminescent sources Table 3 7 Imaging Wizard fluorescence imaging options Option Description See Page Filter Pair Choose this option to acquire measurements of one or more fluorescent probes Spectral Unmixing Acquires an image sequ

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