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IVIS Spectrum CT manual

1. 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 Wel Plate Type EREEREER Well Plate 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 b
2. Make a selection from the Image Attributes drop down list to include image information in the ROI table 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 23 You can also create a custom table configuration J 1 In the ROI Measurements table click Configure NOTE Preset table configurations cannot be edited You can modify a preset configuration and save it to a new name The Configure Measurements box appears Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data 126 Figure 5 24 Configure Measurements dialog box E Configure Measurements eE 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
3. Pa Hide Browse agar Come Preview Label Set A Add to Lest Browse Wi Tett Gonigare Load as Group hemme Chose Location C Ahar Caliper LG Caliper Data ample Daba fiii 200 dab irie LT SA S57 Seene irio tet Preview picture of the selected data 3 6 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 M NOTE CT image acquisition is not available in batch mode To setup and acquire sequences in batch mode 1 Click Sequence Setup in the control panel 2 Choose the Batch Sequences option Figure 3 34 Figure 3 34 Control panel 4 MS Acquisition Control Pane ES Imaging Mode Exposure Time inning F Stop Excitation Filter Emission Filter Meam le Standard One Mol v a Field of View System Status X Rays will be produced when energized Idle Acquire 113 4 cm Imaging Wizard Subject height 1 50 cm Sequence Setup Focus use subject height v Temperature AN Locked Le MS Acquisition Control Panel fax Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter E Display Photographic Settings Subject mto ed E Cc O sa D meam va iy al al Field of View System Status X Rays will be produced when energized Idle Acquire 13 4 cm Imaging Wizard Image Setup
4. 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 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 a 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 CT 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
5. 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 CT Chapter 11 3D Reconstruction of Sources 200 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 235 8 In the Analyze tab click Start 9 The Data Preview window appears and displays the image data that will be included in the reconstruction Usually no data adjustment is required However it is possible to exclude or include user selected pixel data from the analysis For more details see page 195 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
6. A 7 User Settings LP 23 Currently User Accounts are Unlocked 2 add User 2 Change Password 2 deleteuser Security Currently User Accounts are Unlocked Enter Master Password E Lock ver Account Enter a Master Password to Lock the User Account settings Master Password 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 2 add user 2 change Password 2 peleteUser Security a r 7 User Settings 7 ss 7 User Settings Currently User Accounts are Locked E Unlock User Accounts Change Master Password Old Password New Password Confirm Password 2 Add user Change Password 2 deleteuser Security Ni Currently User Accounts are Locked Enter Master Password Enter the Master Password to Unlock the User Account settings Master Password 22 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 2 Getting Started 23 2 6 Tracking System and User Activity Activity Window The Activity window shows the imaging system activities Figure 2 13 The software create
7. 0 000 eee es 9 Overview of Image Acquisition eee 10 Auto Exposure Feature naia mawa aa ede RR DEDDEL e 10 Imaging Modes on the IVIS Spectrum CT es 11 Overview of Living Image Tools and Functions dpe Managing User Accounts 0 ee 20 Adding USGS gecaeueeancuaeae AA ae ose a ae 20 Changing or Adding Passwords 00 eee eee eee 20 Deleting USerS 2226224 ahs eeeeeesecesedeeea AY 21 LOCKING User Accounts 2 36 kaan Rh thee ob Oe eee dee wwe oes 21 Tracking System and User Activity 0 00 cece eee ee eee eee 23 POUVITY VWVINGOW 4 6 6 5 605 ty awe Wd PEPE arae rE Ee hee Sa 23 Image Acquisition 0 0002 ee es 24 CTIA aaa aah EN Erer eso LA PAANDAR ADAM MAA ANA ee ee Ma NG 24 Acquiring CT ImageS 2x na BKA KA 45 S64 we PO oe Ko SON eed 24 Acquiring Reference Images aaa 28 Checking the Instrument Stage Alignment aa 28 Luminescent Imaging 0 ee 30 Quick Guide Acquire a Luminescent Image 31 Acquire a Luminescent Image 0 ccc ees 32 Fluorescent Imaging With Epi Illumination aa 36 Quick Guide Acquire a Fluorescent Image With Epi lllumination 37 Acquire a Fluorescent Image With Epi Illumination 38 Fluorescent Imaging With Transillumination aa 42 Acquire a Sequence Using the Imaging Wizard 0 000 eee ees 48 Set Up a Sequence vau8u sed
8. Display Photographic Settings AcE Cl z Probes 7 Subject Mouse B sa B J Sea3 Seg 4 Seg 5 Mode Exposure amag 1500 Emission FOV Height ug Auto 8 Block 1 GN Ca 3 EEG ato e 1 Block 4 MG Auto 8 1 Block 5 tm Auto 8 1 Block 1 50 600 c 1 50 Copy rows 620 c 1 50 Select All 640 c 1 50 Delete rows Replace Rowvis Paste Row s Number of Segments 1 Delay 0 0 min Apply to All XE Remover A Update Insert Add Table 3 12 Sequence table shortcut menu edit commands Chapter 3 Image Acquisition Command Description Copy row s Copies the selected row s to the system clipboard Select All Selects all rows in the sequence table Delete row s Deletes the selected row s from the sequence table Replace Row s Replaces the row s selected in the sequence table with the rows in the system clipboard Note The Replace function is only available when the number of rows in the system clipboard is the same as the number of rows selected in the sequence table Paste Row s Adds copied rows to end of the sequence 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 40
9. Subject height 1 50 ai cm Focus use subject height v Temperature AN Locked E Number of Segments 1 Delay 0 0 E min Apply to All XM Remover dd Update Insert Add Batch Sequences option 3 To set up the first sequence do either of the following Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition m Click Imaging Wizard and step through the wizard see page 48 for details OR m Set up the sequence manually see page 57 for details 4 To set up the next sequence 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 35 Multiple sequence tabs Three sequences are specified in this example Adds a new tab use with Sequence tabs Removes the active tab and its sequence manual sequence setup E Display Photographic Settings Subject Probes B Exposure Binning FStop Excitation Emission FOY Height Auto Medium 1 Black Still T 1 50 Block 560 1 50 Block 600 1 50 Sequence table Block 620 1 50 Black 640 1 50 Auta Medium Auta Medium Au
10. Tool Palette hal Ca Image Adjust l H Romrections Filtering a gt ROH Tools a Spectral Unmixing and DyCE Sy De eit Select Images Tyra f x o Excitation wavelength alai ba Hire 7 Lab Mine 1 4Le Pad kaa Emission wavelengths of the sequence F E Lee ee ef we Mehak G in 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 3 Select Guided from the Methods drop down list and click Start Unmixing The Unmixing window appears Figure 8 3 Chapter 8 Spectral Unmixing Figure 8 3 Unmixing window See Table 8 3 on page 159 for more details on the toolbar buttons Image cube shows a pseudo color image composite of colorized sequence images No data available Spectrum List aby X Note Background Name Color Pick 1 TissueAF Mime A died 2 AF680 Hired AF a 3 F750 DE FF Image Cube Viewer Y Overview 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 E HX20120419114703 sEQ aa Sequence View Unmixing GS V Normalized Legend me a i 3 pa 3 5 144 Livi
11. l gt Surface Topography 3D Multi Modality Tools Volume Process Slice Results Render Slice Using Slice Color Table Volume Color Table Color Table a 7 Lad Reverse Opacity 100 Color Scale Min 0 Max 337291 Slice Viewer Orientation Coronal v Gi 3D Optical Tools gt DLIT 3D Reconstruction Tool Palette E bana E gt Planar Spectral Imaging o _ Spectral Unmixing and DyCE Volume Slice Viewer A Volume Slice Viewer Orientation Coronal X Slice Spacing 1 50 lt mm Total Slices 21 Q A Dy B Render Thick Slice _ Max 15 00 3mm Min 15 00 mm Click the button to view the slices in a separate window See Figure 13 12 on page 253 for more details on the Volume Slice Viewer Figure 13 12 Volume Slice Viewer Multi View Single View KC Volume Slice Viewer Orientation Coronal Slice Spacing Render Thick Slice 1 50 mm Total Slices 21 a Q A Double click a slice to display the single view 253 Living Image 4 3 1 User s Manual IVIS Spectrum CT Table 13 4 Volume Slice Viewer Chapter 13 3D Multi Modality Tools 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 inc
12. 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 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 134 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 6 3D ROI Tools for Volumetric Data Figure 6 9 Configure Measurements dialog box E Configure 3D ROI Measurements User Lists Name Counts Sort Available Items Average Hounsfield Average Value
13. Save Results Name MULTIMODALITY 1 v 5 30 optical Toos gt DLIT 3D Reconstruction La Surface Topography a Saving the registered and classified data provides a convenient way to share data The software saves the following m Level of detail setting m Crop settings Managing Results Saving Registered Results Color tables for the opacity map and slices Histogram tool control settings and the resulting color opacity map a 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 l 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 255 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools 256 Loading Results 1 Select the results from the Name drop down list 2 Click Load 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 information Optical and volumetric CT data acquire
14. 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 118 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 17 ROI Properties dialog box fe 7 ROI Properties m 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 119 Living Image 4 3 1 User s Manual IVIS Spectrum CT Editing the ROI Line 1 Double click the ROI that you want to edit The ROI Properties box appears Figure 5 18 Chapter 5 ROI Tools for Optical Data Figure 5 18 Editing ROI properties F RotProperties o mE Label BEG 1 Shape Cirde Type Manual Background ROT C U
15. 40 20 0 20 40 Position mm Position 4 2 204 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 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 7 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 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 11 6 Measuring Sources Horizontal Profile The y axis position of the crosshairs horizontal line Vertical Profile The x axis position of the cros
16. 60 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition 3 8 Manually Saving Image Data When you acquire the first image s of a session you are prompted to enable the autosave feature If autosave is enabled all images acquired during the session are automatically saved to 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 GH Alternatively select File gt Save on the menu bar 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 9 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 41 Figure 3 41 Exporting an image to a graphic file IG 11720050524145507_006 ex NG n TENG A NAS v Otsptay Over v T go Ir p Favorites e Organize a Orsktop AJ Libraries Downloads e3 Homegroup 872 byt Computer uns Counts e sg Fe Favorites
17. 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 CT Chapter 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 5 Full Auto Result Color Scale Limits O Full Auto E Min 50 Result A B k M k 1 00 Compute K from ROI v V with Photo from A M Display Result For Measuring GEA Sequence TLT20060510114512 SEQ Radiant Efficiency v Radiant Efficiency E7 TLT_M20060510114512_005 Units Radiant Efficiency Display TLT20060510114512_005 TLT20060510114512_001 1 00 Options gt 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 s
18. Item Description Draw Choose this option to display the pencil tool g 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 CT 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 48 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 location m Fluorescent image at the remaining transillumination locations a CT 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 F5top Excitation Emission Lamp Le
19. Note You can adjust the appearance of the photographic image using the Bright and Contrast controls see Adjusting Image Appearance page 75 Living Image 4 3 1 User s Manual IVIS Spectrum CT Appendix A IVIS Acquisition Control Panel 269 Table A 1 IVIS acquisition control panel continued Item CT Choose this option to acquire a CT image Select a CT imaging mode from the drop down list See Table 3 1 on page 25 for details on the imaging modes 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 well above the noise 5600 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 camer
20. Select DyCE results from the Name drop down list and click Load 2 Click the Image Cube tab Figure 9 15 173 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 ao by X Note Select Name Color Pick Temporal 1 Kane Mime 2 spectra o Ga 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 w
21. Description 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 179 Adds a component to the spectrum list A Deletes the last spectrum in the spectrum list 175 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 Temporal spectra show signal time course of different anatomical regions RKG20110210131022 SEQ h O Sequence View Unmixing Show Labels V Individual Scale Normali E ormalized Legend Kidney 1 00 oo o Unmixed 1 ee 7 0 40 Min 0 00 0 400 800 1200 Max 2 90e4 Time Point s Bladder Spectrum List gt py Xx Note Select Name Color Pick 1 V kidney Hime vifr 2 V Brain mr 3 V Bladder Move z f Min 0 00 Max 2 45e4 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 signa
22. 2n 268 Control EEH AA 268 Manually Setting the Focus 0 aaa 271 PETE AGES nse Fort cow eee OMe ee eee or Oa ere ee eS 273 General Preferences ee ee eee eens 273 OOUONS cacacauoceeeouket ep otbee er d eae d ohee eens 4 45404 oe Ss 275 PCOUISINON p20 5 pend mediena Me a PE Re ee we 276 UNGING ca Gm AN KAKA ee eee ee ows COO ASS eRe eee oe ew eas 277 Optical Propart 2 44 44 600 566 8 64 ent Gare ease eas oe ee Meat an acne oon 280 Menu Commands Toolbars and Shortcuts 200 281 v 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 CLS135292 is only for use with the IVIS Spectrum CT instrument This user manual explains how to acquire optical and volumetric image data on the IVIS Spectrum CT 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 Kinetic please see the Living Image Software User s Manual specific for the IVIS Kinetic Table 1 1 Living Image 4 3 1 software manuals Living Image Software Manual for the Part No
23. 3 Sequence View EP 3D View gt Spectral Unmixing and DyCE gt i gt Surface Topography _ 3D Multi Modality Tools Coronal z 10 7 Sagittal x 2 5 Z 3D Optical Tools Surface Source Registration Select Source Original Display Source Surface i 4 Display Voxels Maximum Intensity Projection Threshold 0 0034 NATOK NG YE i ae A ME e A 4000 0 Gradation lal 50 BA Voxel Size 10 31 Display Voxels As Smoothing 5x5 v Texture Color Scale Min 379 6641 Color Table Transaxial y547 7 usein Reverse al Log Scale Key Value Quantification 5 09e6 photons sec Volume 8 60 mm 3 Depth 4 18 mm Center of Mass 2 5 47 7 10 7 Host Organ Unknown photons sec Source Intensity Subject Height 19 4mm P Perspective Export Voxels Center of Mass La 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 207 Source Description Measurement Quantification The integrated intensity within the selected sources Volume The total volume of the selected sources 206 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Source Description Measurement Depth The
24. Color Scale See Table 3 4 on page 35 for more details on the image window Tool Palette The Tool Palette includes the Image Adjust tools page 75 Corrections Filter tools page 77 Image Information tools page 71 ROI Tools page 97 31 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter3 Image Acquisition 32 Acquire a Luminescent Image This section provides detailed instructions for image acquisition M NOTE The IVIS Spectrum CT should be initialized and the temperature locked before setting the imaging parameters in the control panel See page 7 for more details 1 Put a check mark next to Luminescent and select Auto exposure click the 2 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 Table A 1 on page 268 for details on these parameters Figure 3 7 Control panel IIS Acquisition Control Panel k Imaging Mode Exposure Time inning p Excitation Filter Emission Filter System Status 13 4 Imaging Wizard Subject height 1 50 Sequence Setup Focus use subject height v Temperature AY Locked o 2 Put a check mark next to Photograph 3 Select a Field of View size of the stage area to be image
25. Default x Configure Load as Group Load Remove jl Close Location KATHERINE PC Share Caliper LS Caliper Data Sample Data IVIS200 data TraserBeadsPC TLT 20050624145507_SEQ TLT20050624145507_003 ClickInfo txt Images loaded in the browser as part of a sequence highlighted yellow in this example These images can also be selected for grouping into another sequence 2 In the browser select the images that you want to group together 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 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data 96 Macintosh users Press and hold the Cmd key apple key while you click the images of interest in the browser 3 Click Load as Group The image thumbnails are displayed together in an image window 4 To save the images as a sequence a Click the Save button bf Alternatively select File gt Save on the menu bar a In the dialog box that appears select a folder and click OK 5 ROI Tools for Optical Data About ROIs Quick Guide Drawing Measurement ROIs on an Optical Image or Sequence on page 100 ROI Tools for Optical Images on page 101 Measurement ROIs on page 103 Mirr
26. IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data 100 5 2 Quick Guide Drawing Measurement ROls 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 103 for details on measurement ROIs ag el Open an image or sequence and click ROI Tools in the Tool Palette Select Measurement ROI from the Type drop down list Click the 5 button and select Auto All on 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 2 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 ROIs in a known order for easier comparison between images To renumber the ROIs ascending order from right to left right click the image and select Sort ROls on the shortcut menu If the Apply to Sequence option is selected in the ROI tools choose Sort ROIs in Sequence to sort all of the ROIs in the sequence The sort options are only available if the ROIs have not been sorted Figure 5 2 ROI intensity measurements Genar z R E TLI20050624145507 sEQ amar Sequence View Units Radiance Photons v CJ Use Saved Colors Options Info R NG a ROI 3 50 2 853e 08 ROI 1 50
27. 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 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data 63 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 7 Gose renew Label set 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 SEQ Image sequence DEM Image exported as DICOM file due DyCE image sequ
28. V Normalized Legend a 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 158 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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
29. a 75 Correcting Optical Image Data es 77 Viewing Intensity Data and Making Measurements 79 Viewing X Y Coordinates and Intensity Data 0 00 cee ees 81 Image Histogram sas menarem cued bee ea be ene eee oe oe kn Boh 81 Line FONE 24 045 nae oe bee ea MA ER ee a ee WA 82 Viewing 3D Signal Intensity 0 aa 84 Making Measurements 0 00 ees 85 Creating a Transillumination Overview cece eee eee 87 Overlaying Multiple Images 0 0 cc eee eee 88 Rendering Intensity Data in Color 0 0 0 ete 91 Exporting or Printing Images 0 es 92 Editing an Image Sequence cc ees 94 Creating an Image Sequence from Individual Images 95 ROI Tools for Optical Data 0 440 00 aa 97 ADOUR 242 sebatenee ee NGA FM ee ee ee PLEMA AA MSG wee eae 97 Quick Guide Drawing Measurement ROls on an Optical Image or Sequence 100 ROI Tools for Optical Mages 22 424 DA KG NG KAWA NAGANA AA DBA NAN NG eho 101 Measurement ROIS xxx 0504 06 iw oe wa NA DA ND KG eae ew by ae BN LA 103 Drawing Measurement ROIs Automatically 104 Drawing Measurement ROIs Manually 000 eee eee 106 Drawing ROIs Using the Free Draw Method 106 WON ROIS waavac costar TPP 107 Measuring Background Corrected Signal 2000 eee eee eee 110 Subject ROIS ese ae eee ae eka d AKA eee eee ee daa eee GDS e
30. a PerkinElmer company Living Image Software User s Manual Version 4 3 1 For the IVIS Spectrum CT 2012 Caliper Corporation All rights reserved PN CLS5135292 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 Living Image 4 3 1 User s Manual IVIS Spectrum CT Contents Chapter 1 Chapter 2 Chapter 3 MWOlCOMO 226223268 Gee eae awe ANAN AA AA NA AAAH NG mGa 1 About This Manual 4 xah haa tie pet ANG AMA eed BAKAL DONA NAAT WAAAAA 1 What s New in the Living Image 4 3 1 Software 0 0 cee eee ee 2 Living Image Help 6 64 2 0006 ceo do DENG nnana 3 Caliper Technical Support 0 00 ccc ees 4 Getting Started 2 6465 sk Shh Beene teem unwind en ede MGA 5 Starting the Living Image Software 0 ees 5 Initializing the System and Checking Temperature 7 Initializing the IVIS Spectrum CT ce ee eens 8 CCD Temperature 0 0 eee eens 8 Imaging Platform Temperature
31. 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 TLI20050624145507 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 eae A Reset Zoom TC TLI20050624145507 005 E TLI20050624145507 005 Units Counts v Display Overlay v Options r
32. the imaging parameters See page 7 for more details 1 Click Imaging Wizard in the control Panel Figure 3 28 2 If necessary click Restart in the Imaging Wizard to show the first page of the wizard 3 Double click Bioluminescence Ag Fluorescence p imaging Gianna Pammtrala Chapter 3 Image Acquisition Figure 3 28 Opening the Imaging Wizard IC Ms Acquisition Control Panel Select bha opbon for imaging bislumnaiwant ot Chairman reportecs tach at bry cena chick beete oeme rend oe badal lucifer Standard One Mo T Field of View System Status X Rays will be produced when energized Idle Acquire 13 4 cm Imaging Wizard Subject height 1 50 21 cm Sequence Setup Focus use subject height v Temperature fa cocker 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 8 and Table 3 9 on page 50 for more information on the imaging options 49 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition 50 Figure 3 29 Choose the type of image sequence Imaging Wizard Bioluminescence options Imaging Wizard Fluorescence options j Imaging Wizard Bioluminescence wd j Imaging Wizard Fluorescence Bioluminescence Open Filter Fluorescence Filter Pair Imag
33. 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 cm2 sr uW cm Transillumination fluorescence Fluorescence emission radiance per incident excitation power p sec cm sr mW Efficiency epi fluorescence 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
34. 1 50 1 00 0 50 0 00 O Line Orientation Horizontal Width 1 Position 139 L a bai HHY Min 26 Y Max 117 TLT20050624145507_006 Overlay Tool Palette Luminescence Binning amp Width 12 6 cm Height 12 6 cm Image X Y 12 541 2750 om Image Data 27 counts Crop Distance Counts Color Scale Min 1122 Max 19546 5 X Full Scale Logarithmic Scale 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 Position Line position pixels Enables you to choose the grid line pattern to display in the line profile window Exports the line profile data to a csv or txt file Copies the line profile graph to the system clipboard 83 Living Image 4 3 1 User s Manual IVIS Spectrum CT Table 4 8 Line Profile window Chapte
35. 1 5564 400 8 Time Point s Bladder Spectrum List wo py Xx Note Composite of 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 CT Chapter 9 DyCE Imaging and Analysis 164 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 CT 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 ei WIS Acquisition Control Panel pas Imaging Mode Exposure Time Excitation Filter Emission Filter Imaging Made Bioluminescence Imaging Sadoc ti the option for magng biolumniraani cr chemtluminascenit reporters such as frefly luciferase Gk beetle biciferase neni or bocterial bioferace Pumescerce IMNO Field of View System Status Acq
36. 2 278e 06 ROI 2 50 J 1 017e 07 ROI 4 5095 9 24684 07 a Min x 9 54e7 f3 Max ROI 5 50 J 5 753e 08 ROI 9 50 2 552e 09 ROI 6 50 2 581e 08 ROI 10 50 J 1 361e 09 g Ry a A gi ma 2e7 Min 1 46e8 Max 2 5489 4 Use the Threshold slider or a arrows to adjust the ROI boundaries J NOTE After the ROIs have been created right click an ROI to view a shortcut menu of ROI commands Ctrl click for Macintosh users The shortcut menu provides easy access to many functions for managing ROls and viewing ROI properties 5 Click the Measure button e4eROls in the ROI tools to show the ROI Measurements table Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data Figure 5 3 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 0
37. 3D Multi Modality Tools 2 In the Volume Information dialog box that appears Figure 13 26 enter the m Data width height and the number of slices m Slice row column pixel size and the slice spacing in millimeters Figure 13 26 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 27 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 27
38. 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 image 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 141 3 Spectral Unmixing About Spectral Unmixing Spectral Unmixing Methods on page 143 Correcting Spectra on page 156 Spectral Unmixing Results on page 158 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
39. 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 14 page 230 for details on the animation tools KE So IG 3DAnimation Preset Animations Presets Spin CW on Z Axis z Frame Factor 1 Animation Setup Time Scale Select a preset animation l 100 5 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 CT 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 v Frame Fac
40. 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 HheF 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 on page 227 r Select this tool from the drop down list to display the perspective view 4 Rotates the 3D reconstruction results in the 3D view window 3D scene Click the or key to increase or decrease the rotation speed To stop the rotation click the 3D scene or the ga button 27 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Table 3 2 3D View toolbar continued Item Description Pra Displays measurement cursors in the coronal sagittal or transaxial views Uv Not available for volumetric data Reserved for use with DLIT or FLIT Eo reconstruction results voxels i Not available for volumetric data Reserved for use with DLIT or FLIT B3 i reconstruction results voxels Enables you to save the 3D view to a graphic file for example jpg Acquiring Reference Images During CT imaging a series of X ray transmission images is acquired as a specimen rotates on the stage Each X ray transmission image is
41. Max Hounsfield Max Value Min Hounsfield Min Value ROI Depth mm ROI Height mm ROI Width mm Total Hounsfield Total Value Selected Items Average Counts Min Counts Max Counts Remove lt Column headers in the active ROI table Move Up Move Down 2 Select a configuration from the User Lists drop down list and click Customize Add To add column header to the ROI table make a selection from the Available Item list and click 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 7 Select the custom configuration from the Measurements Unit drop down list Figure 6 10 Figure 6 10 Select a custom configuration for the 3D ROI Measurements table mng S E ROI Measurements a ROI Measurements 3D ROI Measurements Data Types 30 Volumetric Data v Measurements Unit My Custom Configuration Refresh Counts l panes ROI Depth Sequence Number ROI Voxels Total Counts AvHounsfield 5 is Min Counts Max Counts P My Custom Configuration wn mm pa BI20111027132749 SEQ ROI1 8000 8 987e 07 1 123e 04
42. 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 data 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 CT Chapter 13 3D Multi Modality Tools Figure 13 24 Drag volume data from Windows Explorer to the Volume Data Viewer Lg L 4 a WALE i Seo NOT MOLE ag AMAG Go Ie TIFF mouse 3_3 20100528 140051 FOV60m Bs Open jo ALI20110120171831 SEQ Dmouse3 3 20100528 140051 b p AutoHuorBkgCorr z b d BkgSub So dem 20110306103654 gt wi DLIT Datasets gt 120110114162356 SEQ d Katherine Quantum gt Kidney b di KSA20110305104918 SEQ H i LognitudinalData b di Lung di MAT20090302190433 gt Multi Modality Datasets Slide show E mail Burn FEU Print Organize Share with E Volume Data V
43. Spectrum CT Chapter3 Image Acquisition 52 Figure 3 31 Enter information to include with the image optional pan Ha UserID ary Living Image Universal Saved Labels LABELS _ 1 AER V User 7 croup 7 Information entered here appears in the Bi cso Saan image label see Figure 3 33 on page 53 Male nu nu day 8 Mouse 4 7 Commenti _ E Comment2 v Time Point v V Animal Number 4 v Animal Strain v Animal Model x Sex v View w Cell Line X C Reporter v Treatment v E Luc Injection Time v T IACUC Number If this is the first image of the session you are prompted to enable the autosave function Figure 3 32 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 32 Autosave prompt Fa E Living Image 4 3 64 bit Auto Save E3 a E Do you want to enable auto saving of acquired data for this session This can be changed anytime from the Acquisition menu 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 61 for details Image acquisition begins and the upper area of the control panel changes to red color During acquisition
44. 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 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 258 Click the Fiducial Registration button IB The multi modal data are automatically registered and cropped Figure 13 18 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 260 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools Figure 13 18 Registered 3D optical and 3D volumetric data A EL20101119144617 SEQ Sequence View LA 3D 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 19 3D Multi Modality tools Results 3D Multi Modality Tools Key StudyDate NameOfVolume PatientsName Modality Manufacturer BitsAllocated SamplesPerPixel Rows Columns PixelSpacing
45. The maximum Hounsfield unit value for any single voxel in the 3D ROI Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 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 235 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 yie
46. The software alerts you if the number of images in 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 45 sec 3 Dee Auto 2 640 700 High C o 150 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 cli
47. User X v Group v Experiment 87 MG uc2 Intracranial Implantation v Male nu nu day 8 Mouse 24 V Commenti es v Comment2 v 7 Time Point x V Animal Number 4 T Animal Strain F Animal Model E Sex v M View v Cell Line X Reporter Treatment v Luc Injection Time IACUC Number m 3 Click OK when finished The image information is updated 4 Save the image to save the updated image label select File Save or File Save As on the menu bar 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 Hh 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 CT Chapter 4 Working With Optical Image Data Figure 4 12 Add a comment to an image fa TE TUT20050624145507 006 v Display Overlay v See Info
48. V TissueAF Mime x Sr 2 V AF680 l E zf 3 WI AF750 NG 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 158 for information about the results Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 8 Spectral Unmixing 156 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 158 for more Unmixed images details show the 0 50 extracted signal See page 161 for Min 0 00 p Mins 0 00 deta l Is on 004 a T Max 9 83e7 Mge analyzing these Hanna avelenn cih hini AF750 Composite Images Spectrum List See Table 8 30n LL SUGA amp kan PA detallson GG Composite of Select Name Color Pick the unmixed images See page 160 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
49. in the ROI Measurements table Several preset categories are available in the Measurement Types Click Attributes and ROI Dimensions drop down lists 1 Drag a column 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 23 ROI Measurements table a ROI Measurements o 88 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 pixels pixe HX20070420121444 001 ROI1 Overlay 1 425e 06 4 167e 02 6 726e 02 8 462e 00 2 545e 03 3420 2 189e 05 4 141e 02 1 020e 03 2 87 HX20070420121444 001 ROI2 Overlay 1 489e 06 3 309e 02 6 061e 02 8 642e 00 2 545e 03 4500 2 880e 05 4 168e 02 9 001le 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 2 825 4 m p Customized Selections Measurements Types Image Attributes ROI Dimensions Copy Select All Counts v All Possible Values v Pixels r Configure Export
50. 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 268 Imaging Platform Temperature The imaging platform 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 platform may be set to a temperature from 20 40 C The actual surface temperature of the padded rotating animal stage may be lower than that of the imaging platform The imaging platform does not have active cooling The platform may require up to 20 minutes to passively cool from 37 C to ambient temperature Figure 2 5 Set the imaging platform temperature in the control panel 8 v Standard One Mow 7 Demand R g 3 Camera Temp 90 Gi 0P imaging wizard tag m io Ee kamo ey IP Subject height 1 50 H cm 1 Sequence Setup Focus use subject height Temperature I Locked Field of View System Status Acquire Measured 9 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 2 Getting Started 10 2 3 Overview of Image Acquisition The control panel provides the image
51. on page 281 Toolbar File Edit View Tools Acquisition Window a BRS Ww Help Standard One Mo v al 7 Field of View c nd System Status Excitation Filter Emission Filter eck open Acquire System needs initialization Subject height 1 50 Click on Initialize button to proceed be Jem Focus use subject height v Imaging Wizard Sequence Setup Type D Information D Information D Information D Information Activity Activity window Description Living Image 4 3 0 15210 64 bit RC Dec 13 2011 13 13 19 gt gt KS Logged IN lt lt IVIS configuration file found and loaded Reading user preferences a aaa M NOTE The Living Image software on the PC workstation that controls the IVIS Spectrum CT 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 CT must be initialized each time Living Image software is started or if the power has been cycled 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 Spectrum CT The items available in the control pane
52. 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 33 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Figure 3 33 Image window and Tool Palette NG ter20050624145507_s6q soli TooDaterte a CI Sequence View Co Image Adjust A Corrections Filtering n gt Image Information ROI Tools Tool Palette Units Counts X Use Saved Colors Opos Bino sm Jj a aaa Aa Te T1T20050624145507 006 le Dahan a ag Click Info to show Image TUT20030620145507 006 elie Wikia the Image Label 24 00502 57 18 Ani OUNAE tn Paw Open NON ONL fa ag EONO DO 03 210 information Living Image Version 2 50 1 5 20 2005 lt Camera IVIS 200 Beto I SI6NEEY Comment dorsal INO DA f Ay i Mine 2230 Mra 547 Mare 34 Maya Bk a yy i AA Merve 2949 Mina EAF Maxe 51182 5 Maxm 1048 Sequence View 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 Sequence View Displays the image sequence m 3D View Displays the 3D volume if the acquisition
53. 0 00 660 700 740 780 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 bet X page 159 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 154 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 8 Spectral Unmixing 155 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 i
54. 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 CT Chapter 11 3D Reconstruction of Sources 193 5 To view the tissue properties u from the Plot drop down a Hep W for the tissue and source you selected make a selection 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 235 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 195 Figure 11 4 Data Preview window Tool Palette Tl pp0111027132749 SEQ Sequence View kL 3D View Data Preview Sequence S720111027132749_SEQ Source Frefy Threshold 600 106 620 68 c40 31 7 Image Label 7 Median Filter Restore Threshold Data Adjustment E Select All Cancel Reconstruct Data Preview window 8 In the Data Preview window cli
55. 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 04 130 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 6 3D ROI Tools for Volumetric Data 131 Table 6 1 3D ROI Measurements table 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 grayscale 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 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 Vol
56. 5 09e6 photons sec Volume 8 60 mm43 Depth 4 18 mm Centerof Mass 25 477 107 Subject Height 19 4mm f Perspective Host Organ Unknown Export Voxel gt DLIT 3D Reconstruction lt 3 To move a plane put the mouse cursor over a line in the coronal sagittal or transaxial windowpane When the cursor becomes a ji or arrow drag the line The view is updated in the windowpanes as you move the line Figure 11 22 Moving the transaxial plane Transarial 47 7 Subject Height 19 4 mm 209 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 210 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 pa button and select Copy source voxels 3 In the other reconstruction click the button and choose Paste source voxels l NOTE Pasted voxels can be measured For more details on measuring sources see page 205 11 8 Comparing Reconstruction Results Multiple DLIT or
57. 512 lplot Roas Eg S TLT20050624145507 005 Overlay 3 000 2 000 j 0 000 1 00 2 00 3 00 4 00 Bins 113 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data 114 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 15 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 13 Figure 5 13 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 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 3150
58. 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 results 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 CT 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 t
59. 68 4 2 About the Image Window and Tool Palette Image Window An image image sequence or kinetic data set is displayed in an image window Multiple image windows can be open at the same time Figure 4 6 Image windows sequence view and single image The options available in the image window depend on the type of active image data ayo a mr gt pores Cole BI20111027132749 SEG Sequence View aD view spectra Spectra Units Counts r E use saved Coo Optens eine Yg a CF 820111027132749 005 BREA Units Counts Josey Overy Options v paano iy aa Image BI20111027132749_005 User kor Thu Oct 27 2011 13 36 43 Group Em Filter Open Ex Filter Block Bin 15 16 Experiment U87 MG uc2 Intracranial FOV 13 5 f1 3s Implantation E Living Image Version 4 3 0 14942 Oct 27 2011 eae dav 8 Mew Camera Everest B2 Andor ikon mar a Livan F Bo r Li Animal Number 4 X Luminescence Min 44 Max 526 Counts Color Scale Min 44 Max 526 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 wa
60. 800 51 ia 800 51 800 m dk nn 3D Animation Tools 229 E 3DAnimation Select Tools 3D Animation on the menu bar Preset Animations 1 Presets 5pin 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 0 eo Frames Per Second 10 Total Duration secs 5 ata Ss Longitudinal Study 210 Select Tools Longitudinal Study on the menu bar Multiple DLIT and or FLIT reconstruction results can be viewed side by side in the Longitudinal Study window The Longitudinal Study window provides a convenient way to compare different results for example results obtained at different time points or results from different types of reporters Voxel intensity within the entire surface or a user selected area can be measured in all results in the Longitudinal Study window 17 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 2 Getting Started Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum CT continued Living Image Tools and Functions See Page pakanan an z Select Tools Well Plate Quantification for ge Coe tes Dye molecules
61. About DyCE Dynamic Contrast Enhancement M NOTE The DyCE acquisition and analysis features 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 m 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 m An unmixed image An image that represents the peak signal time point for a particular temporal spectrum m 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
62. Acquire a luminescent image 1 Start the Living Image software fa and initialize the IVIS Spectrum CT page 5 Note See the V S Spectrum CT Hardware Manual part no 133577 Rev XX for more information on the instrument 2 Place the anesthetized subjects in the imaging chamber and close the door Imaging Mode 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 6 Click Acquire E Field of View Subject height 1 50 E ME Acquisition Control Panel Exposure Time cm cm Ee Excitation Filter Emission Filter Binning meam ve v System Status Idle Imaging Wizard Sequence Setup Focus use subject height r Temperature I Locked 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 Edit Image Labels dialog box that appears optional The image window and tool palette appear when acquisition is finished Image Window E E TLT20050624145507_001 oer 8 Options 7 info gg Units Counts v Display Overlay X Luminescence 3000 2000 1000 Counts
63. 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 259 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools Figure 13 17 3D optical and 3D volumetric data loaded but not registered C L20101119144617 seq 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
64. CT FOV Setting FOV cm A 4 B 6 5 C 13 D 19 5 4 Select a focus option Figure 3 15 39 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition 40 The focal distance to the camera is set a stage 7 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 211 for manual focus instructions Figure 3 15 Choose a focus option in the control panel C WIS Acquisition Control Panel So Imaging Mode Exposure Time inning D Excitation Filter Emission Filter auto ec v edun gt a aag a medum vle v Field of View System Status Idle Imaging Wizard 13 4 Sequence Setup Subject height 1 50 Focus use subject height Temperature AN Locked 5 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 6 Click Acquire when you are ready to capture the image a
65. CT AA l NOTE If necessary click mage 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 page 48 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 72 for details on adding information to an image after acquisition Living Image 4 3 1 User s Manual IVIS Spectrum CT Figure 3 16 Enter information to include with the image optional Information entered here appears in the image label see Figure 3 18 on page 42 TE Edit Image Labels a 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 Treatment E Luc Injection Time v T IACUC Number Chapter 3 Image Acquisition If this is the first image of the session you are prompted to enable the autosave function Figure 3 17 When Autosave is enabled all images acquired d
66. Cels lt sequence name gt on the menu bar Measurement Sample Wels 3D 3A O set 65 Generate a database of luminescence or 7 Background Wels 6D 6A 1 set 9 fluorescence signal intensities by analyzing cial l images of known serial dilutions of luminescent or jellies Quantfcaton Pots anamda fluorescent cells or dye molecules Click EL20090414101005 001 F Use the quantification database to extrapolate the 5 Ta 7 7 210 Total Efficiency ca number of cells in a DLIT source or the number of dye molecules or cells in a FLIT source 4 0 x10 0 0 1 0 2 0 3 0 40 5 0 well plate population Sequence ARW20050826 124002_SEQ Units Radiant Efficency B a rere ae Select Tools Image Overlay for lt sequence CSE name gt on the menu bar v ARW20050826124002 002 View multiple fluorescent or luminescent signals T in one 2 dimensional image in the Image Overlay BE window o D C in 8 Hi zeoe eee Palette Label Max E L 47s Green x a Scales per Column 3 Opacity B 70 gt E Reverse V Logarithmic 18 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 2 Getting Started Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum CT continued Living Image Tools and Functions See Page T nono7o420122818 SEQ Sao Colorize View 91 equence View ctra Colorize View z Csepevewen basaan Select Tools Colorize for lt sequence name gt
67. Creating a New Image Using Image Math 137 Subtracting Tissue Autofluorescence aa 139 Chapter 8 Spectral Unmixing 24 0002 ee eee an 142 About Spectral UNMIXINg 244645 waw na ba NA KAWDEA NG bed HON MG a Sas 142 Image Requirements 142 Spectral Unmixing Methods 0 00 cee ee 143 Guided Method 022 cee eens 143 Library WICUNOG vitae rue Cede Rona ae Dee eae ee ee Chae eae 146 Automatic Method mapamwa nA ease AG BAKA APAN BAD ERIKA nae bee eee he 149 ME PCB ACE 153 Correcting Spectra bocca bee ae eee oye o GP AA AA ee ig Gaus Lee o 156 Spectral Unmixing ResultS aaa 5440405009555 8524 ea be shoe Gm 158 SPEC a e mawa naa bh68 bee ook eee ewe eee eee eo eee ee eee es 158 Analyzing Images zan DBA DANGWA LANA oe eben eee es eu eae LAG Gees ee 161 Managing Spectral Unmixing Results 0 00 ce aa 161 Chapter 9 DyCE Imaging and Analysis 20208 02 eee een e nee 163 About DyCE Dynamic Contrast Enhancement 0 00 c eee eee es 163 Acquire a DYCE Sequence cn sl dig a KAN MG BAWA GA Pee eee te A 164 DYCE AN IVSIS paa 048 2000 GA OR MG ee GG a AA 170 Automatic DyCE Analysis 0 ee ee 170 Manual DyCE Analysis 0 00 ees 173 DYCE OSS a settee Ar ace be ie ee eA ee A Ba dodo AL we ane ae ee ee 176 Viewing Unmixed Images a aaa 176 Viewing the Composite Image 0 00 cc es 177 Correcting Temporal Sp
68. FLIT reconstruction results can be viewed side by side in the Longitudinal Study window Voxel intensity within the entire surface or a user selected area can be measured in all results in the Longitudinal Study window The Longitudinal Study window provides a convenient way to compare different results for example results obtained at different time points or results from different types of reporters Viewing Results in the Longitudinal Study Window 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 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 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 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Figure 11 23 Longitudinal Study window no results displayed 7 Longitudinal Stu
69. Figure 5 25 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 402e402 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 127 6 3D ROI Tools for Volumetric Data About 3D ROIs Drawing a 3D ROI on page 129 Managing the 3D ROI Measurements Table on page 134 6 1 About 3D ROIs A 3D region of interest ROI can be drawn on a a CT volume a DLIT reconstruction of a luminescent source a FLIT reconstruction of a fluorescent source A 3D ROI measures the signal intensity within a user specified bounding box Figure 6 1 Example 3D ROI BI20111027132749 SEQ Lelea CADERA ESE JEEE 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 measuremen
70. 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 Figure 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 CT Chapter 13 3D Multi Modality Tools Specifying a Color Opacity Map m After the surface and vo
71. 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 CT Chapter 4 Working With Optical Image Data Figure 4 20 View a line profile of pixel intensities 7 1L120050624145507 006 Units Counts X Min 0 Hx Max 239 107 Counts
72. No of Slices Displayed 0 5X 256 1X original resolution 512 1 5X 768 2X 1024 2 5X 1280 3X 1536 247 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools 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 B120111027132749_SEQ o E ROL Jonis 2 i Sequence View LA 3D View gt Spectral Unmb ng and DyCE gt Peo PP F w la amp w ft os lw A gt Surface Topography 4 R e M ia Ee a 17 3D Multi Modality Tools Sironale 10a Sagittal x 2 5 maa Volume Process Slice Results HO On og Y Display Volume Level Of Detail B o Performance Quality Color Opacity Map 1 0 Opacity Xo H Reverse Al Transaxial y 47 7 i Tae eeR eee is ay pcs SS Sg Ai POE LT Baca Subject Height 19 4mm Perspective 0 00 Counts v 6 556 4 Logarithmic Histogram Maximum Intensity Projection MIP Gradient Illumination gt 3D Optical Tools i gt DLIT 3D Reconstruction _ al 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
73. Scale g amp Normalized Legend a 1 00 0 80 0 60 0 40 Min 0 00 0 400 800 1200 Max 1 58e4 Time Point s Spectrum List wo by Xx Note Select Name Color Pick 1 V Kidney Mime aL 2 V Brain i E AF dad 3 V Bladder blue ze Min 0 00 Max 2 45e4 C RKG20110210131022_ SEQ Sequence View Unmixing Composite ko Lo Jea Bees V Bladder Photograph Image Adjust Brightness f 100 Logarithmic Scale Label Bladder Close 177 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 174 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 Color Sho
74. Tools for Optical Data 122 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 ROls 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 Figure 5 20 Delete ROls from an image Tool Palette E A L HE CH W Measure ROIs Apply to Sequence ype Measurement ROI All Save ROIs j Selected Name ROI 2 KSA All Measurements Delete Load Save All Autos Auto ROI Parameters All BEGs Threshold Sa All Subjects All Mirrors 50 ROI1 To permanently delete ROIs from the system 1 Select the ROI s that you want to delete from the drop down list of saved ROIs 2 Click Delete Figure 5 21 Delete ROls from the system Tool Palette C oO BE Wf Measure ROls x Apply to Sequence Type Measurement ROI Y Save ROIs Name ROI 1 KSA Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data 123 5 9 Managing the ROI Measurements Table The ROI Measurements table shows information and data for t
75. Units Counts x E Use Saved Options 7 Info BP R y nG O pov on the menu bar The colorize tool renders each luminescence or fluorescence image of a sequence in color and combines them into a 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 OOK vale NG Mak 9267 Images of Quantum dot nanocrystals 700 or 800 nm were acquired using different combinations of excitation and emission filters E HIGALA BSE Coma NA Cokr sss Nar Sanga ry Sal Aral Como By L Eg Colorize view of the combined images Transillumination Overview 87 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 19 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 2 Getting Started Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum CT continued Living Image Tools and Functions See Page NG pan
76. a reference spectrum library for use with the library method of spectral unmixing See page 146 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 156 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 154 for more details 159 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 O Hx20120419114703 sEQ Co o GC om 20 n1aMo3 KO secon x F sequence View Unmixing C Sequence ve
77. 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 optical 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 CT Chapter 11 3D Reconstruction of Sources 192 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 181 3 In the Tool Palette choose DLIT 3D Reconstruction The Analyze tab shows the data that the algorithm automatically selects for the reconstruction Figure 11 2 For more details about the Threshold 9o see page 195 Figure 11 2 Analyze tab Sequence 5120111027132749 SEQ Source Firefly Threshold 4 Inthe Properties tab make a selection from the Tissue Properties and Source Spectrum drop down lists Figure
78. click the E 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 Figure 11 25 Selection boxes around each surface 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 following graphs are available in the Plots tab Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 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 Figure 11 26 Example Quantification plot 3D View Plots Analysis Type FLIT jReduce quared a Voxel Size he Quantification
79. corrected signal 110 112 batch mode 55 56 binning 78 browse optical image data 62 66 browser optical image data 65 volumetric data 258 C Caliper Corporation technical support 4 cascade images 67 classifying 3D volumetric data 243 control points 244 colorize data 91 92 color opacity map 244 composite image 137 139 control panel 268 269 copy ROI measurements 124 correction filtering tools binning 78 cosmic correction 78 dark background subtraction 78 flat field correction 78 smoothing 78 cosmic correction 78 crop box 86 CT imaging 24 28 reference images 28 D dark background subtraction 78 Data Preview window 195 197 DICOM Viewer 216 264 DLIT sequence requirements 191 DLIT results 201 203 manage 202 DLIT FLIT troubleshooting 234 DyCE 163 180 E edit image label 72 sequence 59 60 94 95 export images 61 92 94 surface 187 Living Image 4 3 1 User s Manual IVIS Spectrum CT F fiducial registration 256 261 264 flat field correction 78 FLIT sequence requirements 198 FLIT results 201 203 manage 202 fluorescence reconstruct 3D sources 198 201 fluorescent imaging quick guide epi illumination 37 fluorescent imaging epi illumination 36 42 fluorescent imaging transillumination 42 48 focus manually 271 272 G gradient illumination 248 H histogram ROI 113 Image adjust appearance 75 76 cascade 67 export 61 92 94 information 71 measurements 85 print 92 94 tag 74 tile 67 image
80. data colorize 91 92 open 66 image histogram 81 image layout window 93 94 image math 137 139 image overlay tool 88 90 image sequence create from individual images 95 96 edit 94 95 image window 68 imaging acquire a sequence 48 55 acquire a sequence batch mode 55 56 acquire a sequence manual 57 60 CT 24 28 fluorescent epi illumination 36 42 fluorescent transillumination 42 48 fluorescent quick guide epi illumination 37 luminescent 30 36 luminescent quick guide 31 imaging modes 11 13 import Index 286 organ atlas 227 surface 187 information about an image 71 L line profile 82 Living Image browser 65 Longitudinal Study window 211 luminescence reconstruct 3D sources 191 197 luminescent imaging 30 36 quick guide 31 M manual focusing 271 272 maximum intensity projection 248 measurement ROI automatically draw 104 105 measurement ROIs 103 105 free draw 106 measurements 85 mirror ROI 107 Mouse Imaging Shuttle 260 multiple reporters per photograph 88 90 N NTF Efficiency 124 268 O open image data 66 optical image data browse 62 66 organ atlas import 227 organ display 224 227 organ registration tools 222 228 overlaying images 88 90 P photon density 203 photon density map measured 205 simulated 205 photon density maps 204 preferences 273 280 print images 92 94 O quantification database 235 240 create 236 239 manage results 240 samples 235 R RAW volumetric da
81. 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 114 View 3D ROI Properties Displays the 3D ROI Properties dialog box see page 132 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 Image Math for Open
82. gt ROI Tools Spectral Unmixing and DyCE Surface Topography 3D Multi Modality Tools _ DLIT 3D Reconstruction 70 Living Image 4 3 1 User s Manual IVIS Spectrum CT 4 3 Viewing Image Information Chapter 4 Working With Optical Image Data At acquisition the software captures image information that includes all of the text information that is associated with an image for example camera parameters and any image label information you enter at acquisition time Figure 4 8 Figure 4 8 Image window displaying image information Click Info to display the image label and acquisition information Ar Fi TLT20050624145507_006 Units Counts O s Image label fi 2724 2005 07 57 18 Camera IVIS 200 Beta II SI620EEV v Display Overlay v J 000 Em Filter Open Bin M 8 FOV 12 6 f4 1s Living Image Version 2 50 1 5 20 2005 Series Options v Male Nn nu Experiment DOB 03 21 05 Label kidney Comment dorsal Luminescence 15000 10000 5000 Counts Color 5cale Min 1122 Max 19546 oo Sa G ib Another way to view information about images is available in the View menu 1 Open an image or sequence 2 Select View Image Information on the menu bar The Image Information window appears 3 Choose an image by making a selection from the Sequences drop down list and
83. 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 TF RotProperies ROI Roi Label Shape Type Background ROI Subject ROI Image Number TLT20050624145507 006 Label ROI 1 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 115 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data Figure 5 15 ROI properties Subject tab image For more details see Table 5 6 page 116 The items in the ROI Properties box depend on the type of ROI selected in the Subj ROI tab ROI Properties 5 nga Label Subject 1 Type Manual Subject ROI Enter information about the selected ROI optional E Lock Position Xe pix 122 2j444 Yel pix 127 40694 Lock Size Width pix 78 65097 Height pix 197 84919 Line Size 2 Line Color Done ROI bj Drop down list of subject ROIs in the image shape square ROI label name Edit the label here Table 5 6 ROI Properties Item Description or free draw ROI A drop down list of ROIs in the
84. 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 is 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 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 203 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Viewing Photon Density Maps or NTF Efficiency 1 After the reconstruction is finished or results are loaded click Photon Density Maps or NTF Efficiency 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 A Photon Density Maps Wavelength AI Waves v AngleofView PRMD www phot
85. 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 windows Ctrl W Closes
86. 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 143 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 149 Manual If necessary perform a manual analysis after an automatic analysis to 153 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 CT Figure 8 2 Sequence for spectral unmixing D wamo seo LI Segre Views nets kadant Efficeney OT Mire 1 2587 Max 1 0088
87. list to overwrite that spectrum with the computed spectrum when you click Apply 180 10 Reconstructing a 3D Surface Generating a Surface Managing Surfaces on page 186 Export or Import a Surface on page 187 A surface is a 3D reconstruction of the animal surface topography derived from X ray images 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 191 page 198 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 10 Reconstructing a 3D Surface 10 1 Generating a Surface 1 Load the image sequence for the reconstruction a sequence that was acquired for DLIT or FLIT analysis Select a subject Nude Mouse or Phantom M NOTE Furred mice are not recommended for DLIT or FLIT The Surface Topography tool can appropriately generate the surface of a furred mouse However the optical data pattern can be grossly shifted by the fur Select the data for surface reconstruction by adjusting the Threshold slider or entering a threshold value in the adjacent box Figure 10 2 Hover the mouse cursor over the Threshold slider handle Left click when the handle turns blu
88. nm Maxe 1 00e8 y a 6 5108 Spectrum List See Table 8 3 on YY fb Hsk X f fth page 159 for detailson t Composite of the this toolbar ning Name Color a unmixed Images 1V TissueAF Mime zZ See page 160 for 2 V aF6so Li aL 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 143 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 CT Chapter 8 Spectral Unmixing Figure 8 6 Sequence for spectral unmixing Sequence View Units Radiant Efficiency E Use Saved Colors Options x Info j a PR i NG a Min 9 70e6 Max 1 8
89. 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 CT 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 Autofluoresc
90. or photons 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 E irmas a KAN ee d il hg Fluorescent image Overlay Fluorescent image on photograph CT imaging A series of radiographic exposures of the subject acquired using the X ray energy source on the IVIS Spectrum CT analyzed by computed tomography CT to generate a 3D volume Gumana sg clog CI bbaaree m cs Di CT volume classified 12 Living Image 4 3 1 User s Manual IVIS Spectrum CT Table 2 1 Imaging modes on the IVIS Spectrum CT continued Chapter 2 Getting Started Imaging Mode Description Photograph A short exposure of the subject A illuminated by the lights located in the ceiling of the imaging chamber The photographic image is displayed as a grayscale image 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 CT If analyzing data acquired on a different type of IVIS instrument say for example the Lumina XR please see the Living Image Software
91. or texture Color Scale Color Scale Min la 2 10e3 5 Color Table kei 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 the left and right sliders to set the minimum and maximum colors Reverse Choose this option to apply the colors of the selected color table in reverse order to the 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 221 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Table 11 12 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 205 Quantification FLIT For uncalibrated sources the fluorescence yield measured for the voxels selected using the Measure Source tool s
92. page 139 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 on creating a sequence from individual images see page 95 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
93. 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 are located This information is available if organs are displayed with the reconstruction For more details on displaying organs see 3D Tools Registration page 222 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 g 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 information on planes button to display slice planes through the center of mass See page 209 for more 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 Tool Palette L gt
94. right or left side of the window and release 274 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 kot 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 275 Living Image 4 3 1 User s Manual IVIS Spectrum CT Appendix B Preferences 276 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 Preference Third Preference Target Counti Minimum Luminescent 3000 Exposure Time F Stop ka Epi fl
95. sequence choose this option to 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 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 45 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition NOTE The lamp may be set to Low for certain applications such as long wavelength data through thin tissue 6 Select a Field of View size of the area to
96. subtract compute pure spectra by subtracting unmixed spectra which overlap 156 Export unmixing results as an image or sequence which can be analyzed using image 161 analysis tools ROIs Mirror ROIs for optical data support measurements on the left and right views of images 107 acquired using the Side Imager accessory View a histogram of measurement ROI pixel intensities 113 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 VIS Acquisition Control Panel Ti j IIS Acquisition Control Panel DR Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter Luminescent 1 00 sec DE jfi x Block v fopen Mi fluorescent q F Stop Excitation Filter Emission Filter 1 v Exposure Time auto S sec W medum v po oen w Field of view C System Status Field of view D v System Status Initializing Acquire C xFov 24 12 5 cm Imaging Wizard Subject height 11 50 Slem Sequence Setup Focus use subject height w Temperature AN Locked 12 8 naging Wizard Stage temperature controller Service 8 Subject height 1 50 sequence Setup Focus use subject height Temperature NANG Locked Initialize Save a batch sequence setup xsq 56 Living Image 4 3 1 User s Manual IVIS Spectrum CT C
97. tables CEDERE LT IR Render Slice Using Slice Color Table Volume Color Table Color Table a Reverse Opacity Color Scale Subject Height 19 4mm Perspective 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 a P P a F 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 slices are due to the black color level at the low end of the color palette To correct this go to Sources tab in the 3D Optical Tools and move the low end colorbar slider up from the black level Living Image 4 3 1 User s Manual IVIS Spectrum CT Viewing Slices Chapter 13 3D Multi Modality Tools Figure 13 11 Viewing slices Choose a slice orientation Use the slider to move through the selected slices
98. tebek 3 Musik FOV 2 4 f2 is Liig Image Version 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 bar 3 4 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 19 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 CT Chapter 3 Image Acquisition Figure 3 19 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 i This
99. 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 98 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data 99 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 110 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 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 KO 22 Jed ea Living Image 4 3 1 User s Manual
100. 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 284 Index Symbols 3D Multi Modality tools adjusting image resolution 247 classifying 3D volumetric data 243 color opacity map 244 control points 244 fiducial registration 256 gradient illumination 248 loading data 258 260 manual registration 261 264 maximum intensity projection 248 requirements 242 242 243 volume display options 246 3D quantification database 235 240 create 236 239 manage results 240 samples 235 3D reconstruction fluorescent sources 198 201 luminescent sources 191 197 reconstruct particular regions 196 3D reconstruction results DLIT or FLIT 201 203 3D ROI draw 129 132 Measurements table 134 136 properties 132 133 3D signal intensity 84 3D tools 217 234 Animate tab 229 234 Registration 222 228 Source tab 220 222 Surface tab 218 219 3D Volumetric Browser 258 A acquire a sequence batch mode 55 56 acquire a sequence manual 57 60 acquire a sequence 48 55 animation 229 234 custom 231 233 edit an animation setup 233 234 preset 231 animation tools 229 234 autofluorescence 110 See tissue autofluorescence autoluminescence 110 automatically draw ROIs 104 105 average background ROI 110 B background
101. to long wavelength The two sliders determine the lower and upper limits of the color range that is used to render color The parts of the color map outside the selected range are not used in the color rendering process By default the entire color range is selected 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 a Annotate and
102. 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 6 7 BBB L2 NAG Sb fe ae Select this tool from the drop down list to display the perspective view ar Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 10 Reconstructing a 3D Surface 184 Table 10 1 3D view tools continued Tool Description Click to show or hide measurement cursors in the coronal sagittal or transaxial views Ka Click and drag the green handle I at either end of a measurement cursor to resize and reposition it H If DLIT or FLIT results are loaded click a voxel in the 3D reconstruction then click this Had button to display measurements for the voxel in the 3D tools source voxel measurements ag Enables you to save the 3D view to a graphic file for example jpg Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 10 Reconstructing a 3D Surface 185 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 the view Figure 10 5 m Click
103. 0 3 Height cm 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 CT Chapter 5 ROI Tools for Optical Data see Table 5 6 page 116 Figure 5 14 ROI Properties Background ROI tab LO a Units Radiance Photons v Display Overlay v Options x Info NG igi BKG 1 1 278e 05 ROI 1 BKG 1 25 4 825e 09 ROI 2 BKG 1 25 2 394e 09 Luminescence ay Radiance p sec cm3 sr Color Scale Min 1 46e8 Max 2 54e9 ej TLT20050624145507 006 e ss 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 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 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
104. 0110210131022 SEQ SequenceInfo txt 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 Mina 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 Mina 71 Mina 68 Mins 68 Max 5747 Methods Automatic v Start Unmixing 170 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 9 DyCE Imaging and Analysis 171 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
105. 1 3D Tools Surface Chapter 11 3D Reconstruction of Sources Use the Surface tools to adjust the appearance of the reconstructed animal surface and photon density maps Figure 11 30 Surface tools and example DLIT reconstruction with photon density or NTF Efficiency maps 7 BI20111027132749 sEQ sequence view LE 3D View Gronal z 10 7 faa Tool Palette ROI Tools L gt Spectral Unmixing and DyCE p amp ss Ay L g EH a Mm ar E bs o _ Surface Topography H Help gt 3D Multi Modality Tools 3D Optical Tools Sagittal x 2 5 Surface Source Registration Y Display Subject Surface A Q Opacity Apply Simulated Wavelengths 600 Intensity Color Table Rainbow V Reverse Log Scale Transaxial y 47 7 Subject Height 19 4 mm Table 11 11 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 Wire frame amp a Point cloud surface face Pah Wire frame ih Surface face 218 Living Image 4 3 1 User s Manual IVIS Spectrum CT Table 11 11 3D Surface tools continued Chapter 11 3D Reconstruction of Sources Item Description Cone a Shading styles for the surface Smooth surface face
106. 10 No23 745 No23 500 Block 500 ND2 500 430 500 520 Block 520 9 ND2 520 430 520 465 520 560 Block 560 8 ND2 560 430 560 465 560 500 560 580 Block 580 8 ND2 580 430 580 465 580 500 580 535 580 600 Block 600 ND2 600 430 600 465 600 500 600 535 600 620 Block 620 ND2 620 430 620 465 620 500 620 535 620 570 620 Block 640 ND2 640 430 640 465 640 500 640 535 640 570 640 660 Block 660 ND2 660 430 660 465 660 500 660 535 660 570 660 605 660 Block 680 ND2 680 430 680 465 680 500 680 535 680 570 680 605 680 640 680 Block 700 ND2 700 430 700 465 700 500 700 535 700 570 700 605 700 640 700 720 Block 720 ND2 720 430 720 465 720 500 720 535 720 570 720 605 720 640 720 740 Block 740 ND2 740 430 740 465 740 500 740 535 740 570 740 605 740 640 740 760 Block 760 ND2 760 430 760 465 760 500 760 535 760 570 760 605 760 640 760 780 Block 780 ND2 780 430 780 465 780 500 780 535 780 570 780 605 780 640 780 s Omen He Sm Pn a s 710 760 710 780 800 Block 800 ND2 800 430 800 465 800 500 800 535 800 570 800 605 800 640 800 675 800 710 800 745 800 C i c k S e e ct by S p e ct rum Li b ra ry 820 Block 820 ND2 820 430 820 465 820 500 820 535 820 570 820 605 820 640 820 675 820 710 820 745 820 840 Block 840 ND2 840 430 840 465 840 500 840 535 840 570 840 605 840 640 840 675 840 710 840 745 840 Blocl iC ND2 Open 430 Open 465 Open 500 Open 535 Open 570 Open 605 Open 640 Open 675 Open 710 Open 745 Open and choose a spectrum library in the dialog
107. 1022_007 5 2 2012 3 42PM _File folder Ji RKG20110210131022_008 5 2 2012 3 42PM File folder Ji RKG20110210131022 009 5 2 2012 3 43 PM File folder Ji RKG20110210131022 010 5 2 2012 3 43PM File folder Ji XML 5 1 20128 56AM File folder _ Sequencelnfo 4 16 2012 10 03 AM Text Document TIFF Image Files tif tiff All Files Select the file type s 66 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data 67 Table 4 2 File filters 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 CT Chapter 4 Working With Optical Image Data
108. 11 3D Reconstruction of Sources 191 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 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 48 for more details on the Imaging Wizard If you plan to manually set up the sequence the sequence must include m A CT image m Optical data from
109. 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 217 229 For details on managing results for example save load or delete see page 202 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 194 3D View toolbar E 000060407145405 SEQ Li Sequence View D View Tool Palette _ ROT Tools so Spectral Uamixing and DyCF Surface Topoyraphy 3D Mult Modality Toots gt 3D Optical Tools FLIT 3D Recoestrection AryTE Properties kesdu FIT Readia AIT 49405 foie Key Value Pinal vosel sae mm 3 1 25 2 Number of vowels Reduced Chi 2H2e 03 Starting wie 5 00 Neurf best Total surf samp
110. 18 ROI tools 101 transillumination overview 87 troubleshooting DLIT FLIT 234 U user preferences 273 280 V volume slices information and results 255 volumetric data classify 243 244 color opacity map 244 display options 246 information and results 255 rendering slices 252 smoothing 251 viewing slices 253 254 vsize starting 202
111. 1L120050624145507 006 oO 8 Units Radiance Photons v Display Overlay v Options Y Info NG igj Luminescence Contour 2 5 Auto 2596 Background ROI Subject ROL Image Number TLT20050624145507 006 v ROI BKG 1 v BKG 1 1 278e 05 x10 ROI 1 BKG 1 25 4 825e 09 IV Lock Position 1 0 ROI 2 BKG C gi a Height cm 0 89250 p sec cm2 sr Color Scale Line Size 3 2 Min 1 46e8 Max 2 54e9 Line Color y aao 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 19 Save Load or Delete ROIs The software automatically saves ROIs with an image The ROI measurements are saved in the AnalyzedClickInfo txt file associated with the image ROIs are saved per user and can be applied to other sequences Additionally ROI parameters can be saved per user and applied to other sequences To save ROls to the system 1 In the Name drop down list confirm the default name or enter a new name for the ROI s 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 121 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI
112. 2 Quantification Database 238 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 highlighted 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 Pa D LC Well Plate Quantification Window oS Ka For Sequence EL20090414101005_SEQ Image EL20090414101005 001 v Fluorophore Type o Dye mendes Ca Measurement 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 e W For Image EL20090414101005 001 Quantify 15 Click Quantify The results are displayed Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 12 Quantification Database Figure 12 6 Example fluorescence quantification plot and results
113. 3 369e 03 3084e 03 3 34le 04 6 4 TO Configure Export To delete a custom table configuration Select the configuration from the User Lists drop down list and click Delete 135 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 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 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 a 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 136 7 Image Math Creating a New Image Using Image Math Subtracting Tissue Autofluorescence on
114. 4 an Image Data 15 counts Crop Distance B Z209 6 96 Distance 1 62 am 1 49 0 64 ROI Tools gt Planar Spectral Imaging _ Surface Topography gt 3D Multi Modality Tools gt DUT 3D Reconstruction Luminescence 15000 10000 5000 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 po button 85 Living Image 4 3 1 User s Manual VIS Spectrum CT Chapter 4 Working With Optical Image Data Table 4 10 Measurement cursor position and length Description 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 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 Open an image and in the Image Information tools click t
115. 5 8 456e 03 1 784e 03 6 007e 03 1199c 04 z Customized Selections Me ts Types Attributes ROI Dimensions vesatenens let mrave Abibmesom cm 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 123 6 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 4 Figure 5 4 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 ima
116. 50 1 50 1 50 Auto Medium 1 Block 580 600 DA 3 DBA Auto Medium BIDO O O 4 WG Auto Medi N 620 1 50 Field of View System Status X Rays will be produced Cs when energized 5 4 m Auto Med 640 1 50 Idle Acquire 13 4 cm Imaging Wizard Subject height 1 50 cm Image Setup min Apply to All Md Remover A Update Jl Insert Jl Add Focus use subject height v Temperature IAN Locked Initialize Number of Segments 1 2 Enter anew 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 row 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 Living Image 4 3 1 User s Manual Method 2 VIS Spectrum CT 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 40 Sequence table edit commands in the shortcut menu
117. 6e 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 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 123 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 7 ROI create tool G a AN NAN chad AA MPD 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 121 105 Living Im
118. 7e8 f3 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 V 700 V 720 9 740 V 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 158 for information about the results 147 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 8 Spectral Unmixing Figure 8 7 Select a reference spectral library CG Load Spectrum Library Spectrum Libraries show qualified only Available spectrum libraries 0 0 660 FOO Spectrum List Note
119. 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 117 and page 118 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data 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 ell NOTE If the image was acquired using the Side Imager draw a background ROI on each view Figure 5 11 Cli Figure 5 11 Draw a background ROI on each view in an image acquired using the Side Imager 7 1v20120412111353_004 a Units Radiance Photons v Display Overlay Habana p1 on Luminescence pai ys ROI 2 50 1 931e 06 3 Miror 1 3 7102 06 x10 a A Mirror 2 2 447e 06 u 4 T 3 0 AL e ROI 1 50 8 593e 05 ae 2 0 nik BKG 2 1 430e 05 Na ri BKG 3 1 293e 05 BKG 1 1 306e 05 A F l Radiance pi secicm2fsr Color Scale Min 1 29e5 Max 6 13e5 5 Associate each background ROI with a measurement ROI s or mirror ROI s using one of the methods in Table 5 5 111 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data Table 5 5 Methods for associating measurement or mirror ROIs with a background ROI Method Descriptio
120. DLIT or FLIT reconstructions m Make source measurements page 205 m Export voxel measurements csv Figure 11 31 Source tools and example DLIT reconstruction IT BI20111027132749 sEQ Tool Palette _ Sequence View 4 3D View gt ROI Tools ae Bi eee AI Ca Spectral Unmixing and DyCE Ry NG BF Gl z gt Surface Topography Gronal z 10 7 Sagittal x 2 5 gt 3D Multi Modality Tools 7 3D Optical Tools Surface Source Registration Select Source Original Display Source Surface 4 m Opacity Display Voxels Color Scale Color Table Transaxial y 47 7 _ Measured Sources Key Value Quantification 0 00 photons sec Volume 0 00 mm 3 photonsimm 3 Depth 0 00 mm Subject Height 19 4mm Photon Density Center of Mass 0 00 0 00 0 00 Host Organ Unknown gt DLIT 3D Reconstruction Table 11 12 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 210 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 wrapp
121. 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 orientation flipped or reversed along the x y or z axis As a result the slice views transaxial coronal sagittal may be flipped or rotated so that the actual view that is displayed does not match the 3D View windowpane name for example the 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 Manually rotate the data using the Transformation tool for more details see page 264 266 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools 267 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 28 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 271 A 1 Control Panel The control panel provides the image a
122. I 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 117 and Editing ROI Dimensions page 118 7 Click the Measure button W MeasweRols The ROI table appears For more details on the table see Managing the ROI Measurements Table page 123 109 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data 110 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 background 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 t
123. IVIS Lumina II CL5135289 IVIS Lumina XR CL5135290 IVIS Kinetic CLS135288 IVIS 200 CLS135287 IVIS Spectrum CLS135291 IVIS Spectrum CT CLS135292 Please see the IVIS Spectrum CT Hardware Manual part no 133557 Rev 00 for information on the IVIS Spectrum CT instrument Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 CT 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 163 Note DyCE acquisition and analysis features require a separate license DyCE acquisition supports Cerenkov radioactive luminescent or fluorescent imaging DyCE acquisition and analysis tools enable real time pharmacokinetic Spatio temporal biodistribution studies of probe or dye signal Improved Spectral Unmixing Tools 142 Choose from four methods of spectral unmixing depending on your knowledge of the probe 143 spectral response and the probe location If a user created spectrum library reference spectral data from known probes at known locations is available it can be selected in the Imaging Wizard during sequence setup This provides a convenient way to select filters Ability to
124. 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 242 If the appropriate license is not installed or the GPU does not meet these specifications the 3D Multi Modality tools will not appear in the Tool Palette 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 Shared Recommended 1GB Dedicated Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools 243 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
125. Is 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 CT Table 1 2 Tech Notes continued Chapter1 Welcome 14 Transillumination m 14a Transillumination Fluorescence m 14b Raster Scan a 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 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 Initiali
126. 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 124 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 106 ROI size dimensions or position 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 ROI so that it becomes a 4 arrow 2 Drag the ROI 3 Release the mouse button when the ROI i
127. NG pen i minimum counts required for reconstruction Keep the ELLS Painting size Segment Opacity minimum counts gt 200 To select particular regions for reconstruction 1 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 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 197 M NOTE If the pencil tool markings are applied to the image only the marked pixels are included in the analysis Figure 11 8 Selecting regions to include in reconstruction Threshold Tools Region Selection Tools Adjustment 10 5 Draw Erase Min counts 29 Painting size Segment Opacity i Use these tools to select particular image data to include Choose the Draw option then mark in the analysis See Table 11 4 for details on the tools the area to include in the reconstruction using the pencil tool In this example the green area marked with the pencil tool is will be reconstructed Table 11 4 Region Selection Tools
128. 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 Wew Era ribs Couns oe Use Saved Colors Goto sin gt GF i a p Dap Pos 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 33 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 54 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Table 3 10 Sequence View window continued Item Description wj Creates a preview picture snapshot of the image or thumbnails that the Living Image Browser displays when the data are selected For more details on the browser see page 62 T igoa kaosi S00 i IEK 3 i ICFT50 dye in pillowes gt ER CKINGLAIGLADOSE SOO j EE Jina LMOOSTI DUT subjects phantom Seg TLTAMGOSTMISI ISE f AT igea TLS SEQ f iTLT a i a p
129. 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 O Solid Color Gradient Colors Color Theme Midnight r Top o P Bottom 4 is Rest
130. Palette EJ Image Adjust lle Apply corrections or filters to the raw data Corrections Filtering E Read Bias Subtraction Flat Field Correction v Cosmic Correction Lens Distortion Correction Adaptive FL Background Subtraction Normalization Threshold Counts 100 anng smeoting None gt _ Image Information _ ROI Tools _ Surface Topography gt 3D Multi Modality Tools gt FUT 3D Reconstruction 77 maga Bifoanatian MAG ols Units Cm View information about an optical image for tunog example a pixel histogram or a 3D plot of pixel Binning 8 Width 12 6cm Height 12 6 cm intensities peat AAO NO Make measurements in the image Image Data 2715 counts 71 yROITols sid ROI Tools for Optical Data O Q W Measreros 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 Delete Load sae Auto ROI Parameters Threshold 26 B 2t 6 3D ROI Tools for Volumetric Data E WA Measure 3D ROIs x 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 bg 37 128 14 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 2 Getting Started Table 2 2 Living Image tools av
131. ROI Tools E BI20111027132749 sEQ f N J 3 Sequence View 14 3D View La Spectral Unmixing and DyCE Surface Topography e L gt 3D Multi Modality Tools 17 3D Optical Tools Surface Source Registration Select Source Original Display Source Surface i G V Display Voxels oR ity Projection Threshold lal 0 0034 Gradation fal 50 Voxel Size 0 31 Y s Display Voxels As Smoothing 5x5 Texture v Color Scale Min 379 6641 Color Table BlueYellow Reverse Log Scale Measured Sources Key Value Quantification 5 09e6 photons sec Volume 8 60 mm 3 Depth 4 18 mm Center of Mass 2 5 47 7 10 7 Host Organ Unknown Export Voxels Lo DLIT 3D Reconstruction Gronal z 10 7 Sagittal x 2 5 J DA Transaxial y 47 7 Subject Height 19 4mm Perspecti Arima on EEA H Help 2000 0 Transaxial 547 7 Subject Height 19 4 mm 207 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 208 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 po
132. Reflect smooth ka Surface face surface face qy Reflect surface a 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 DLIT 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 219 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 220 11 12 3D Tools Source Use the Source tools to a Adjust the appearance of sources in
133. Restart Wizard Cancel Back Next m Bioluminescence DyCE Choose Manual Settings and set appropriate exposure parameter 7 Set the exposure parameters 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 J 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 J images 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 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 166 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 9 DyCE Imaging and Analysis J NOTE
134. Select Name 1 Tissue AF 2 AF680 3 AF750 Information LLE pem s 1n LE e E Pon e Tas LOE LE Por A E L Je 000 PE E m L B 740 605 760 605 780 605 800 7 Index 2 Name AF680 Origin HX20120419114703 SEQ WavelengthPairs 605 660 605 680 605 700 605 720 605 740 605 760 605 780 605 800 Index 3 Name AF 750 Origin HX20120419114703_5EQ WavelengthPairs 605 660 605 680 605 700 605 720 605 740 605 760 605 730 605 300 740 Emission Wavelength rm Normalized U Legend Tall Color Choose this option to show only spectrum libraries with excitation emission filters that match the data set being analyzed Ca 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 Co LO fata E HX20120419114703 sEQ LO Sequence View Unmixing V Show Labels 7 Individual Scale Normalized E Legend a g Tissue AF 8 7 x10 6 E 5 Spectra plot See page 158 for more 3 details 2 1 Min 0 00 0 Max 9 1367 660 700 740 730 AF750 Emission Wavelength nm Spectrum List See Table 8 3 on t _ aw It X page 159 for details Note on this tool ba r Select Name Color Pick 1 V TissueAF Dime zi M E 2 V AF680 E v 4r 3 V AF750 W z P Min 0 00 x 2 34e8 TT Min 0 00 x 6 4666 Composite Unmixed images show th
135. 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 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 261 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools 262 Figure 13 20 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 258 The software determines a default air noise boundary for the 3D volumetric data Figure 13 21 If you need to remove noise from the 3D volumetric data move the air noise boundary to the right in the histogra
136. Spectrum CT Chapter 10 Reconstructing a 3D Surface 187 Item in the Surface Description Topography Tools Name Name of the selected surface Delete Removes the selected surface from the system Load Opens the selected surface Save Saves a surface to the selected name Overwrite Saves the surface and overwrites the previous surface results 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 273 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 227 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
137. T Ch 817 Color Scale Limits Auto Full Manual Individual Color Table 600 400 200 Counts Color Scale Min 44 Max 817 Color scale Min and Max Table 4 4 Image Adjust tools Item Description co 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 T Click this button to incrementally zoom in on the image incrementally magnifies the image in the image window 75 Living Image 4 3 1 User s Manual IVIS Spectrum CT Table 4 4 Image Adjust tools continued Chapter 4 Working With Optical Image Data 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 77 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 im
138. The data mask includes the entire subject by default 4 If necessary 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 x 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 173 for details on manual analysis 8 Click Finish The Unmixing window shows a time plot of the temporal spectra unmixed images and a composi
139. Tranclumnasonlocion Opiom inde cA wj Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data 88 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 95 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 CT Cha
140. User s Manual IVIS Spectrum CT Contents Chapter 13 Appendix A Appendix B Appendix C Index 3D Multi Modality Tools aan aman 242 About the 3D Multi Modality Tools 0 00 aaa 242 3D Multi Modality Tool Requirements 2 00000 es 242 Classifying 3D Volumetric Data aaa 243 Specifying a Color Opacity Map cc es 244 Volume Display Options 0 000 cc ee ees 246 Adjusting the Image Quality 0 0 0c cc ee 246 Adjusting Volume Opacity na 6 ie ice ee awd bad oa ww alain ee eS bare ee 248 Maximum Intensity Projection MIP 0 00 ee es 248 Gradient Illumination n on anaa ees 248 Modifying Volume Resolution nananana anaana nanana 249 View a 2D Projected Image of the Volume 000 ce eee ees 249 Smoothing a Volume Xx sasaka ak 6d eRe WA HAD AA ed dete ee aS eee ed bee e as 251 Rendering and Viewing SIICES 2 0 ees 252 Rendering SliceS 22 Ka eed Pha KAKA MG BAR KG BKA ede teed eae eee e ds 252 Viewing SINCGS sama AMG benssgeteendceueueedeetagatdees aeeeavec 253 Volume Information and Results 000 ee eee eee 255 Managing Ba PAP AA 255 Registering Optical and Volumetric Data 00 cee ee ees 256 Loading Data for Registration 0 ccc ees 258 Registering Multi Modal Data 00 0 es 260 Volume Data Viewer a 264 Viewing RAW Volumetric Data 0 es 265 IVIS Acquisition Control Panel
141. User s Manual specific for the Lumina XR instrument M NOTE The tools available in the Tool Palette or menu bar depend on the active image data Figure 2 7 Living Image tools are located in the menu bar and Tool Palette Tools Window Help J 3D Animation Longitudinal Study Well Plate Quantification for BI20111027130508 SEQ Image Overlay for BI20111027130508 SEQ Colorize for BI20111027130508 SEQ Image Math for BI20111027130508 SEQ 027130508 SEQ Sequence View l2 3DView Spectra Units Counts ir E usesevedcol Optens sine gea i Min 47 Min 24 Max 241 3 Max 27 Min 44 Max 5695 User KSA 13 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 2 Getting Started Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum CT Living Image Tools and Functions See Page a i Image Adjust am Q a EE Tune the photograph brightness contrast or Photo Adjustment Brightness f 100 opacity Contrast Bi 1 5 Opacity 100 Color Scale Min f 2140 Max lef 36446 Color Scale Limits Auto O Full Manual Individual Change the minimum or maximum of the color scale applied to the optical image data 4h 4h ale Select a color table for image display abt Color Table Rainbow 7 E g V Reverse E Logarithmic Scale 75 Corrections Filtering Optical Data Tool
142. VIS Spectrum CT Chapter 2 Getting Started Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum CT continued Living Image Tools and Functions See Page 3D Multi Modality Tools ag Volume Process Slice Results Set color and opacity values for different intensity HOA O g ranges of a CT volume so that the color opacity 7 Display Volume map shows the volume regions you are interested Level Of Detail in opaque in the map and hides unimportant regions aaa ony C ister 3D tructi f lumi t a o register reconstructions of luminescent or fluorescent sources biological information with a gt CT volume to provide anatomical context for 1 0 Opacity _ lt Ela E Reverse interpreting biological functional information Cai Note The 3D Multi Modality tools require a separate license Pa 0 00 Intensity 6 55e4 V Logarithmic Histogram Maximum Intensity Projection MIP T Gradient Illumination eb Opucal Took KR Surface Surface tools Adjust the appearance of the F Display Subject Surface reconstructed animal surface and the photon A M density maps Opacity 100 Source tools Adjust the appearance of Display Photon Density Map reconstructed sources make source POH measurements export voxel measurements Wavelengths Registration tools Display organs on the Intensity 10027 reconstructed surface adjust the location or scale Color Table of o
143. a 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 5 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 The aperture size controls the amount of light detected and the depth of field A larger f stop number corresponds to a smaller aperture size and results in lower sensitivity because less light is collected for the image However a smaller aperture usually results in better image sharpness and depth of field A photographic image is taken with a small aperture f 8 or f 16 to produce the sharpest image and a luminescent image is taken with a large aperture f 1 to maximize sensitivity For more details on f stop see 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 I
144. a graphic file 35 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Table 3 4 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 browser see page 62 D Living Imaget Browser rec C 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 3 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 loca
145. 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 the Edit Image Labels dialog box see page 26 and other information automatically recorded by the software Living Image 4 3 1 User s Manual IVIS Spectrum CT Table 4 3 Image window continued Chapter 4 Working With Optical Image Data Item Description Opens all of the images in a sequence Closes all open images of a sequence Opens the Edit Sequence dialog box that enables you to add or remove images from the sequence display x JA Opens a dialog box that enables you to export the active view as a graphic file Tool Palette Takes a snapshot that is displayed with the data in the Living Image Browser See page 62 for more details on the browser Living Image Browser TLT20050624145507 SEQ Click Number EX Filter EM Filter Illumination Mode User ID Experiment Commenti Becsgpecccessssssssssesssseossscossossssosssoessosssssssodoesssosssossosssssoessesossossessossessscosssssosssisososssossssossssssessssessoesseosssoessssssissosesssooes
146. acquisition functions Figure 2 6 See Appendix A on page 268 for details on the imaging parameters in the control panel M 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 x Imaging Mode Exposure Time Binning FiStop Excitation Filter Emission Filter Imaging modes o Pao fev meam fi gt See Table 2 1 on page 11 medum ve v for an overview of imaging modes Standard One Mol v a Field of view System Status Auto exposure selected Idle 13 4 cm Imaging Wizard Subject height 1 50 cm Sequence Setup Focus use subject height v Temperature IG Locked 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 the minimal required target max count The max count is permitted to exceed the user specified target count as long as it does not saturate If the resulting image has too little signal or saturated pixels the software adjusts the parameters and takes
147. 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 ROI Label Click to edit the selected ROI label name Image Number A drop down list of open images Background ROI The Background ROI tab shows a drop down list shows all average background ROIs in tab 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 116 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data 117 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 ID User entered information about a subject ROI Label Label name of the selected subject ROI
148. age 1 Click the db button 2 When the pointer becomes a nd 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 soft binning to the image data TIP See these technical notes for helpful information select Help 5s Tech Notes on the menu bar m Detection Sensitivity includes information about binning and smoothing u Luminescent Background Sources and Corrections a Fluorescent Imaging for more about fluorescent background Figure 4 15 Tool Palette Corrections Filtering tools Tool Palette 3 Corrections Filtering gt Read bias supt Tacuo Fiat Field Correction Note Read Bias Subtraction and CLUU Baran Flat Field Correction are default Lens Distortion Correction mandatory corrections in Radiance Adaptive FL Background Subtraction units mode In counts mode these Normalization Threshold Counts 100 corrections can be cleared Binning 2 6 bmoothing None 4 _ Image Information _ ROI Tools _ Surface Topography gt 3D Multi Modality Tools gt FUT 3D Reconstruction ens Distortion Correct Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data Table 4 5 Corrections Filt
149. age 4 3 1 User s Manual IVIS Spectrum CT 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 reconstruction 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 Thres
150. age 4 3 1 User s Manual IVIS Spectrum CT Table 11 10 Living Image 3D Browser DICOM viewing controls Chapter 11 3D Reconstruction of Sources 11 10 3D Tools Overview After you reconstruct or load a surface or 3D sources the Tool Palette includes the 3D Tools which are used to modify the source display parameters Item Description Start Index Specifies the first image slice for viewing Auto Preview Select this option to automatically play back the images End Index Specifies the last image slice for viewing Load Opens the DICOM data in a 3D View window Load data in new Ifthis option is selected DICOM data are opened in a new 3D View window when you window click Load 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 220 measurements export voxel measurements Registration Tools Display organs on the reconstructed surface adjust the location or 222 scale of organs on the surface import an organ atlas Animate Tools Display preset animations of the 3D View scene Enables youtocreate 229 217 Living Image 4 3 1 User s Manual IVIS Spectrum CT 11 1
151. age 4 3 1 User s Manual IVIS Spectrum CT Appendix A IVIS Acquisition Control Panel 272 7 Click OK when the image is focused Appendix B Preferences General Preferences Options on page 275 Acquisition on page 276 Theme on page 277 Optical Properties on page 280 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 Ja 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 Measurement Show Advanced Options Counts Tatal Count bd Radiance Photons Tatal Flux Efficiency Tota
152. age 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data 106 Drawing Measurement ROIs Manually 1 Open an image or image sequence and in the ROI tools select Measurement ROI from the Type drop down list 2 Select the ROI shape a Click the Circle OJ Square 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 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 all of the ROIs in the sequence The sort options are only available if the ROIs have not been sorted 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 ove
153. age 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 right to adjust the gamma of an image displayed in overlay mode Alternatively enter a gamma value Gamma is related to image contrast Opacity Click and move the slider left or right to adjust the opacity of the pseudocolor luminescent data of an image displayed in overlay mode Alternatively enter an Opacity value Color Scale Min The minimum pixel intensity associated with the color scale for an image Pixels less than the minimum value are not displayed Max The maximum pixel intensity associated with the color scale for an image Pixels greater than the maximum value are displayed in the maximum color Color Scale Limits Auto If this option is chosen the software sets the Min and Max values to optimize image display and suppress background noise The Min and Max settings can be manually adjusted to further optimize the image display for your needs Full Choose this option to set the Max and Min values to the maximum and minimum data values in the image Manual Choose this option to enter Max and Min values for the image display Individual Applies a separate color table to each image in a sequence Note This option is only available when an image sequen
154. age 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 STs APAN REST RES 5 A EEE E TLT20050624145507_005 f o Units Counts v Display Overlay Options 21b nG ig Image TLT20050624145507_005 Series Male Nn nu Fri Jun 24 2005 07 56 46 Experiment DOB 03 21 05 Em Filter 640 Bin M 8 FOV 12 6 f2 1s Label kidney Camera IVIS 200 Beta II SI620EEV 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 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 72 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data 73 Figure 4 11 Edit Image Labels E Edit Image Labels x UserID B Living Image Universal Saved Labels LABELS 1 Ab V
155. ailable for data acquired on the IVIS Spectrum CT continued Living Image Tools and Functions See Page ai Spectral Unmixing 119 Analyze luBemiins Use spectral unmixing to EMAN hee Extract the signal of one or more fluorophores kare from the tissue autofluorescence 2 a Analyze luminescent or fluorescent images oo when more than one reporter is used in the 720 M7 same animal model 740 v 760 V ba Methods Library v Start Unmixing DyCE Dynamic Contrast Enhancement 163 Analyze Use DyCE to Select Images Type Time hh mm ss m Determine real time pharmacokinetic spatio Int temporal biodistribution of a probe or dye signal Extract temporal spectra signal intensity asa function of time from particular anatomical regions Note DyCE acquisition and analysis tools require a separate license Methods Start Unmixing Surface Topography 181 Spectrum CT Surface Reconstruction Generate 3D reconstruction of the animal surface Threshold topography derived from the CT image D0 FE aa A surface is a required input for ee Ee res DLIT diffuse light tomography analysis which as ES generates a 3D reconstruction of luminescent Name SURFACE 4 WMIC_Right Mouse x Sources ene FLIT fluorescence imaging tomography analysis which generates a 3D reconstruction of fluorescent sources Living Image 4 3 1 User s Manual I
156. ally acquired one after another without user intervention See page 55 for more details Temperature L E a 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 8 for more details Acquire Or X Rays will be produced when energized Acquire Click to acquire an image using the settings and options selected in the control panel or to acquire an image sequence specified in the Sequential Setup table Sequence Setup Click to display the sequence table so that you can specify and manage sequence acquisition parameters or open sequence acquisition parameters xsq See page 48 for more details on setting up an image sequence Imaging Wizard Click to start the Imaging Wizard Sequence Setup Click to open the sequence table Living Image 4 3 1 User s Manual IVIS Spectrum CT Appendix A IVIS Acquisition Control Panel 271 Table A 1 IVIS acquisition control panel continued Description Image Setup Click to close the sequence table Initialize Click to initialize the IVIS Spectrum CT Se
157. ama tala Image Math Window 137 l jaji PR Select Tools Image Math for lt sequence TUDO LOE aL OA TUES Oe name gt on the menu bar Mathematically combine add multiply subtract or divide two user selected images mama For example subtract a blue shifted background filter image from the primary excitation filter image to remove tissue autofluorescence signal ms Photo kom a 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 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 7 User Settings LP 23 amp Add User amp Change Password P 2 Delete User fe Security Add New User User ID Password Confirm Password Deletes all entries 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 Living Image 4 3 1 User s Manual IVIS Spectrum CT Figure 2 9 User Settings Change Password r A User Settings Change Password User ID HLA New Password Confirm Password Deletes all entries 2 Add User 2 change Passwo
158. 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 225 for more details If necessary adjust the opacity of the organs using the slider or enter a number in the box The organs are easier to view if you uncheck Skin in the Organs list To clear all organs from the surface click the Deselect All button 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 224 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Manually Adjusting the Scale or Location of Organs Load reconstruction results and confirm that the surface is in the perspective view click the 1 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 th
159. 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 276 for more details Living Image 4 3 1 User s Manual IVIS Spectrum CT Imaging Modes on the IVIS Spectrum CT Chapter 2 Getting Started Table 2 1 briefly explains the types of images that can be acquired on the IVIS Spectrum CT Table 2 1 Imaging modes on the IVIS Spectrum CT 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 ma asa aly Lee Cptors Se E Putannanaayanso one olog gt ors m Luminescent image Overlay Luminescent image on photograph 11 Living Image 4 3 1 User s Manual IVIS Spectrum CT Table 2 1 Imaging modes on the IVIS Spectrum CT 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
160. ans to the surface using the iat or pii tool if necessary you can click 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 224 2 bhe Drawing styles for the organs see Display Organs 223 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Table 11 13 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
161. are 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 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 48 139 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 7 Image Math 140 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 a IC KSA20110305104918 SEQ So al 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 measure
162. ase the rotation speed To stop the rotation click the 3D scene or the ga button Displays measurement cursors in the coronal sagittal or transaxial views Click this button then select a source or a point in a source to obtain source measurements total flux volume center of mass host organ in the 3D tools Source tab For more details see page 205 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 210 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 a 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 requir
163. associated with a corresponding angle The Living Image software subtracts dark bias no X ray from each transmission image then uses an X ray reference image to normalize the transmission image on a pixel by pixel basis The natural logarithm of the result is used to produce the CT projections images the final processing step before the series is reconstructed Fresh reference images X ray and dark bias must be acquired every five days to ensure that the X ray transmission images are correctly normalized The system prompts you acquire reference images Acquisition cannot proceed until the reference images have been acquired To acquire reference images 1 Confirm that the imaging chamber is empty and that the IVIS Spectrum CT is ready to acquire a CT image 2 Select Acquisition 5 CT Acquisition Acquire Reference Images on the menu bar Checking the Instrument Stage Alignment On rare occasion the instrument stage may become misaligned with the X ray source and detector If this occurs CT images may display artifacts for example double images as shown in Figure 3 4 blurring or shadows Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Figure 3 4 Example of image artifacts To check stage alignment 1 Remove all objects from the imaging chamber 2 Insert the alignment tool into the rotating stage See the Rotation Stage Alignment Kit instructions for deta
164. atus 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 CT Chapter 2 Getting Started Figure 2 4 Instrument temperature status in the control panel pag T MIS Acquisition Control Pane Imaging Mode Exposure Time Standard One Mol v P Field of View System Status Focus use subject height Y Temperature Locked Temperature box color White System is not initialized Ha Red System is initialized but CCD camera Field of View Demand Measured 7 9no om i pi Subject height 1 50 2 cm Sequence Setup Gubject height 11 50 Jem Click the temperature box to display the demand and measured temperatures temperature is out of range and not ready for imaging a Green System is initialized and CCD camera is at or within acceptable range
165. based on the idea that light is reflected at boundaries between different voxel intensities but 1s 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 248 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools 249 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 Edit Space and Orientation button Imr 2 In the dialog box that appears Figure 13 7 edit the pixel or slice spacing Figure 13 7 Volume Information dialog box A Volume Information Slice Information Pixel Spacing Row Column 0 1000 3 0 0016 1 mm Slice Spacing 0 0016 mm Subject Orientation Invert X Axis Invert Y Axis Invert Z Axis o cane View a 2D Projected Image of the Volume For DLIT or FLIT sequences a 2D image of the maximum intensity projection can be extracted from the 3D volume The extracted image is simila
166. be imaged Table 3 7 Field of View FOV settings on the IVIS Spectrum CT FOV Setting FOV cm A 4 B 6 5 C 13 D 19 5 7 Select a focus option Figure 3 15 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 211 for manual focus instructions Figure 3 23 Choose a focus option in the control panel A WIS Acquisition Control Panel o mi Imaging Mode Exposure Time inning D Excitation Filter Emission Filter Subject height 1 50 cm Focus use subject height w Temperature NG Locked 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 i NOTE If necessary click ag 5eup jin the control panel to operate in single image mode In single image mode the SequenceSetup button appears in the control panel Use this button to set up s
167. box that appears Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 8 Spectral Unmixing 143 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 143 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 to generate a spectrum
168. 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 el NOTE To open a recently viewed file select File Recent Files on the menu bar 1 Click the Open button pp on the toolbar Alternatively select File Open on the menu bar 2 In the box that appears choose a file type filter 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 W Favorites E Desktop m Downloads Recent Places ov Libraries Documents a Music E Pictures E Videos Fil Homegroup Computer Local Disk C th Network File name Go di CaliperLS Caliper Data CerenkovDyCE RKG20110210131022 SEQ v Search RKG20110210131022 5 J m Name Date modified Type Size RKG20110210131022_001 5 2 2012 3 42PM_ File folder Ni RKG20110210131022_002 5 2 2012 3 42PM_ File folder J RKG20110210131022_003 5 2 2012 3 42PM_ File folder J RKG20110210131022 004 5 2 2012 3 42PM File folder Ni RKG20110210131022 005 5 2 2012 3 42PM Filefolder Ni RKG20110210131022_006 5 2 2012 3 42PM_ File folder Ni RKG2011021013
169. butes ROI Dimensions Copy Select All Counts v All Possible Values v Pixels Configure Export Close 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 Counts Includes Total Counts Avg Counts Stdev Counts Min Counts and Max Counts in luminescence the table Total Counts the sum of all counts for all pixels inside the ROI Avg Counts Total Counts Number of pixels or super pixels Stdev Counts standard deviation of the pixel counts inside the ROI Min Counts lowest number of counts in a pixel inside the ROI Max counts highest number of counts in a pixel inside the ROI 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 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data Table 5 7 ROI Measurements table continued Item Description Radiance Photons fluorescence Total Flux photons sec the radiance photons sec cm steradian in each pixel summed or integrated over the ROI area cm
170. ce 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 5 Automatically drawing measurement ROls detected by the software ot 7 TG SS ee AA E TLI20050624145507 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 C gt 3D Multi Modality Tools C gt DLIT 3D Reconstruction 3 Click the Measure button Me ueROls in the ROI tools to show the ROI Measurements table 104 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data Figure 5 6 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 53
171. ce View Unmixing Units Counts 7 Use Saved 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 CT 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
172. ce is active Color Table aa Click the drop down arrow to select a color table for the image data 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 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 76 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data 77 Magnifying or Panning in the Image Window To incrementally zoom in or out on an image Click the E or Cal 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 84 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 im
173. chnical note DLIT and FLIT Reconstruction of Sources for more details on the DLIT or FLIT 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 192 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 198 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 CT Chapter 11 3D Reconstruction of Sources 189 Reconstruction Inputs NOTE Use the Imaging Wizard to set up the DLIT or FLIT image sequence See page 48 for more details DLIT The input data to the DLIT algorithm for a 3D reconstruction of luminescent light sources includes A surface topograp
174. ck No in the prompt and manually save the image data See 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 167 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 9 DyCE Imaging and Analysis 168 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 v User v Baa 7 Information entered here appears in the ooo POTE image label see Figure 9 9 on page 168 Male nu nu day 8 Mouse 4 J Comment1 S Comment2 z Time Point v 7 Animal Number 4 T Animal Strain v T Animal Model v Sex v View v Cell Line z C Reporter v 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 Sequen
175. ck 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 displays the animal surface and the reconstructed sources a 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 217 229 For details on managing results for example save load or delete see page 202 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 194 Figure 11 5 DLIT reconstruction results 3D View toolbar A B120111027132749_SEQ Sequence View b 3D View Tool Palette gt ROI Tools gt Spectral Unmixing and DyCE a Surface Topography ba 3D Multi Modality Tools gt 3D Optical Tools DLIT 3D Reconstruction Sagittal x 2 5 Analyze Properties Results PLIT Results DLIT 1 Loaded Key Value Final voxel size mm 1 25 Number of voxels 8 Reduced Chi2 1 31e 02 Index Of Refraction 1 40 Angle limit deg 60 Damping reduce 100 Data range Top Side view L Curve Mirror XOffset NNLS solver Photon Density Maps amp Export Results Save Results Transaxial y 47 7 Na On i v Delete Subject H
176. cquisition 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 Background Opens a dialog box
177. cquisition functions Figure A 1 Figure A 1 IVIS acquisition control panel auto exposure selected I MS Acquisitio Imaging Mode Expo F al Field of View A Subject height 1 50 To acquire an image using auto exposure click the arrow and select Auto Control Panel al auto fac v Medium r lz w Focus use subject height v Temperature M Locked Binning System Status 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
178. cy fit only appropriate for the mouse phantom 234 1 2 Quantification Database Preparing and Imaging the Samples Creating a Quantification Database on page 236 Managing Quantification Results on page 240 It is possible to determine the number of cells in a DLIT source or the number of dye molecules or cells in a FLIT source if a quantification database is available The database is derived from an analysis of images of known serial dilutions of luminescent cells or fluorescent cells or dye molecules 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 a 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 s
179. d TIP See the technical 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 on the IVIS Spectrum CT FOV Setting FOV cm A 4 B 6 5 C 13 D 19 5 4 Select a focus option in the control panel Figure 3 8 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition The focal distance to the camera is set a stage 7 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 211 for manual focus instructions Figure 3 8 Choose a focus option in the control panel A WIS Acquisition Control Panel Field of View System Status Idle 13 4 cm Subject height 1 50 cm Sequence Setup Focus use subject height w Temperature NG Locked Ng Imaging Mode Exposure Time inning D Excitation Filter Co la Emission Filter Imaging Wizard 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 Luminescen
180. d on the IVIS Spectrum CT are automatically co registered when the SequencelInfo file is loaded in the Living Image software This section explains how to register optical data with volumetric data not acquired on the IVIS Spectrum CT Two methods are available a Automatic fiducial registration For experiments in which the optical data are acquired on the IVIS Spectrum CT 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 260 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 261 for more details Figure 13 14 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 243 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools 257 Figure 13 14 Steps to register multi modal data volumetric data not acquired on the IVIS Spectrum CT 1 Load the optical data s Bioluminescence or fluorescence image sequence and structured light surface m 3D source reconstruction DLIT or FLIT results page 191 or page 198 2 Load 3D volumetric data CT or MRI page 258 3 Registe
181. de the Sequence Setup button appears in the control panel Use this button to set up optical sequence acquisition see page 48 for more details 6 Enter information about the image in the Edit Image Labels box that appears optional Click OK Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition l 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 72 for details on adding information to an image after acquisition Figure 3 2 Enter information to include with the image optional IC Edit Image Labels ese UserID Br x Living Image Universal Saved Labels LABELS_1 vA a v User V Group v Experiment 87 MG uc2 Intracranial Implantation v Information entered here appears in the image label see Figure 3 11 on page 35 Male nu nu day 8 Mouse 4 V Commenti 5 nel bud Comment2 4 Time Point V Animal Number 4 Animal Strain Animal Model F 4 4 4 4 4 4 4 4 4 4 4 If this is the first image of the session you are prompted to enable the autosave function Figure 3 10 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 Click Yes in the prompt to enable autosave then ch
182. display with each image D mexani Cla D Sequence view 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 169 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 View Default x Configure Load as Group Load Remove Close Location KATHERINE PC Share Caliper LS Caliper Data CerenkovDyCE RKG2
183. dy Window boo ea ca Angle Of View 1JGNE NG Select a Sequence 3D View Plots E DLIT EL20100601160926 sEQ sequences E DLIT EL20100615094528_SEQ E DLIT TLT20050624145507_SEQ Select Analysis Result Results saved with TT a the sequence DLIT 2 selected in the upper box Select the results to display from this list Unit DLIT photons sec cells Display Voxels DLIT Color Scale La v Display Surface E Opacity CJ 50 amp 4 Display Photon Density Map M NOTE After the Longitudinal Study window is open more sequences can be added to the window by clicking the Open button cae and selecting sequenceinfo txt files found in the sequence data folder 2 To show particular results a Select a sequence in the upper box b Select one or more analysis results in the lower box To choose multiple adjacent results press and hold the Shift key while you click the first and last result To choose non adjacent results press and hold the Ctrl key while you click the results c Click Load 3 To show more results repeat step step 2 4 Toremove 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 X Remove and choose Selected Result To remove all results click the Remove button X Remove and choose All Results 5 To view a particular image in a sequence a Click t
184. e The pink mask indicates the CT volume intensity data that is above the threshold The Surface Topography tool will generally provide a surface that very closely resembles a surface shrink wrapped around the pink mask Slide the handle to larger or smaller values until you are satisfied with the selection Figure 10 2 Data selected for surface reconstruction pink Fi B120111027132749_SEQ Sequence View lA 3D View Cronal z 11 0 Sagittal x 0 0 Tool Palette gt ROI Tools Le hyfk amp s B amp B amp 7 gt Spectral Unmixing and DyCE v A r 7 a L maa f Surface Topography Spectrum CT Surface Reconstruction Threshold A 33 5 Subject Nud Mouse x Generate Surface Surface Smoothing Level Low v Save Results Name SURFACE 1 Delete li Load 3D Multi Modality Tools 3D Optical Tools DLIT 3D Reconstruction Transaxial y0 0 Subject Height 26 4mm Perspective 4 To apply surface smoothing optional select a smoothing level low medium or high and enter the number of times to apply the smoothing operation Click the 6 button to remove smoothing and restore the original surface Click Generate a Surface The surface appears in the 3D View See page 217 for more details on the 3D Optical Tools 182 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 10 Reco
185. e 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 112 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 12 Viewing the ROI histogram E7 1L120050624145507 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 jsr 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
186. e the concept tech note Image Display and Measurement for more details on measurement units select Help Tech Notes on the menu bar 89 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 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
187. e ee od 110 Measuring Background corrected Signal 000 eee eee ee eee 110 ROLAM aaa BAEK soa DBA WG WA OOS SHEE eee ea NG NAA 113 Managing ROI Propenies aka na butane eed DA KA news NG GA O84 ee ew 114 Viewing ROI Properties i2 sma mGa ADA LAKAD ud ce betwateaeceaead 114 Moving an ROI zxoaaa saan BAK BADIAN A NINA NAADA ASE Ee eee 117 Editing ROI Dimensions 0 00 cc eee 118 Editing the ROI Line i620066 40 0600 et dove db oan ob tea BALAG eee Oe 120 Move or Edit the ROI Label 0 0200 aaa 121 Living Image 4 3 1 User s Manual IVIS Spectrum CT Contents iii Save Load or Delete ROIS 00 cc eee 121 Managing the ROI Measurements Table 0000 cece eee eee ees 123 Viewing the ROI Measurements Table 0 000 eee eee eee 123 Configuring the ROI Measurements Table 0000 eee eens 125 Copying or Exporting the ROI Measurements Table 126 Chapter 6 3D ROI Tools for Volumetric Data 2 0002 wees 128 About 3D ROIS 7a eta ae bo a owe eG bed KAG ee ee a ae eh eh we 128 Drawing a JD ROL zx aan kaha ha daoedes ee soe Waa BA dee kA hoa 129 ROI Properties ee ees 132 Managing the 3D ROI Measurements Table 000 eee eee eee eee 134 Configuring the 3D ROI Measurements Table 134 Copying or Exporting the ROI Measurements Table 136 Chapter 7 mage Wath a xx ee ee RR ew RRR REE OO OOO Ee OO 137
188. e extracted signal See page 161 for details on analyzing these images Composite of the unmixed images See page 160 for more details 148 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 Tool Palette _ Image Adjust _ Corrections Filtering gt ROI Tools 7 Spectral Unmixing and DyCE Analyze Results Select Images Type Emission 7 HX20120419114703 seQ E23 52 Sequence View Units RadiantEfficency T Use Saved Colors Options z Info BP R NG aa Excitation wavelength Min 1 25e7 Mins 1 4167 Max 1 00e8 92 ax 1 39e8 13 Max 2 7168 Emission wavelengths of the sequence ERARA REKS Methods Automatic v 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 Automatic fr
189. e mouse when the hand appears 2 In the 3D registration tools choose the Display Organs option and select an organ atlas The organs in the selected atlas appear on the surface Only Skin is selected in Figure 11 33 3 Click the Transform tool button The transform tool appears Figure 11 34 explains the tool functions Figure 11 33 Displaying the transform tool Hoo Tool Palette E BI20111027132749 SEQ Sequence View L 3D View gt ROI Tools Fu A na gt ral Unmbang and Qvyk KG Be es Sy ig spect bang and DyCE ZI Surface Topography a gt 3D Multi Modality Tools 3D Optical Tools Surface Source Registration ation Tools Bo v Display Organs dr G v Opacity Organ Atlas Female Dorsal Sagittalfx 2 5 Cronal z 10 7 Organs O adrenal WB bladder bone brain cecum O colon MB eyesExterior Transaxial y 47 7 CAE Use Tab key to switch between transformation toc Scaling On XYZ Subject Height 19 4 mm gt DLIT 3D Reconstruction Gray surface of the imaged mouse Pink skin surface 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 225 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 226 Figure 11 34 Transform tools ni
190. e n4da5044 55 h5006 HERG NG CER WES DES NG PAGA 49 Acquire the Sequence 2 eens 51 Acquire Multiple Sequences in Batch Mode 55 Manually Set Up a Sequence 2 tees 57 Editing Image Parameters 460455 KAPAKANAN CHS ANGARA HSE BAKA 59 Inserting Images in a Sequence 1 a 59 Removing Images From a Sequence 0 eee ee eee 60 Manually Saving Image Data cc teens 61 Exporting ImageS chucdeecea tc eucuscateeedees sian shun Pees ee eeue sees 61 Living Image 4 3 1 User s Manual IVIS Spectrum CT Contents Chapter 4 Chapter 5 Working With Optical Image Data 4 0 aan 62 Loading Optical Image Data aaa aw DA KA KAKA BKA KA NAGA KAKA KG 62 Loading Optical Images From the Living Image Browser 62 Opening Data from the Menu or Toolbar 2 000 cee eee es 66 Organizing Images ees 67 About the Image Window and Tool Palette 2 eee eee eee 68 Image Window Hagaaapaaa aba MA 58584530858 G8 bee GAS ee ee KA 68 OO BALETE oeie AG ae Ade re Gn Pe oS E GA ee GAGA Be 70 Viewing Image Information o i ee ic665444 KAG ded eneeudouke Fae teagan 71 Editing the Image Label 0a adam a ooo 4 Ge bees PLEDGE SS DANG 72 Adding Comments or Tags to an Image ee 73 Adding COMMENTS g2a ccamecatargnenceoene DAG HAN AMANG MGA 73 Tagging an Image L A a e455 ee HAY BALANGA eee Gm ka hun ee HA AB a 74 Adjusting Image Appearance
191. e of organs in the surface page 225 Check the organ fit page 226 Import an organ atlas page 227 You can check the organ fit in the 3D View window page 226 222 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Figure 11 32 3D registration tools and surface with fitted organs skin not displayed E BI20111027132749_SEQ Sequence View LA 3D View Gronal z 10 7 olo ia Tool Palette E L gt ROI Tools Be gt Spectral Unmixing and DyCE la i Surface Topography 4 3D Multi Modality Tools Z 3D Optical Tools Surface Source Registration Registration Tools a a Z Display Organs amp Opacity Ljo9 6 Organ Atlas f Female Dorsal x Sagittal x 2 5 Organs z Z E adrenal 7 bladder v bone v brain v LIN cecum 7 E colon 7 WB eyesExterior Transaxial y 47 7 Subject Height 19 4 mm gt FUT 3D Reconstruction i Table 11 13 3D Registration tools Description TEE Use this tool to manually adjust the scale of location of organs For more details see page 225 Fits the organs to the surface using a linear transformation that keeps the shape of the atlas surface Fits the organs to the surface using linear transformation and volume deformation BET p a ka z After fitting org
192. e page 7 for more details on initializing the system Table A 2 Optical 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 Di Mansi Focu Window O Step Fistop Lg Levet suba Maeght m A MIS Acquisition Control Panel Imaging Mode Exposure Time inning p Excitation Filter Emission Filter Field of View System Status Idle Acquire 13 4 cm Imaging Wizard Subject height 1 50 cm Sequence Setup e subject height Temperature M Locked Initialize To mark the center of the camera in the window put a check mark next to Display CCD Center Select the size of the step increment that the stage moves Coarse Normal or Fine Click Up or Down to move the stage and change the focus O sa 2 IN If necessary select another F stop setting from the drop down list and adjust the light level using the arrows 6 Click Update to apply the settings The resulting focal plane cm above the stage is automatically entered in the Subject height box Living Im
193. ea 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 205 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 the 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 227 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 205 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 224 Manually adjust the location or scal
194. ectra 0 ccc eee tees 179 Chapter 10 Reconstructing a 3D Surface 0 0 0 cee a 181 Generating a Surface aahh AGA KA h5eGeh ewe hh dh bau eiendeesdu bates ts Swed 182 Living Image 4 3 1 User s Manual IVIS Spectrum CT Contents Chapter 11 Chapter 12 Managing SurfaceS xa aaa 6665 ha 54 wen ease eh a KKK GA NG oe Se ko wes 186 Export OF Import a Surface paaa AG KA AN Gaon AN KAMALAYAN AA 187 3D Reconstruction of Sources 2 000 022 an 188 Overview of Reconstructing Sources eee 188 Overview of Workflow for 3D Reconstruction of Sources 190 Reconstructing Luminescent Sources eee ee ee 191 General Considerations 00 aa 191 DLIT Image Sequence Requirements 000 ee eee eee eee eens 191 Steps to Reconstruct Luminescent Sources Using DLIT 192 Including or Excluding Data for 3D Reconstruction 195 Reconstructing Fluorescent Sources 0c cee eee eee eee 198 Image Sequence Requirements cee eee ees 198 Steps to Reconstruct Fluorescent Sources 0 0000 eee es 198 3D Reconstruction Results 62248cedeewebed eee eb eee chan de we Gave en 201 DLIT or FLIT Results gw tw 6 40 kao BIKE mek wa ow Me A Se Bawa 201 Checking the Reconstruction Quality 2 000 cee ee ee 203 Viewing Photon Density Maps or NTF Efficiency 204 Measuring Sources es 205 Determining the Source Ce
195. ectral 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 153 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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
196. ectrum CT Chapter 2 Getting Started 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 Sagu REM a 4 Di Select Add UserID Add New User User ID Password Confirm Password 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 2 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 CT Figure 2 2 For more details on the control panel see Appendix A on page 268 a b c d NOTE The Living Image software has optional password protection for user accounts See page 20 for more details 6 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 2 Getting Started Figure 2 2 Living Image main window and IVIS Acquisition Control Panel Menu bar for more details see Appendix C SAAE
197. ed 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 bhe Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Table 11 12 3D Source tools continued Item Description 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 DLIT or FLIT 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 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
198. ed for reconstruction Keep the minimum counts gt 200 m Region selection see page 196 Use the pencil tool to mark particular regions to include in the reconstruction This may be useful for noisy images with high intensity pixels where changing the Threshold value is not helpful You can also use this method to focus on particular sources to reconstruct and ignore others To change the Threshold for a selected image 1 Click Start in the Analyze tab Figure 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 195 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 196 5 To reset the Threshold 90 to the default value for the selected images click Restore Threshold Figure 11 7 Adjusting the Threshold Data Preview window Tool Palette Sequence 8720111027132749_SEQ Source Firefiy Threshold 10 6 The red outline indicates the image selected for data adjustment Set the Threshold here Note Min Counts translates ma KANA the Threshold to the Adjustment E 10 5 E
199. ed 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 mouse key while you drag the header left or right Release the mouse key to set the new position 65 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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
200. ed 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 or grid shape on the image 100 Free draw Draw line segments that define the ROI 106 103 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 Sequen
201. ee Table 7 1 page 138 2 E 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 Measuring 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 138 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 7 Image Math Table 7 1 Image Math window continued Item Description Color Ra
202. egments 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 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter3 Image Acquisition 59 Table 3 11 Sequence table Item Description Apply to Al Applies the selected cell value to all cells in the same column Remove Remove Selected Deletes the selected row from the sequence table Remove All Removes all rows from the sequence table Updates the selected row in the sequence table with the acquisition parameters in the tf Update control panel asset Inserts a row above the currently selected row using the information from the control panel 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 39 Figure 3 39 Control panel and sequence table n A MIS Acquisition Control Panel mi Mode Exposure Time inning F Stop Excitation Filter Emission Filter BE Display Photographic Settings Subject Mouse Probes z v E j Seg 1 Seg 2 se T cCCHCCU eEA Mode Exposure Binning FStop Excitation Emission FOY Height 1 HR Auto Medium 1 Block 560 C 1
203. eight 19 4mm f Perspective Figure 11 6 3D View toolbar Cao go 2 De Table 11 3 3D View tools Tool Description Image Tools A drop down list of tools for viewing and working with the surface or DLIT results fy or Rotates or spins the surface in the x y or z axis direction h Moves the surface in the x or y axis direction 34 px pg Zooms in or out on the image To zoom in right click Cmd key apple key click for Macintosh users and drag the D toward the bottom of the window To zoom out right click and drag the Ek 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 E AE Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Table 11 3 3D View tools continued Tool Description ka y 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 227 ny Select this tool from the drop down list to display the perspective view e Rotates the 3D reconstruction results in the 3D view window 3D scene Click the or key to increase or decre
204. ence Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data 64 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 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 ti
205. ence 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 151 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 8 Spectral Unmixing 152 Figure 8 13 Principle component analysis E Principle Component Analysis 3 Suggested of components 3 Explained variance 26 of Components 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 158 for information about the resu
206. equence select an image and click Move Up or Move Down M NOTE The Move Up and Move Down buttons are only available when the sequence view window displays images in the default sort order If the TimeStamp or UserID sort order is selected the images cannot be reordered 7 When you are finished editing the sequence click Close The updated image sequence is displayed 4 13 Creating an Image Sequence from Individual Images You can create a sequence from images acquired during different sessions 1 In the Living Image Browser browse for the images of interest See page 62 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 Individual images highlighted blue in this example that may or may not be part of a sequence can be selected for grouping into a new sequence I Living Image Browser secs TLT20050624145507_003 Click Number EX Filter EM Filter Illumination Mode User ID User Group Experiment EQ CK20090729114835 SEQ MB231D3H2LN luc MB231D3H2LN luc MB231D3H2LN luc MB231D3H2LN luc B231D3H2LN luc M TLT20050624145507 560 TE ete TLT20050624145507 580 i TLT FUME TLT20050624145507 600 TLT ji ek TLT20050624145507 620 TLT Hr AA APA AA AA AA ATA AA AA Close Preview Label Set All ba jw Add to List Browse View
207. equence View Unmixing Senra Unmiring nod OVCE ee sisal l Anal ze Results V Show Labels Y Individual Scale NG amp ig sey ani Dad dte V Normalized T Legend a aj TissueAF j 3 v 1 00 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 161 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 8 Spectral Unmixing 162 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 164 DyCE Analysis on page 170 DyCE Results on page 176 9 1
208. equence acquisition see page 48 for more details on sequence setup 10 Enter information about the image in the Edit Image Labels box that appears optional Figure 3 24 Click OK 46 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition l 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 72 for details on adding information to an image after acquisition Figure 3 24 Enter information to include with the image optional Comment2 v Time Point v V Animal Number 4 v Animal Strain x Animal Model v Sex View x Cell Line X Reporter v Treatment v F Luc Injection Time v F IACUC Number X T Edit Image Labels UserID B1 x Living Image Universal Saved Labels LABELS 1 Ab V User kd T Group Information entered here appears in the ma qe aaa image label see Figure 3 26 on page 48 Male nu nu day 8 Mouse 4 J Commenti ECE v If this is the first image of the session you are prompted to enable the autosave function Figure 3 25 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 25 Autosave prompt LC Living Image 4 3 64 b
209. equence 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 CT 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 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 236 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 12 Quantification Database 237 Figure 12 3 Well plate image with grid ROI rc CF EL20090414101005 001 unis aan ole js Options y info ity ie o x0 5 Adjust the grid ROI to closely fit the plate wells 6 I
210. ering 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 Mask 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 Cor
211. export an image for example bmp m Print an image Copy an image to the system clipboard oS SS To open the Image Layout window select View gt Image Layout Window on the menu bar To paste the active image into the Image Layout window click the 2 button To resize the image drag a handle at a corner of the image To reposition the image in the window drag the image 92 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data 93 Figure 4 30 Image Layout window pam TLE image Layout Window DO m z ee JJ O Pa vy amp v X F7 LayoutStyle Freestyle v TE w a mo r Annotation TAN oe Table 4 14 Image Layout window Item Description i Clears the Image Layout window Note If you do not clear the layout click the button before you close the Image Layout window the same window contents are displayed the next time the window is opened Opens a dialog box that enables you to save the Image Layout window contents to a graphic file Pastes 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 Arrow and line drawing tool Select an the item in the Image Layout window To m
212. f 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 Transillumination Choose this option to display the transillumination setup window that enables you to select the locations for image acquisition using bottom illumination that originates beneath the stage 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 perfo
213. first then stores the triangle information in terms of indexes into the vertex list AutoCAD DXF dxf Drawing exchange format that is compatible with most 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 191 Reconstructing Fluorescent Sources on page 198 3D Reconstruction Results on page 201 Checking the Reconstruction Quality on page 203 Measuring Sources on page 205 Viewing Luminescent and Fluorescent Sources in One Surface on page 210 Comparing Reconstruction Results on page 210 Exporting a 3D Scene as DICOM on page 215 3D Tools Overview on page 217 3D Tools Surface on page 218 3D Tools Source on page 220 3D Tools Registration on page 222 3D Animation on page 229 DLIT FLIT Troubleshooting on page 234 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 te
214. frame 4 Time stamp 75 frame occurs 7 5 seconds after the start of animation Key frame 5 Time stamp 100 last frame of the animation Presets A drop down list of predefined animation setups Key frame A 3D view The software interpolates the key frames to create intermediate frames in real time then generates an animated sequence from all of the frames Each successive key frame in a sequence should differ slightly from the preceding one so that motion is smoothly depicted when the frames are shown at a proper frame rate frames second The Living Image software provides preset key frames or you can specify the 3D views for the key frames Preset Key Frame Factor Determines how many key frames are used to generate one revolution in a spinning animation No of frames 4 x Key Frame Factor 1 Increasing the key frame factor reduces the time period between key frames and creates the appearance of finer movement Decreasing the key frame factor increases the time period between key frames and creates the appearance of coarser movement Frames displayed per second in the animation sequence Creates a new key frame from the current 3D view Updates the selected key frame to the current 3D view Deletes a selected or all key frames from the key frame box Moves a 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 an
215. 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 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 IC Imaging Wizard Fluorescence Spec Unmix Filter Scan Open the Filter Section Configuration dialog box IC Filter Selection Configuration Excitation Filters Filter Scan Type Excitation Scan Emission Scan Both Block ND2 430 465 500 535 570 605 640 675 710 745 ND3 Block ND3 ND2 ND3 430 ND3 465 ND3 500 ND3 535 ND3 570 ND3 605 ND3 640 ND3 675 ND3 710 ND3 745 ND3 No540 Block No ND2 No540 430 No540 465 No540 500 No540 535 No540 570 No540 605 No540 640 No540 675 No540 710 No540 745 No540 Noll Block Noll ND2 Noll 430 No11 465 No11 500 Nol1 535 Nol1 570 Nol1 605 Nol1 640 Nol1 675 Nol1 710 Nol1 745 No11 No22 Block No22 ND2 No22 430 No22 465 No22 500 No22 535 N022 570 No22 605 No22 640 No22 675 No22 710 No22 745 No22 No23 Block No23 ND2 No23 430 No23 465 No23 500 No23 535 No23 570 No23 605 No23 640 No23 675 No23 7
216. g a session is named ROI 1 by default A tooltip shows the ROI name when you put the mouse pointer over an ROI 129 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 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 132 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
217. g 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 i MP Piz Click to export the slice view as a graphic file for example bmp 254 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 13 13 Figure 13 13 Volume information gt ROI Tools gt Spectral Unmixing and DyCE is 7 3D Multi ModalityTools Volume Process Slice Results Volume Information Unsaved Key Value StudyDate 20111027 NameOfVolume BI20111027132749 dcm PatientsName DevpPhase Modality OT Manufacturer Caliper Life Sciences BitsAllocated 16 SamplesPerPixel 1 Rows 100 Columns 400 NumberOfFram 400 PixelSpacing 0 300000 0 300000 ImagePositionP 6 0000 0 8400 6 0000 ImageOrientati 1 0000 0 0000 0 0000 0 0000 PatientsBirthDate 20111027 l4 NI
218. ge 264 Figure 13 15 Opening the 3D Volumetric Browser File kat Yaw Tee 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 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 Pi vaid E Beene Dala wil ba levecker 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 16 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 17 The software converts loaded volumetric data into an 8 bit representation to reduce memory overhead and for easier color mapping The 3D Mult
219. ges Item Description Click to select the number of circle ROIs to add to the active image mr 101 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data 102 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 104 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 122 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 intensit
220. gnal is positive Autoscaling 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 157 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 aaa 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
221. gs 279 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 luminescent source spectrum Th
222. hapter 1 Welcome 1 3 Living Image Help 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 anitem Click the k toolbar button then click the item in the Living Image user interface The Living Image Software Press F1 or select Help User Guide on the menu bar and select the User s Manual manual specific for your imaging system Living Image technical notes see Select Help 5 Tech Notes on the menu bar Table 1 2 on page 3 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 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 RO
223. he Image Crop button Figure 4 23 Using a crop box to make measurements Tool Palette a MI merooosos2eass706 8 Ceea ia enn Adit dG Units Counts Display Overlay X Options ifo NG i gt Corrections Filtering 2 Image Information Luminescence Image Image Data 15 counts Crop Distance ROI Tools w A P IF Units cm n Binning 8 Width 12 6 cm Height 12 6 cm Image X Y 12 051 11 831 an 1 62 1 48 Distance 2 19 an 15000 6 81 8 10 10000 Ee Planar Spectral Imaging gt Surface Topography 5000 _ gt 3D Multi Modality Tools DLIT 3D Reconstruction Counts Color Scale Min 1122 Max 19546 2 When the mouse pointer changes to a draw a rectangle on the area of interest 3 To change the size or position of the crop box drag a handle I at a corner or side of the box 4 To delete the crop box from the image click the 777 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 86 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data 87 4 8 Creating a Transi
224. he 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 22 ROI Measurements table 7 ROI Measurements GEA 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 a ccd Pixels pixels pixels pixe HX20070420121444 001 ROI1 Overlay 1 425e 06 4 167e 02 6 726e 02 8 462e 00 2 545e 03 3420 2 189e 05 4 141e 02 1 020e 03 2 87 HX20070420121444 001 ROI2 Overlay 1 489e 06 3 309e 02 6 061e 02 8 642e 00 2 545e 03 4500 2 880e 05 4 168e 02 9 001e 02 2 82 HX20070420121444 001 ROI3 Overlay 1 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 2 825 4 r Customized Selections Measurements Types Image Attri
225. he 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 75 and Magnifying or Panning in the Image Window page 77 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 Measuring Background corrected Signal Draw one or more measurement ROIs on the subject see page 103 for more details 4 Draw an average background ROI on the subject a Select Average
226. he 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 Quantification Plots Copy Select All Export Results Sequence Database WPQUANT_1 w Name WPQUANT_1 X Name Overwrite Overwrite 241 13 3D Multi Modality Tools About the 3D Multi Modality Tools Classifying 3D Volumetric Data on page 243 Volume Display Options on page 246 Smoothing a Volume on page 251 Rendering and Viewing Slices on page 252 Volume Information and Results on page 255 Registering Optical and Volumetric Data on page 256 Volume Data Viewer on page 264 Viewing RAW Volumetric Data on page 265 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 a Refine the appearance of the volume volume processing m Register optical and imported volumetric data for example optical data acquired on the IVIS Spectrum CT with volumetric data not acquired on the IVIS Spectrum CT for example MRI or PET data M NOTE Optical and volumetric CT data acquired on the IVIS Spectrum CT are automatically registered when the data are loaded 3D Multi Modality Tool Requirements The Living
227. he 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 211 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Figure 11 24 DLIT and FLIT results in the Longitudinal Study window Choose an image to display from the selected results Use the thumb wheel to rotate the surfaces Pa la Select a Sequence eya A Longitudinal Study Window DLIT EL20100601160926 SEQ aa DLIT EL20100608105326 SEQ a DLIT EL20100616161949 SEQ DLIT EL20100621094608 SEQ Cl Angle ofvew POOT Ram ay 3DView Plots Wavelength 580 X Remove Select Analysis Result DLIT 1 Ca Unit DLIT Select the units photons sec of the results See Table 11 8 V Display Voxels DLIT V Display Surface Opacity l l Display Photon Density Map fo r more Color Scale detai Is Min 8 98e 5 Max 7 Rainbow Reverse Log Scale MIP For MB details on these display controls see Table 11 8 Voxel color scale Click a surface to select it Table 11 8 Longitudinal Study window Item Description pagkaen DLIT Select photons sec or cells results calibrated using a ks Pa quantification database CJ cells FLIT Select pmole M cm or pmoles results calibrated using a Hing Er abi quantification database
228. hen the mouse pointer is over the image cube 4 To add another component 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 174 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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
229. hold 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 Table 11 6 Reconstruction results Item Description Name The name for the active DLIT or FLIT results Select results from this drop down list Delete Deletes the selected DLIT or FLIT results Load Opens the selected reconstruction results in the 3D View 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 202 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Table 11 6 Reconstruction results continued Item Description Overwrite If you reanalyze saved results sa
230. hy of the subject generated from a CT image m A sequence of two or more images 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 CT 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 CT 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 235 for more details on generating a database Using a quantification database is optional Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Overview of Workflow for 3D Reconstruction of Sources Figure 11 1 Basic 3D reconstruction workflow 1 Set upa DLIT or FLIT sequence using the Imaging Wi
231. i 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 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools Figure 13 16 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 los 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
232. ic 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 273 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 273 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 enables you to save the voxel inf
233. iewer mouse 3 3 20100528 140051 in J RAW State BR Shared Date taken Specify date taken mouse 3 3 20100528 140051 TIF File To view a particular slice move the slider or enter a slice number 20100528 140051 F D m a nk ee rer Hi Starts playback of the DICOM files 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 25 M NOTE Only single raw or vox files consisting of multiple slices of a 3D volume can be loaded into Living Image Figure 13 25 Opening RAW volumetric data di lung d KANONG NN mean Eba ISa Cuar madi NAGA AT AA MUA 511 Pd Sees alo Tool Palette ka go di il Shure Calpa lb ColperDiets b KAY ap 5 D Image Adjust a Crgence 7 Ogan Bum Higa Folder 7 i G HO Took de Cabpar L5 ot Specbral Unmizing and DytE Mice es areponm bai bala a Sarac Topography A MI TI ma ere F3 T a Auto eigen eo dh myt Wimme Process She Reidi di Corg Derme mi al m H E do dme Ma H Aj AM mi Diner Daglay Youre bo emiiri berd Of Oria E pers 9g E d ahime Ghetto Kain F ki as Diii Snot Mag E ARIE SEQ P Legret odra Cate oon MIT 30 Bec bree tacos 265 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13
234. ils part no 134379 3 Select Acquisition gt CT Acquisition gt Generate Alignment data on the menu bar 4 Click Update Alignment Database in the dialog box that appears Figure 3 5 The Y FIT curve blue and Y Intensity curve red should be closely superimposed If not contact Caliper technical support for assistance with instrument alignment see page 4 Figure 3 5 Alignment results plot H 7 Results Plot x Alignment Plot 3000 Target Y Position pixels 25 20 15 10 Angle deg Alignment Plot 745 Target Z Position pixels Angle deg Results Normalized Crop Positions Left 0 05 Top 0 66 Right 0 95 Bottom 0 93 Binning 1 Axis Tilt deg 0 0640443 Center Row 770 Center Column 1635 Update Alignment Database m Y FIT m Y Intensity Z FIT m Z Intensity 29 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter3 Image Acquisition 30 3 2 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 6 on page 31 m Detailed instructions See page 32 See page 48 for information on acquiring a luminescent sequence Living Image 4 3 1 User s Manual IVIS Spectrum CT Quick Guide Acquire a Luminescent Image Chapter 3 Image Acquisition Figure 3 6 Quick Guide
235. imation sequence Play Click to view the animation sequence defined by the current key frames and animation parameters Record Displays a dialog box that enables you to save the current animation to a movie mov mp4 or avi mpg Animation Setup Load Displays a dialog box that enables you to open an animation setup xml Save Displays a dialog box that enables you to save the current key frames and animation parameters to an animation setup xkf 230 Living Image 4 3 1 User s Manual IVIS Spectrum CT Viewing a Preset Animation Chapter 11 3D Reconstruction of Sources 231 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 14 page 230 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
236. included CT mode TIP See the tech note Saturated Pixels In an Image for information on pixel measurements Table 3 10 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 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 53 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Table 3 10 Sequence View window continued 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
237. indow 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 215 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Table 11 9 3D Scene Exporter dialog box Item Description Save DICOM as Single Frame DICOMs Exports multiple files that contain a single frame each Multi Frame DICOM Exports a single file that contains multiple frames Note Choose the Single Frame or Multi Frame DICOM option depending on the third party software you will use to import and view the 3D scene Some applications cannot reconstruct multi frame DICOM files Slice Orientation Choose transaxial coronal or sagittal slices for the export Export voxels using Original resolution Choose this option to export source voxels without any smoothing or binning The original resolution of the source voxels is the resolution obtained after DLIT or FLIT reconstruction approximately 1mm resolution Slice Resolution Sets the number of slices required to accommodate the slice orientation with good slice sampling spacing Total Slices Parameters that determine the number and resolution of the slices to export Slice spacing Pixel spacing Solid mesh If this option is chosen voxels generated inside the hollow mesh are assigned an intensity so that they are d
238. ing Type Epi Illumination Trans Illumination For bioluminescent imaging an open filter image gives the most sensitivity since none of the emission light is being Open Filter filtered This measurement technique is the most Filter Pair commonly used bioluminescent measurement This option selects the best excitation and emission filters for a specific fluorescent probe The fluorescent signal is then detected on the surface of Spectral unmixing Spectral Unmixing the subject ban l Filter Scan Table 3 8 Imaging Wizard bioluminescence imaging options Option Description See Page Open Filter Acquires a luminescent image at maximum sensitivity Spectral Unmixing Acquires an image sequence for analysis using the Spectral Unmixing 142 tools to analyze luminescent or fluorescent images when more than one reporter is used in the same animal model DyCE Acquires a time series of optical images following a bolus injection of 163 radiotracer to enable detection of radiotracer distribution by tracking Cerenkov emission from charged decay products Note DyCE imaging and analysis requires a separate license DLIT Diffuse Light Acquires 191 Imaging Tomography ACT image that is used to segment the surface of the animal a An image sequence for analysis with the DLIT algorithm that reconstructs the position geometry and strength of 3D luminescent sources Table 3 9 Imaging Wizard f
239. ing System 200 Series and IVIS Spectrum Mouse Imaging Shuttle Choose this option if the subject will be contained in the Mouse Imaging Shuttle during image acquisition Load Moves the stage from the cleaning position back to the home position Subject height cm Sets the position of the focal plane of the lens CCD system by adjusting the stage position The subject height is the distance above the stage that you are interested in imaging For example to image a mouse leg joint set the subject height to a few mm To image the uppermost dorsal side of a mouse set the subject height to the 1 5 2 0 cm The default subject height is 1 5 cm 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 Image window so that you can manually adjust the stage position For more details on manual focusing see page 271 Batch Sequences Choose this option if you want to specify multiple separate image sequences for batch acquisition multiple image sequences are automatic
240. ion 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 108 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data Figure 5 10 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 ROI Tools O O Q Q Measure ROIs x Apply TO sequel Type Mirror ROI v V Photo Mask Save ROIs Name ROI 2 KSA sf 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 all 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 RO
241. is 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 280 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 66 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 File Browse 3D Volumetr
242. isplayed 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 The intensity of pixels inside the surface is set to zero so that the exported surface appears as a hollow empty structure Viewing the DICOM Data The 3D scenes exported to DICOM can be viewed in the Living Image 3D Browser 1 Select File Browse 3D Volumetric Data on the menu bar 2 In the dialog box that appears select the DICOM data dcm or dc3 and click Open The 3D Browser window appears Figure 11 29 Living Image 3D Browser A Browse 3D Volumetric Data Col Ha Add to list Browse Name Folder Path Slices Dimension Modality Pixel Spacing Slice Spacing Data Type Type iBEM dcm 20110306103654 C Share Caliper LS Caliper Data 256 128 x128 OT 0 28906310 289063 0 3977 8 bit DICOM dcm 20110306103654 094 dcm a dem 20110306103654 095 dcm dcm 20110306103654 096 dcm dcm 20110306103654 097 dcm dcm 20110306103654 098 dcm dcm 20110306103654 099 dcm dcm 20110306103654 100 dem dcm 20110306103654 101 dcm dcm 20110306103654 102 dcm dcm 20110306103654 103 dcm dem 20110306103654 104 dcm E E StartIndex 1 M Auto Preview M EndIndex 256 ipang a fa 100 Data will be loaded in TLT20050624145507 SEQ Move the slider to select a particular slice for viewing or click an image 216 Living Im
243. it Auto Save xa 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 the image data See page 61 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 26 See Table 3 4 on page 35 for details on the image window 47 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Figure 3 26 Overlay fluorescent image on photograph in the image window ag x a E 7 ALI20110615093703 001 CE we CG ata Tool Palette Units NTF Efficiency Display Overlay X al Fluorescent x on 4 Photograph M 7 Transillumination Location Options v g igi Adjust E Corrections Filtering Image ALI20110615093703 001 User A Wed Jun 15 2011 09 37 41 ba a Lo Image Information Level High Em 800 Ex 745 Trans illumination Bin 2 FOV 13 f2 0 5s 3 eee ROIT Living Image Version 4 2 0 13627 Jun 9 2011 Pi
244. j Info nG igj Options zji Info nG wj Luminescence 10000 8000 6000 4000 2000 Counts Color Scale Min 586 Max 10348 74 Living Image 4 3 1 User s Manual IVIS Spectrum CT To move a tag 1 Position the mouse pointer over the tag 2 When the hand tool appears use a click and drag operation to move the tag then click the mouse to set the tag location 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 Chapter 4 Working With Optical Image Data lt NOTE Not all tools are available for all image display modes Figure 4 14 Tool Palette Image Adjust tools Thu Oct 27 2011 13 29 30 USO KAPE AAP NG mag ete sng NAIA NG TAN BI0111027132749 00 Image BI20111027132749 001 Em Filter Open Ex Filter Block Bin H5 16 FOV 13 5 f1 5s Living Image Version 4 3 0 14942 Oct 27 2011 Camera Everest B2 Andor iKon Ko Ca fa l Tool Palette splay Overy El Luminescentie on eroian optere Game am a a waga E User Group Photo Adjustment Experiment U87 MG uc2 Intracranial Implantation Brightness f 100 Comment Male nu nu day 8 Mouse 4 Spectrum CT RES B 1 5 Animal Number 4 Opacity 100 Color Scale Luminescence in F 44 800
245. l Bi Exposure Time F Excitation Filter Emission Filter medum vje v Standard Wne lol YY Skandard One Mouse Standard Two Mice Fast AG Res System Status X Rays will be produced High Fes T when energized EU Acquire Lo cm Imaging Wizard Subject height 1 50 cm Sequence Setup Field of View C Table 3 1 CT acquisition modes CT Acquisition Voxel Resolution FOV Filter No of Binning Est Dose Total File Mode Size LxWxH Views mGv Time Size um cm sec MB Standard One 150 425 12x12x13 440 Al 720 4 52 8 140 256 Mouse Standard Two Mice 150 425 12x12x13 120 Cu 720 4 23 150 256 Fast 300 850 12x12x13 440 Al 360 4 13 2 90 32 Medium Res 75 225 6x6x3 440 Al 720 2 13 2 210 512 High Res 40 150 2 4x2 4x2 0 120 Cu 1440 1 46 300 360 for imaging the bottom 2 cm of the field of view High Res Top 40 150 2 4x2 4x2 0 120 Cu 1440 1 46 300 360 for imaging the top 2 cm of the field of view 1Times are approximate and include overhead acquisition and reconstruction time m NOTE If you want to check the subjects inside the chamber before acquisition take a photograph uncheck the CT option choose the Photograph option and click Acquire Be sure to select the CT option after taking the photograph 5 Click Acquire when you are ready to capture the image m NOTE If necessary click mageSeup in the control panel to operate in single image mode In single image mo
246. l 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 CT 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 locations Show Advanced Options If this option is selected advanced features are available in the menu ba
247. l 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 CT Hardware Manual part no 133577_Rev A 7 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 2 Getting Started 8 Initializing the IVIS Spectrum CT 1 Start the Living Image software double click the 4 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 E MS Acquisition Control Panel Imaging Mode Exposure Time Binning Ge l Luminescent 1 00 sec 8 AE J Fluorescent _ Transillumination amp Level Hic Auto 2 vis l C CE Red color appears in the control panel during initialization The color p turns blue when System States initialization is finished Acquire i W Service 15 5 Imaging Wizard Ga Emissor amp Excitation filter wheel motors Subject height 1 50 cm Sequence Setup CCD Temperature The IVIS Acquisition Control Panel indicates the temperature st
248. l for a temporal spectrum a Double click an unmixed image to view it in an image window 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 176 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 Sequence View Normalized 1 00 0 80 0 60 0 40 0 Spectrum List Note Select 1 V Name Kidney 2 V Brain 3 V Bladder TC RKG20110210131022 sEQ V Show Labels V Individual Scale kola faa Unmixing oe Gd lx Kidney O o Q E a p 1 o be lt E Mine 0 00 Min 0 00 400 800 1200 Max 2 90e4 Max 1 58e4 Time Point s Bladder Composite eye x Colgr Pick lime wl dii Bf red x Z ff blue X Sr Min 0 00 Max 2 45e4 Close 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 I 7 RKG20110210131022 sEQ aoe e Sequence View Unmixing V Show Labels V Individual
249. ld 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 235 for more details Total pmol Total picomoles of fluorescent probe within the 3D ROI Average pmol Total picomoles Number 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 134 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
250. les 1296 Threshold angle 70 00 Damping Reduction 10000 Median Filter TRUE Uniform Surface Sam TRUE Proton Density Mans M Epot Araits Save Rests Name FLIT 45405 Oetetr Load 11 4 3D Reconstruction Results The Results tab displays information about the photon density voxels and algorithm parameters DLIT or FLIT Results ell NOTE For more details on DLIT see the see the reference article DLIT and FLIT Reconstruction of Sources select Help gt 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 _ ROI Tools _ Spectral Unmixing and DyCE Surface Topography gt 3D Multi Modality Tools gt 3D Optical Tools DLIT 3D Reconstruction Analyze Properties Results DLIT Results DLIT_1 Loaded E Key Value z Final voxel size mm 1 75 Number of voxels 34 E 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 Photon Density Maps a Export Results Save Results Name DLIT_1 Delete toad Overwrite Results name 201 Living Im
251. level is changed from 8 to 16 the new binning is indicated as 8x2 Width Height The FOV dimensions Note If Pixels is selected from the Units drop down list the dimensions are provided in terms of binned pixels Image X Y The x y pixel coordinates of the mouse pointer location in the image Image Data The intensity counts or photons at the pixel location of the mouse pointer Crop Distance 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 85 and 86 80 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 Chapter 4 Working With Optical Image Data The x y coordinates and intensity data are displayed in the Tool Palette Figure 4 18 x y coordinates and intensity data at the mouse pointer location ey KAI ISI ee ke a 7L720050624145507 unis ons Joo Tool Palette Luminescence Counts Co
252. list Click Unmix after deleting a spectrum to a Enter a name in the Results tab of the tool palette Figure 9 13 b Click Save 172 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 9 DyCE Imaging and Analysis 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 Delete Load Results name Manual DyCE Analysis 1 Load a DyCE image sequence Alternatively load DyCE results obtained from an automatic analysis Figure 9 14 M NOTE This section illustrates manual analysis of DyCE results obtained from an automatic analysis Figure 9 14 Load DyCE results e Fi RKG20110210131022_SEQ Sequence View Unmixing Units Counts X F Use Saved Colors cee Tool Palette B E Image Adjust 2 X fo m MM i E ROL Toots Options In Ba ja Spectral Unmixing and DyCE i 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 Min 68 Max 57193 Delete
253. llumination Overview The transillumination overview tool combines the images of a FLIT sequence a fluorescence sequence acquired in transillumination mode into a single image All of the individual fluorescent signals are stacked over one photograph and the intensity is summed One overview is created per filter pair If two filter pairs were used during acquisition then two overview images will be created All transillumination locations are displayed simultaneously a tool tip displays the transillumination position when you mouse over a transillumination point An overview image is displayed 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 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 Puarescent om E hanga 7 ov
254. 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 r una Radiant EfSoency Rapley Oueriay opo il ao gg am image 7 TITJ90402 17513344 Sores Puwescent Background Tests Tue Feb 17 200403 4 57 Egerek 2 kemale Ny My Ti Level regh Gn Cy5 5 Ex CyS Shing Ex Auraton Set Me tabat FOW 12 4 f2 18 Living D Wereon 2 50 2 12 2004 restructured 2 S1620EEV C t ing image Comment Camera MS The Tool Palette includes the Image Adjust tools page 75 Corrections Filter tools page 77 Image Information tools page 71 ROI Tools page 97 See Table 3 4 on page 35 for more details on the image window Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition 38 Acquire a Fluorescent Image With Epi lllumination This section provides detailed instructions for image acquisition M NOTE The IVIS Spectrum CT 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 dete
255. 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 158 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 CT Chapter 8 Spectral Unmixing 146 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 158 for more E See page 161 for details details on analyzing i these images 0 WS mission Wavelength
256. lor Scale Min 1122 Max 19546 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 len Figure 4 19 View a histogram of pixel intensities i Histogram Window Fa mon 112 2 Max Bin 19545 Bins 512 a 4 naag ge g TLT20050624145507_006 Overlay Bins os Jie js 104 Tool Palette gt Image Adjust Image Information a Bae c w os Image Binning amp Width 12 6 cm Height 12 6 cm Image X Y 6 231 6 921 om Image Data 2210 counts 81 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data B 82 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 arrows T 3 To edit the number of bins enter a new value in the Bins box or click the arrows Po M
257. low end of the color scale Red Click the drop down arrow to select a color table for the 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 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 90 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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
258. lts Figure 8 14 Spectral unmixing results Sees E Hx20120419114703_SEQ Cleo e Gi sequence view Unmixing V Show Labels V Individual Scale 3 amp V Normalized Legend IG ol TissueAF o 3 Spectra plot See 100 a page 158 for more Unmixed images details Z show the extracted 0 50 5 signal See page 161 for details aE MI on analyzing these n y MINS g A 00 7 m7 am ar Max 9 83e7 x 2 34e8 Images Emission Wavelength nm AF750 Composite Spectrum List See Table 8 3 on oe eb xX page 159 for details note Composite of the on this toolbar Select Name Color Pick unmixed images 1 V TissueAF min f See page tes for 2 V AF680 red f more detalls 3 V AF750 We AF A Min 0 00 ax 6 4865 Close Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 Sp
259. lume 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 boundary 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 Ma
260. luorescence 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 sequence for analysis with the Spectral Unmixing 142 Filter Scan tools to Extract the signal of one or more fluorophores from the tissue autofluorescence a Determine the optimum excitation and emission filter for a probe Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Table 3 9 Imaging Wizard fluorescence imaging options continued Option Description See Page DyCE Acquires a time series of optical images following a bolus injection of 163 probe radiotracer bioluminescent or fluorescent to track probe biodistribution Note DyCE imaging and analysis requires a separate license FLIT Fluorescence Acquires 198 Imaging Tomography ACT image that is used to segment the surface of the animal a An image sequence for analysis with the FLIT algorithm that reconstructs the position geometry and strength of 3D fluorescent sources 5 Step through the rest of the wizard Fach 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 30 Figure 3 30 Control panel and sequence setup Each row in the sequence table specifies the acquisition parameters for one image in the se
261. luorescent 1 00 CO Manual z 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 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 paa a Text Color B Fluorescent m Text Size 8 Restore Defaults Restore Defaults Cancel Apply Living Image 4 3 1 User s Manual IVIS Spectrum CT Appendix B Preferences Table B 5 Image view preferences Item Description Color
262. m 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 179 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 from the drop down
263. m tool Figure 13 21 Adjusting the air noise boundary in the histogram tool Color Opacity Map Grays v 1 0 Opacity Pa x Reverse Defaultair noise Example noise in boundary the 3D volumetric data 3 28e4 Intensity 3 78e4 Adjust the air noise boundary to reduce noise in the 3D volumetric data Reduced noise 3 Ifthe volumetric data needs cropping for example to remove structures such as the stage from the CT view follow step a to step c below If cropping is not needed proceed to step 4 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 22 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 Regist
264. me of image or sequence acquisition A snapshot can also be captured manually see page 36 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 CT Chapter 4 Working With Optical Image Data Figure 4 3 Image sequences opened loaded Multiple data sets can be loaded at the same time CHAD 100628 141050 SEQ Chek Number EX Filter EM filter Ithuremnation Mode eb CK20100628141050 SEQ CF73O dye in pillows Ceo meena a My 1 sd Yy owe Preview Label Set Al TAY add to bst Gronse Mew Defaut Congre Lox pow Location KATHERINE PC Share Caliper L5 Caliper Data lU87 MG Aac2 OLT 20 27 11812011 10271390508 SEQ Sequencelnfo vet SEQ LE Dee spectra 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 select
265. ment 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 measurement 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 CT 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 138 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
266. n Draw a subject ROI See page 110 for details na Cab a TE Tunasan ass os 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 Radance pisaca ir Right click a measurement ROI and select an average background ROI from the shortcut menu E HATES ON e tel 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 te G1 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 1 5 ber Optons Retate Copy ROI Copy All ROls Dupdicate ROL Hide ROI Tag 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 Typ
267. n the well plate table select the sample cells 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 cells and click the button 8 To apply a color to table cells a Select the table cells and click the ng button Alternatively right click the selected table cells and choose Background Color on the shortcut menu b Choose a color from the color palette that appears Figure 12 4 Select the sample wells and enter the number of cells or molecules TE Well Plate Quantification Window For Sequence EL20090414101005 SEQ Image EL20050414101005 001 Fluorophore Type Dye molecules Cells Measurement Sample Wells 30 3A Background Wells Set Well Volume uL E Apply to Sequence For Image EL20090414101005 001 For Sequence EL20090414101005 SEQ Image EL20090414101005 001 Fluorophore Type Dye molecules gt Cells Measurement Sample Wells 30 34A E Background Wells Set Well Volume uL E Apply to Sequence Well Plate Set position and enter dilution values in nanoMolar units For Image EL20090414101005 001 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 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 1
268. naging 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 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 244 Living Image 4 3 1 User s Manual IVIS Spectrum CT M NOTE The minimum and maximum intensity levels associated with the and O control points Chapter 13 3D Multi Modality Tools cannot be changed The opacity level associated with these points can be changed Figure 13 3 Histogram tool Grays 1 0 Opacity Perspective Color Opacity Map Cd SM W Reverse all TOCCU ioe TTL ee Trill HT TT Each control point specifies a particular opacity intensity color Double click a control poi
269. nerated 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 Managing 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 pa 7 3DAnimation So Preset Animations Presets Spin CW on Y Axis v Frame Factor 1 Animation Setup Time Scale A 0 B Key Frame 2 Key Frame 3 Key Frame 4 Key Frame 5 Play Record Frames Per Second 10 Total Duration secs 5 Ca 7 Living Image 4 3 1 User s Manual IVIS Spectrum CT 3 To add a key frame a saya the position of the recon
270. nformation 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 156for instructions Figure 8 18 Mark the probe locations for unmixing on the image cube Kpa C HX20120419114703_SEQ bol Sequence View Unmixing Ba 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
271. ng Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 8 Spectral Unmixing 145 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 probe locations Figure 8 4 Mark the probe
272. ng the ROI Measurements Table page 123 For information on how to save ROIs see page page 121 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 103 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 8 ROI tools Tool Palette gt Image Adjust gt Corrections Filtering gt Image Information ROI Tools LI YW Measure ROls x Apply to Sequence ype Mirror ROI E Fhoto Mask Save ROIs Name ROI 1 KSA aa a Ca 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 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 illuminati
273. nges 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 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 softw
274. nion fre oe r o7 eas Camera 150702N4088 Andor iKon Commenti 50 ul xen10 injection 1e7 Comment2 FLIT Time Point ojn Animal Number 2 ih Trans fluorescence 1 00 l 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 3 5 Acquire a Sequence Using the Imaging Wizard The Imaging Wizard Figure 3 27 provides a convenient way to set up a sequence for some imaging applications see Table 3 8 and Table 3 9 on page 50 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 57 for details TIP See the Imaging Wizard tech note for a quick guide select Help gt Tech Notes on the menu bar 48 Living Image 4 3 1 User s Manual IVIS Spectrum CT Figure 3 27 Imaging Wizard Takk bha Sport Por imaging biokannawani or Chisi ngaran th ant Arey lucferang ck beste bese men on haters urea NG Ware Set Up a Sequence NOTE The IVIS Spectrum CT should be initialized and the temperature locked before setting up
275. nning 16 Width 13 5 cm Height 13 5 cm Image X Yi 8 774 04m Image Data i counts Image Information Description See Page x y coordinates and The x y pixel coordinates of the mouse pointer location in the image 81 associated intensity and the intensity counts or photons at that location Histogram Histogram of pixel intensities in an image 81 Line profile Plots a line graph of intensity data at each pixel along a user specified 82 horizontal or vertical line in the image 79 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 81 Click this button to display a line profile see page 82 Click this button to display a 3D representation of signal intensity see page 84 Click this button to display the distance measurement cursor in the image window see page 85 Click this button to draw and measure a rectangle on an image see page 86 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
276. nstructing a 3D Surface 183 Figure 10 3 3D view and 3D tools in the toolbar and Tool Palette LA 3D View Sequence View Col os Tool Palette gt ROI Tools T Spectral Unmixing and DyCE 7 Surface Topography Spectrum CT wate Reconstruction Threshold laf 33 5 napo emae Seats Surface Smoothing Gronal z 9 0 Sagittal x 4 0 Level Low v 0 Save Results Name SURFACE 2 gt 3D Multi Modality Tools gt 3D Optical Tools pm gt DLIT 3D Reconstruction Transaxial y 1 0 Subject Height 19 4mm Perspective Figure 10 4 3D View toolbar Ro e NG t Gi 4 amp prg 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 Click and display measurement dimensions in the coronal sagittal or transaxial view in the 3D view window Drag a measurement cursor in the coronal sagittal or transaxial view and display measurement dimensions See page 207 for details on measurement cursors Select eg to zoom in or out on the image use a click and drag operation Select h to move the subject in the window use a click and drag operation Select a to rotate the subject around the x y or z axis use a click and drag operation Click to hide or show the x y z axis display in the 3D view window Click
277. nt to open the color palette 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 245 Living Image 4 3 1 User s Manual IVIS Spectrum CT Saving a Color Opacity Map Chapter 13 3D Multi Modality Tools A color opacity map can be saved and applied to any volumetric data set 1 Click the Save button Gr Figure 13 4 2 In the dialog box that appears select a folder for the file tfn and enter a file name 3 Click Save Loading a Color Opacity Map 1 Click the Open button Ga Figure 13 4 2 In the dialog box that appears navigate to the map file tfn and click Open Figure 13 4 Save or load a color opacity map Tool Palette ROI Tools Co Spectral Unmixing and DyCE _ Surface Topography 3D Multi Modality Tools volume Process Slice Results Ao oR g Povcvecenescscseccccesesecessscoorey Level Of Detail Y Performance Quality Color Opacity Map Loopacty i X 5 E Tree Bi 0 00 Counts z 6 55e4 V Logarithmic Histogram T Ma
278. nter of Mass a tees 206 Measuring Source Depth 0 cc es 207 Viewing Location Coordinates 2 0 00 ccc eee tees 208 Displaying Slices Through a Reconstruction 0 000 cee ees 209 Viewing Luminescent and Fluorescent Sources in One Surface 210 Comparing Reconstruction Results 0 0 00 ees 210 Viewing Results in the Longitudinal Study Window 210 Measuring Intensity aaa 213 MIGWING PIOIS scascaceeacsaceeadadeewed saa cede tae GA MAA are MG a i 213 Exporting a 3D Scene as DICOM 22220 0a 215 Viewing the DICOM Data 0 cc eee 216 3D TOOT OVEIVICW ama neva ede eae Se eee ee eee ow eee en ees 217 3D Tools SOMACe 9a 7208 4c eens eek eee Adee AA 218 SD Tools SOUNCe 2 ma cana eee s sued oes oon ok eee ed oe eee DOH oe eee 220 3D Tools Registration 1 cc es 222 Displaying Organs With the Reconstruction 000 0c eee eee 224 Importing an Organ Atlas 0 0 00 eee 227 SU UAAP ones wees 229 Viewing a Preset Animation 0 000 cee 231 DLIT FLIT Troubleshooting egeuaceidegectesaoergaeeearedeaeeectadaunes 234 Quantification Database 4 4 24 ee es 235 Preparing and Imaging the Samples 0 cee ees 23b Creating a Quantification Database ee es 236 Managing Quantification Results 0 aaa 240 Exporting Quantification Results 0 aaa 241 iv Living Image 4 3 1
279. om 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 149 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 8 Spectral Unmixing 150 Figure 8 10 Auto Unmix window a TE Hx20120419114703_SEQ a 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 unclear 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 151 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 a mask
280. on fluorescent images only a Confirm the purple data mask in the dialog box that appears Figure 5 9 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 10 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data Figure 5 9 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 TIOE 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 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 opt
281. on on page 42 Acquire a Sequence Using the Imaging Wizard on page 48 Acquire Multiple Sequences in Batch Mode on page 55 Manually Set Up a Sequence on page 57 Manually Saving Image Data on page 61 Exporting Images on page 61 3 1 CT Imaging The Living Image software for the IVIS Spectrum CT acquires and reconstructs CT images representing 3D volumes M NOTE The IVIS Spectrum CT should be initialized before setting the imaging parameters in the control panel See page 7 for details Acquiring CT Images 1 Place the subject s in the imaging chamber and close the door 2 Confirm that the IVIS Spectrum CT is ready for X ray acquisition Make sure that the a Key switch on the front of the instrument is turned to the ON position b EMERGENCY Stop switch is in the READY out position If necessary turn the knob clockwise to reset it to the READY out position M NOTE Pushing in the EMERGENCY Stop switch cuts off power to the entire instrument so that all functions stop including the camera and motor driven components c X RAY ARMED button is pushed in The button is illuminated when engaged 3 Put a check mark next to Photograph in the control panel optional Figure 3 1 4 Put a check mark next to CT and select a CT acquisition mode from the drop down list Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter3 Image Acquisition 25 Figure 3 1 Control panel E MS Acquisition Control Pane
282. ons mm Measured 0 00 100 200 Yo Error x Tool Palette sa Log Scale T G gt ROI Tools lel gt Spectral Unmixing and DyCE ka _ Surface Topography a 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 5 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 N Nsurface limits 200 00 200 00 nG Photon Density Maps Tb Export Results Save Results Name DLIT 1 v Delete Load erwrite 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 Figure 11 17 Simulated red and measured blue photon density or NTF Efficiency plots 640 nm wavelength selected A Photon Density Maps photons mm Horizontal Profile m Measured ai Simulated Photon density photons mm 5 0 Position mm Position 8 7 Wavelength 640 Angle of View FEM Ine Log Scale y B3 Vertical Profile m Measured ao Simulated Photon density photons mm
283. oose a location in the dialog box that appears Alternatively click No in the prompt and manually save the image data See page 61 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 The 3D View and Tool Palette appear when acquisition is completed Figure 3 3 M NOTE See Chapter 13 on page 242 for information on viewing and classifying 3D volumetric data using the 3D Multi Modality tools The 3D Multi Modality tools require a separate license 26 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Figure 3 3 CT image i A B120111027132749_SEQ 3D View toolbar k Ea es mee 4s i Subject Height 264mm Perspective Table 3 2 3D View toolbar Item Description Image Tools Ba px A drop down list of tools for viewing and working with the surface or DLIT results fy or b Rotates or spins the surface in the x y or z axis direction h Moves the surface in the x or y axis direction Eg Zooms in or out on the image To zoom in right click Cmd key apple key click for Macintosh users and drag the A toward the bottom of the window To zoom out right click and drag the Ek toward the top of the window
284. or ROIs on page 107 Measuring Background Corrected Signal on page 110 ROI Histogram on page 113 Managing ROI Properties on page 114 Managing the ROI Measurements Table on page 123 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 100 for optical data optical image grid or contour Quick Guide 103 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 CT 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 107 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 the average signal intensity in a Circle or square 110 user specified area of an optical image that is considered background Note Using this type of ROI is optional If
285. ore 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 278 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 3 button to restore the defaults for the selected color theme DP eto1i1027132749_ seq S fa O Sequence View Z JD Ver O Ng w TEE Spe a Mi hee is 4 3 e Or ki ee ah ra i NA i Background Color Settings that modify the appearance of the background in the 3D View window Solid Color Choose this option to apply a non gradient background color to the 3D view in the image window Gradient Color Choose this option to apply a gradient background color to the 3D view in the image window Top the color at the top of the window Bottom the color at the bottom of the window Surface amp Text Color Settings that modify the display of the surface and text in the 3D View window Color Palette Source voxels Choose a color table for voxel display Reverse Choose this option to reverse the min max colors of the selected color table Restore Defaults Click to apply the default settin
286. ormation 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 CT 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 11 on page 73 Edit Preferences Opens the Preferences box see page 273 View Tool Bar Choose this option to display the toolbar View Status Bar Choose this option to
287. ossocsssoseslsoosssssssossssesooessssessssssosesosesiosssssesesssesssessssesosesossessssssosssosssoseee SecasereccccccceccccscccccccsccesevascceccccesceccecsccesdscnccccucccceccsccccsccecheassececccsscccnsescceceuebecscecceceecsccsccesscceseuasccecocceseecccelecesesescceccncecccceccsDeccsvanseeuscceseccnsesccccescccesccccwecceesscesceceseusecascceccccssescscescsecosesceeal p O O E O O m Hide Browse View Close Preview Label Set All nA le Add to List Browse View Default M Configure Load as Group Load Remove Close Location C Share Caliper LS Caliper Data Sample Data IVIS200 data TraserBeadsPC TLT 20050624145507_SEQ SequenceInfo txt Snapshots of an image sequence The Tool Palette appears when you open an image or sequence The options available in the Tool Palette depend on the type of active image data A tool is only available if the data set includes the components that the tool requires to perform the analysis Figure 4 7 Tool palette See page 13 for an overview of the Living Image tools Tool Palette Tool Palette Image Adjust E Click to expand a tool a a mAg a E Ba Photo Adjustment Brightness lef 100 Contrast lel 1 5 Opacity 100 Color Scale Min 2140 Max 36446 Color Scale Limits Auto s Individual Reverse C Corrections Fiterng Image Information
288. ove the item to the front or et back in the window choose an option from the drop down list 4 Bring forward p pn p Y Send backward WY send to back Living Image 4 3 1 User s Manual IVIS Spectrum CT Table 4 14 Image Layout window continued Chapter 4 Working With Optical Image Data Item Description Ng Deletes the selected image Layout Style Layout ene Pol 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 fmatahon To apply notes to an image enter text in the annotation box and press Enter Drag Jila i the 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 62 for more details on browsing 3 In the image window click the Edit but
289. photomw sec 0 0 1 2 Results VA 0 First surface 1 Second surface 2 Third surface and so on p hotons sec DLIT Results 214 Living Image 4 3 1 User s Manual IVIS Spectrum CT 11 9 Exporting a 3D Scene as DICO Chapter 11 3D Reconstruction of Sources 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 Figure 11 27 3D scene De eimina 580 CI Sequence ew he O Wee To export the 3D scene 1 Load the results that you want to export 2 Select File Export 3D Scene as DICOM on the menu bar 3 In the dialog box that appears set the export options and click Export For more details on the 3D Scene Exporter see Table 11 9 Figure 11 28 3D Scene Exporter dialog box E 3D Scene Exporter Save DICOM as Slice Orientation Export voxels using original resolution Parameters Slice Resolution Low Total slices 256 Slice spacing mm 0 3977 2 9 MB Solid mesh Approx size Pixel spacing mm 0 2891 Hollow mesh Transaxial Slice P 88 Single Frame DICOMs ka ka ha Export OK In the Browse For Folder dialog box that appears choose a folder for the DICOM files and click During the export operation the 3D View w
290. pmol M 2 cm t CJ pmol cells Red ka Reverse Log Scale V MIP 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 Reverse is chosen the source intensity photons sec from low to high is represented using the color scale from red to transparent Log Scale Applies a log scale to the color scale MIP When this option is chosen all maximum intensity voxels in the view are projected along the viewing direction into the viewing plane 212 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 213 Table 11 8 Longitudinal Study window Item Description Copies the 3D View tab in the Longitudinal Study window to the system clipboard BE Opens a dialog box that enables you to export the 3D View tab to a 7 graphic file for example png no Enables you to select voxels for measurement Measurements are displayed in the Plots tab Measuring Intensity 1 Load 3D reconstruction results and
291. pply To Sequence If this is the first image of the session you are prompted to enable the autosave function Figure 3 10 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 10 Autosave prompt Fa E Living Image 4 3 64 bit Auto Save E3 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 61 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 11 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Figure 3 11 Overlay luminescent image on photograph in the image window SARAS MA Pine cons e Apply ma ag oa So M11 1027 32749 00 ane nts Counts Depley Overton v E uummescent on ff rhotogrash Opson
292. pter 4 Working With Optical Image Data Figure 4 26 Image Overlay window G Image Overlay Window Sequence ARW20050826124002_SEQ Photograph Units Radiant Efficiency vv ARW20050826124002_001 Fluorescent Images ARW20050826124002 001 F ARW20050826124002 002 rte 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 ARW20050825124002 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 Se
293. quantum dot nanocrystals 700 or 800 nm File Edit View Tools Acquisition Window Help o amp aa R Units Counts Apply to all HX 20070420121444_SEO gt Image Adjust ey Sequence View 4 gt ROI Tools p its Counts w Use Saved Colors gt Spectral Unmixing Units Kapa na ua n CSE Ekg EER SILL fon PS ETRE Fy Mig A565 ak3 31126 ns 694 Ng gt P ED ESPERE LUO S 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 E Hx20070420121444 EQ C Sequence View Spectra Cokrire View E a m Colormap NIR Color Range Fier Range Mm ng amp 91 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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
294. quence See page 58 for details on the sequence table Ka A WIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter F Display Photographic Settings Subject Mouse piace 5 B Seg 1 Mode Exposure Binning FStop Excitation Emission FOY Height 1 WI Auto Medium 1 Block 560 C 150 a 2 EB Auto Medium 1 Block 580 C 1 50 m 3 I uto Medium 1 Block 600 C 150 4 Auto Medium 1 Block 620 C 150 Field of View System Status X Rays will be produced tm when energized Bt Auto Medium 1 Block 640 C 1 150 Idle Acquire 13 4 cm Imaging Wizard Subject height 1 50 cm Image Setup Focus use subject height v Temperature M Locked Initialize Number of Segments 1 gt Delay 0 0 H min Apply to All Add 6 To clear the sequence click the Remove button and select All Acquire the Sequence 1 Confirm that the IVIS Spectrum CT is initialized and the CCD temperature is locked See page 7 for details 2 Click Acquire Sequence in the control panel when ready to begin acquisition 3 Enter information about the image in the Edit Image Labels box that appears optional Click OK Figure 3 31 M 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 51 Living Image 4 3 1 User s Manual IVIS
295. r 4 Working With Optical Image Data Item Description FS Opens the Print dialog box X Min Displays the minimum and maximum value of the x axis Use the arrows to X Max change the x axis min or max If 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 Displays the minimum and maximum value of the y axis Use the arrows to Y Max change the y axis min or max To display the range available for the Y Min or Y Max place the mouse pointer over the Min or Max edit box Click to reset the X and Y Min and Max values to the defaults Full Scale Select this option to display the full X and Y axis scales Logarithmic Scale Viewing 3D Signal Intensity Select this option to apply a log scale to the y axis 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 Figure 4 21 3D intensity signal g 3D Plot Window Perspective For Click TLT20050624145507 005 Plot Full Image Z Max 240 2 9 B 2 Refresh C fa 2000 0 Max 10348 2 Tochange the display make a selection from the Plo
296. r 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 drag it a distance greater than its width To dock the Tool Palette in the main window drag the palette to the
297. r in appearance to an X ray image This enables you to view overlays that include the X ray image for example a luminescent image on X ray Figure 13 8 To view a 2D projected image 1 Load a DLIT or FLIT sequence 2 Click the Rk button in the 3D Multi Modality tools Figure 13 8 3 Double click an image in the sequence and select the types of image to overlay Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools Figure 13 8 Viewing a 2D projected image Click to extract a 2D projected Tool Palette Image Adjust BI20111027132749 sEQ Ga Corrections Filtering ROI Tools _ Spectral Unmixing and DyCE _ Surface Topography 3D Multi Modality Tools Volume Process iF Slice Results fo Rt net v Display Volume Level Of Detail G Performance Color Opacity Map ai Sequence View m73 3D View Units Counts gt E Use Saved Colors Options Info Min 63 Min 63 Max 817 92 Max 191 Max 296 250 image of the volumetric data 1 0 Opacity X o WW Reverse A Min 63 Max 258 95 Min 38 Max 469 b Eee ee eee eee BEER eRe Ree ee 0 00 Counts 6 55e4 Double click an image in the sequence V Logarithmic Histogram Maximum Intensity Projection MIP Gradient Illumination Le DLIT 3D Reconstruc
298. r 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 256 or m Manual registration Match animal surface representations using the Manual Registration tool page 261 4 Classify the 3D volumetric data to help identify and separate objects page 243 Save the color opacity map optional 5 Save the registered 3D multi modality results page 255 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools 258 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 242 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 15 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 pa
299. r the ROI When the pointer becomes a click the ROI b Place the mouse pointer over an ROI handle so that it becomes an 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 117 and Editing ROI Dimensions page 118 5 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 123 For information on how to save ROIs see page page 121 Drawing ROIs Using the Free Draw Method 1 Open an image and in the ROI tools select the type of ROI that you want to draw from the Type drop down list 2 Click an ROI shape button Circle amp _ Square O or Contour 8 and select Free Draw from the drop down list In this example the Contour shape was selected for the free draw method The ROI shapes that are available depend on the type of ROI selected 3 If you selected 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 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 5 ROI Tools for Optical Data 107 4 Click the Measure button Wf MeasueROls The ROI measurements and table appear For more details on the table see Managi
300. ral animal background luminescence and should not be confused with the dark charge and read bias background corrections that are applied by default to the raw CCD data to remove electronic noise before any measurements For more details see page 110 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 100 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 ROl s on the image To do 104 this the software locates the peak pixel intensities in the image and searches the neighborhood around a peak pixel A pixel is includ
301. ration button JA The transformation tool appears Figure 13 23 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 263 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools 264 Figure 13 23 Manual registration tool transformation modes ive Tab here Bi a a AT Use Tab hey ko rtdh babaman trarakarmabon toot Use Tab hey to switch betmeen transformation tools Use X Yor 2 hanes 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 260 M NOTE
302. rd Delete User ae Security 3 Select a User ID enter and confirm a new password and click Submit Deleting Users Chapter 2 Getting Started 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 for 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 6 User Settings 8 adduser By change Password 2 delete user Gi Security Ceia Delete User User ID HLA x Enter master password Enter Master Password Clear Deletes all entries 3 Select a User ID 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 21 Living Image 4 3 1 User s Manual IVIS Spectrum CT To lock user accounts 1 Select Edit User settings on the menu bar Chapter 2 Getting Started 2 Click the Security tab in the dialog box that appears 3 Click Lock User Accounts Figure 2 11 User Settings Security fe Add User 2 Change Password 2 Delete user Searrity
303. rease the number of slices in the viewer or a larger value to decrease the number of slices in the viewer Total Slices The 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 Min 15 00 mm Max 15 00 mm Min The slice coordinate of the first slice being viewed Zero is defined as the center plane of the image Max The slice coordinate of the last slice bein
304. rection 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 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 78 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data 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 Figure 4 17 Tool Palette Image Information tools Tool Palette ABC BB units om Image Bi
305. rgans on the surface import an organ atlas Reverse Y Log Scale DLIT 3D Reconstruction 191 Diffuse light tomography DLIT analysis provides Sequence Al 20 100609165247 SEQ es a a complete 3D reconstruction of the luminescent source distribution within the subject The 3D Threshold reconstruction is presented as volume elements 3 7 called voxels 2 9 0 6 If a luminescent calibration database is available the number of cells per source can be determined 0 6 in addition to source intensity photons sec 16 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 2 Getting Started Table 2 2 Living Image tools available for data acquired on the IVIS Spectrum CT continued Gamot a apan CAST LIBAOGGL LINING NG Pt zepetecn cee NG BB Cur nmana aQ Living Image Tools and Functions See Page FLIT 3D Reconstruction 198 Fluorescent imaging tomography FLIT analysis Sequence CXAOD90 229114835 S50 i 1 nang alee akan provides a complete 3D reconstruction of the Select Image Sources fluorescent source distribution within the subject EmWL Threshold The 3D reconstruction is presented as volume 6 800 51 elements called voxels 800 51 l E a00 51 If a fluorescent calibration database is available i B00 5 0 the number of fluorophore molecules or cells per Mk 800 51 source can be determined in addition to the total 5 ALINA fluorescence yield
306. rmed Overlay If this option is chosen the system automatically displays the overlay after acquisition is completed for example luminescent image on photograph Lights Turns on the lights located at the top of the imaging chamber Living Image 4 3 1 User s Manual IVIS Spectrum CT Appendix A IVIS Acquisition Control Panel 270 Table A 1 IVIS acquisition control panel continued Item Description 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 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 Only available on the IVIS Imag
307. rmines the binning and F Stop settings TIP See the tech note Auto Exposure for helpful information select Help s Tech Notes on the menu bar Alternatively manually set the exposure binning and F Stop See Table A 1 on page 268 for details on these parameters Figure 3 13 Control panel IC MS Acquisition Control Panel Imaging Mode Exposure Time inning FiStop Excitation Filter Emission Filter Field of Yiew System Status 13 4 cm Subject height 1 50 cm Focus use subject height w Temperature fF 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 14 Living Image 4 3 1 User s Manual IVIS Spectrum CT Figure 3 14 IVIS Spectrum excitation and emission filters Transmission Transmission 500 Excitation Filters 560 600 640 680 720 760 800 Wavelength nm Emission Filters 550 600 750 800 Wavelength nm 3 Select a Field of View size of the stage area to be imaged Chapter 3 Image Acquisition 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 5 Field of View FOV settings on the IVIS Spectrum
308. s gt a Image 8120121027132749 user Thu Oct 27 2011 13 29 20 Group Em Filter Open Ex Fiter Ekock Bins MS 16 FOV 13 5 1 Se R Living Image version 4 3 0 14942 Oct 27 2011 oe or Nidan ago CN KA Cane a Everest B2 Andor Kon amment kp mum day 3 Mouse 4 Spectrum CT TIP See the tech note Determine Saturation for information on pixel measurements select Help Tech Notes on the menu bar Table 3 4 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 Note If the acquisition included more than two imaging modes for example luminescent x ray and photograph additional drop down lists appear so you can conveniently choose any two images to overlay Display Overlay C Luminescent oon KA Photograph 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 11 and other image information automatically recorded by the software ig Opens a dialog box that enables you to export the active view as
309. s Y 4 HI Excitation Filters 400 500 600 700 800 900 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 5 165 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 9 DyCE Imaging and Analysis Figure 9 5 Imaging Wizard Bioluminescence DyCE Fluorescence DyCE Ka A Imaging Wizard Bioluminescence DyCE Imaging Subject Mouse T Exposure Parameters Auto Settings Manual Settings C Imaging Wizard Fluorescence DyCE Imaging Subject Mouse v Exposure Parameters Auto Settings Manual Settings Standard One Mouse A Total number of Intervals 3 Exposure Binning F Stop Luminescent 1 00 sec x 8 x la v m Fluorescent p Photograph Auto sec 2 v is z ila Photograph min Gi Standard One Mouse i i CT Field of view Field of View C 13 4cm X C 13 4cm X Focus Focus Subject Height 1 50 2 cm Focus use subject height w Subject Height 1 50 3 cm Options Options Time Series Time Series Total number of Intervals 3 Focus use subject height Y a Interval Duration min Delay sec Max Images Interval Duration min Delay sec Max Images 2 5 A5 IG 2 5 45 6 3 3 5 24 3 2 SE 24 iga Back Next
310. s 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 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 Edit View Tools Acquisition Window Help SARU LVR EOSS Time Binning F Stop sec v medum vfa auto Medium fs Field of View System Status Ba Jen Ineson er Subject height 1 50 cm Sequence Setup Focus use subject height v Temperature M Locked Initialize i 5 Type Description 5 D Information Living Image 4 3 0 15210 64 bit RC Dec 13 2011 13 13 19 z DD Information gt gt KS Logged IN lt lt Activity Wi n dow Information MIS configuration file found and loaded Activity Window Eh x Information Reading user preferences 4 User KS 3 Image Acquisition CT Imaging Luminescent Imaging on page 30 Fluorescent Imaging With Epi Illumination on page 36 Fluorescent Imaging With Transilluminati
311. s of type Import Organ Atlas Organ Files Select Skin Mesh skin iv ka Generate Mesh Co efficients uterus iw 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 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 is created and is added to the Organ Atlas drop down list in the 3D tools Registration tab 228 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 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 View a preset animation generated from a factory loaded animation setup page 231 Create a custom animation created from yo
312. s 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 CT Figure 5 16 ROI Properties dialog box ROI Label Shape Type Manual Background ROI Subject ROL Use as BKG for future ROIs in TLT20050524145507 005 only Entire sequence E Lock Position Xc pix 117 00218 Yel pix 37 93839 Angle deg 0 0000 E Lock Size Width pix 22 06161 Height pix 20 43172 Line Size J Line Color Pag noo Done Position of the ROI selected in the image Chapter 5 ROI Tools for Optical Data 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 a Edit the settings in the ROI Properties box M NOTE You cannot change the size of an ROI that was created using the auto ROI or free draw tool To resize an ROI using a handle
313. s 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 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data 69 Table 4 3 Image window continued Item Description Options Layout Choose a display option for the images 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 gt 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 Coun 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
314. s 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 5 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 282 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 background acquisition A
315. sa Tabi kay bo pith eee trani ormat hii Libi TERET kary ibi Saidh babash Ir arah anmiana bar Lee Tab kry bo skih babaan ameiona bo Sealing Ore XYT Click and drag the organ s Click and drag a handle to scale To rotate the organ s on the x y when the yellow appears increase or decrease the size or z axis click the blue green or of the organ s red circle and drag the mouse Red W Scales on the z axis arrow in the direction of interest Blue W Scales on the x axis Green W Scales on the y axis 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 CT BI20111027132749 sEQ e Es Sequence View b 3D View ey 8 Bey Y Y 2 Ey ig Sagittal x 2 5 Transaxial 47 7 Subject Height 19 4 mm Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 227 3 Press the V key or the ia button to display alternative views of the surface Figure 11 36 Alternate views of the s
316. sc Mode Exposure Binning FStop Excitation Emission FOV Height 1 Pg Auto Medium 1 Block 560 C 150 TATTO 2 NG Auto Medium 1 Block 580 C 150 m 3 ES Auto Medium 1 Block 600 C 1 50 4 Auto Medium 1 Block 620 C 1 50 Field of view System Status X Rays will be produced Hi when energized s BI Auto Medium 1 Block 640 C 150 Idle Acquire 13 4 cm Imaging Wizard Subject height 1 50 cm Image Setup Focus use subject height v Temperature M Locked Initialize Number of Segments i Delay 0 0 min Apply to All Add Table 3 11 Sequence table Item Description Starts the Imaging Wizard Pg Displays a dialog box that enables you to select and open a sequence setup xsq La 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 l If a subject and probe are specified optional the software uses the information to aa 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 s
317. se 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 120 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 at the new location Figure 5 19 Figure 5 19 Move or edit the ROI label EF
318. section explains how to acquire a single fluorescent optical image with transillumination See page 48 for information on acquiring a fluorescent sequence To acquire a fluorescent image with transillumination NOTE Use only the Single Mouse Anesthesia Manifold when imaging with transillumination The Dual Mouse or Five Mouse manifolds cannot be used with transillumination lt 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 20 Control panel ia MS Acquisition Control Panel posure Time Excitation Filter Emission Filter auto see v medun Je 7 Setup maam vje v Standard One Mol Field of View System Status mil Acquire 13 4 cm Imaging Wizard Subject height 1 50 cm w Sequence Setup Focus use subject height Temperature I Lecked i ntilie O Oe 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 21 43 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter3 Image Acquisition 44 Figure 3 21 IVIS Spectrum excitation and emission filters Excitation Filters Transmission 96 Wavelength nm Emission Fil
319. shairs vertical line The x y positions are relative to the center of the FOV where x O andy 0 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 l 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 210 205 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 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 128 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 Po 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 _ 8 E B120111027132749_SEQ le BAN Tools
320. shold specifies the minimum percent of peak pixel intensity that a pixel must have to be included in an ROI identified by the software After ROIs are drawn on an image if you modify the Threshold move the slider or enter a new value the software automatically updates the ROls Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 natu
321. sition 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 Sagittalfx 2 5 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Displaying Slices Through a Reconstruction 1 Click a location on a source Alternatively click the Kon toolbar button draw a box around a source then click Center of mass in the 3D Source tools 2 Click the gff toolbar button The Coronal Sagittal and Transaxial windowpanes show a slice through the surface taken by the associated plane Figure 11 21 Planes cutting a reconstruction Tool Palette O B120111027132749_SEQ koko 2 BAL Tools o F sequence View L 3D View Spectral Unmixing and DyCE Do fz a 8 ty G 7 ge g pa NG La Surface Topography an Cc 3D Multi Modality Tools ee 730 Optical Tools Surface Source Registration Select Source Original v Display Source Surface y G V Display Voxels Maximum Intensity Projection Threshold 0 0034 Gradation Jal 50 Voxel Size 0 31 v Display Voxels As Smoothing 5x5 s vs Texture z Color Scale Min 379 6641 7 Color Table i BlueYellow X T Transaxial y 47 7 paa Reverse a Log Scale Measured Sources Key Value Quantification
322. struction in the 3D view using an image tool for example 47 or For more details on the image tools see page 194 b Click the _ button c To reorder a key frame in the sequence select the key frame and click the or 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 Y button and select Delete Current 11 15 DLIT FLIT Troubleshooting Chapter 11 3D Reconstruction of Sources 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 Metallic objects can scatter the x rays resulting in noisy CT images To avoid the scattered x ray paths which cause spikes adjust the threshold that specifies the CT volume data used for surface reconstruction Tool Palette Spectrum CT Surface Reconstruction Threshold Subject Nude Mouse Surface Smoothing Level O Restore Save Results Name SURFACE 1 gt 3D Multi Modality Tools 3D Optical Tools gt DLIT 3D Reconstruction Bad Photon The optical properties or source spectrum may have been incorrectly chosen For Density of NTF example Mouse Tissue optical property is appropriate or mice but XPM 2 XFM 2 is Efficien
323. t drop down list and click the Refresh button E Refresh 84 Living Image 4 3 1 User s Manual IVIS Spectrum CT Table 4 9 3D Plot window Chapter 4 Working With Optical Image Data Item Description Plot Full Image Displays all signals in the image ROI lt ROI number or names 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 Copies the 3D window to the system clipboard 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 ____ 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 Tr TE TLI20050624145507 006 Units Counts v Display Overlay 8G Tool Palette gt Image Adjust a ernie tty Options Info iy ie pre T rw RAR la 8 pial H units m Image Binning 8 Width 12 6 cm Height 12 6 cm Image X Y 6 628 11 05
324. t option choose the Photograph 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 SS eee aaa aS SSS i NOTE If necessary click mage 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 48 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 72 for details on adding information to an image after acquisition 33 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter3 Image Acquisition 34 Figure 3 9 Enter information to include with the image optional TE Edit Image Labels a UserID B v Living Image Universal Saved Labels LABELS 1 AER V User v 7 croup 7 Information entered here appears in the Bi cso Saan image label see Figure 3 11 on page 35 Male nu nu day 8 Mouse 4 V Commenti SPEE v E Comment2 x Time Point bad V Animal Number 4 E Animal Strain v Animal Model ba Sex kd F View v M Cell Line 7 7 Reporter hd C Treatment v E Luc Injection Time v F IACUC Number be A
325. ta 265 267 reconstruct 3D fluorescent sources 198 201 reconstruct 3D luminescent sources 191 197 Living Image 4 3 1 User s Manual IVIS Spectrum CT reconstruct particular pixels 196 reduced Chi2 202 reference images 28 registering multi modal data fiducial registration 256 loading data 258 260 manual registration 261 264 ROI 97 128 automatically draw 104 105 background corrected signal 110 112 delete 122 edit dimensions 118 free draw 106 histogram 113 managing 114 manually draw 106 measurement ROI free draw 106 measurement ROIs 103 105 Measurements table 101 mirror 107 move 117 move or edit label 121 quick guide 100 ROI line 120 save 121 subject 110 tools 101 102 ROI Measurements table 123 124 configure 125 126 134 136 copy or export 126 136 ROI properties 114 117 ROI types average background 110 S save ROI 121 segment 58 sequence edit 59 60 sequence requirements DLIT 191 FLIT 198 spectral unmixing 142 slices rendering 252 viewing 253 254 smoothing 78 source depth 207 spectral imaging See planar spectral imaging spectral unmixing 142 162 sequence requirements 142 stage alignment 28 29 Index 287 subject ROI 110 surface export or import 187 generate 182 manage 186 T tag an image 74 Tech Notes 3 4 technical support 4 tile images 67 tissue autofluorescence eliminate by spectral unmixing 142 162 subtracting with background filters 139 141 tool palette 70 3D tools 217 234 overview 13
326. ta Medium e e he He C cy oa 03 Auto Medium Ses E Number of Segments i Delay 0 0 HI min Apply to All X Remove A Update Insert Add 5 To remove a sequence click the sequence tab and then click the E button 6 Click Acquire Sequence when you are ready to capture the sequences Image acquisition proceeds with no intervening time delay between sequences The upper area of the control panel changes to red color during acquisition The control panel returns to blue color when acquisition is finished NOTE If the check mark is removed next to the Batch Sequences option in the control panel Figure 3 34 only the sequence in the active tab will be acquired To save the batch sequence setup 1 Click the Save button m 2 Enter a file name xsq and choose a location for the file in the dialog box that appears 56 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter3 Image Acquisition 57 3 7 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 51 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 seq
327. te of the unmixed images Figure 9 12 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 TC Normalized E Legend a ret Kidney 1 00 A a Pa lo Temporal spectra 080 show signal time course of different 0 60 West anatomical regions 0 40 Min 0 00 400 800 1200 Max 2 90e4 Time Point s Bladder Spectrum List pv X Note Select Name Color Pick 1 V Kidney Mime br 2 V Brain mig 3 V Bladder BB O ze Min 0 00 Max 2 45e4 Unmixed images Each image is a representation of a temporal spectrum at the peak signal time point Min 0 00 Max 1 58e4 Composite Composite of the unmixed images 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 179 Adds a temporal component to the spectrum list when performing a manual analysis See page 173 for more details on manual analysis view updated DyCE results 9 To save the results Deletes the last component in the spectrum
328. ted above the stage m Quick guide See Figure 3 12 on page 37 m Detailed instructions See page 32 See page 48 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 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Quick Guide Acquire a Fluorescent Image With Epi Illumination Figure 3 12 Quick Guide Acquire a fluorescent image with epi illumination 1 Start the Living Image software F and initialize the IVIS Spectrum CT page 5 Note See the V S Spectrum CT Hardware Manual part no 133577 Rev 00 for more information on the instrument 2 Place the anesthetized subjects in the imaging chamber and close the door CC ms Acquisition Control Panel Imaging Mode Exposure Time Binning Fi Stop Excitation Filter Emission Filter E meim je E Field of view System Status _ Acquire Idle 13 4 cm Imaging Wizard Subject height 1 50 cm amp Sequence Setup Focus use subject height Temperature I cocker 3 Puta check mark next to Fluorescent and select Auto exposure 4 Select an excitation and emission filter 5 Choose Photograph and Overlay 6 Select Use subject height and enter the height in centimeters 7 Click Acquire 8 When prompted select a
329. ters 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 22 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Figure 3 22 Open the Transillumination Setup dialog box A WIS Acquisition Control Panel Imaging Mode a a Cu Standard One Mol a Field of View C Service 13 4 cm Subject height 1 50 gt cm Focus use subject height v Exposure Time Binning auto sex o medun o Idle Temperature fC Locked Single location mode acquires one image at the location marked by a green square J bos Emission Filter Excitation Filter j Transillumination Setup Single Location Mode FiStop C Move Motors To Selected Spot C Mask Grid Points To Subject C Raster Scan Seis z Grid Type 15x23 System Status Table 3 6 Transillumination Setup box Item Description Move Motors to Selected Spot Transillumination motors will move the excitation light source to the grid location selected in the Transillumination Setup dialog box Mask Grid points To Subject When setting up a transillumination
330. 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 61 4 Working With Optical Image Data Loading Optical Image Data About the Image Window and Tool Palette on page 68 Viewing Image Information on page 71 Adding Comments or Tags to an Image on page 73 Adjusting Image Appearance on page 75 Correcting Optical Image Data on page 77 Viewing Intensity Data and Making Measurements on page 79 Creating a Transillumination Overview on page 87 Overlaying Multiple Images on page 88 Rendering Intensity Data in Color on page 91 Exporting or Printing Images on page 92 Editing an Image Sequence on page 94 Creating an Image Sequence from Individual Images on page 95 4 1 Loading Optical Image Data You can load open optical images from the m Living Image Browser see below Toolbar or menu bar page 66 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
331. 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 67 Window Tile Organizes the open image windows in a tiled arrangement see page 67 283 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 window in the list to make
332. the 3D ROI 132 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 6 3D ROI Tools for Volumetric Data 133 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 Subject 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 3D ROI Properties o For Sequence BI20111027132749 SEQ ROI Name ROI1 Location a Brightness slider Yc Cross hairs in the custom color field UCUNUN 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 1 User s Manual IVIS Spectrum CT 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
333. the Images drop down list Figure 4 9 Figure 4 9 Viewing image information Pa LC Image Information gt fms Images B120111027132749 002 Sequence Info Drop down list of images in the selected sequence Or a list of single images if Individual Drop down list of open Sequences _ BI20111027132749 sequences Choose LI Show Al Sections Individual Images from niea Section the list to show the open single images in the Images drop down list User Label Name Set luminescent image Choose the Show All Sections option to Label Living Image Universal luminescent TIF Images is selected in the Sequences drop down list display all categories of image information Acquisition Date Acquisition Seconds Acquisition Time Adapative FL corrected Background Corrected Binning Factor Cosmic photographic image photograph TIF Camera System Info readbiasonly image readbiasonly TIF Key Value Thursday October 27 2011 3402592204 13 30 04 0 1 4 0 71 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 Im
334. the spectra to subtract in the dialog 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 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 si
335. the surface in the 3D View window then press the V key to cycle through the different views of the surface a Figure 10 6 shows examples of the available views You can view the surface from different perspectives by doing one of the following Figure 10 5 Surface perspective view IU B20111027132749 seq Hoho CJ Sequence View A 3D View Gronal z 9 0 Sagittal x 4 0 Transaxial y 1 0 Subject Height 19 4mm Perspective View name Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 10 Reconstructing a 3D Surface Figure 10 6 Alternate views of a surface Click the surface then press the V key to change the view Top Bottom Back Left Front Right 10 2 Managing Surfaces After the surface is saved it can be shared by the DLIT or FLIT tools Figure 10 7 Tool palette Surface topography tools Tool Palette gt ROI Tools gt Planar Spectral Imaging Co Spectral Unmixing Surface Topography Spectrum CT Surface Reconstruction Threshold 21 G Subject Nude Mouse Generate Surface Surface Smoothing Level Low na 0 G Restore Save Results Surface name Name SURFACE_1 Delete Load gt 3D Multi Modality Tools C5 3D Optical Tools gt DLIT 3D Reconstruction LL 186 Living Image 4 3 1 User s Manual IVIS
336. 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 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 124 Living Image 4 3 1 User s Manual IVIS Spectrum CT Configuring the ROI Measurements Table Chapter 5 ROI Tools for Optical Data 125 You can customize the data and information column headers
337. tion Gama C B120111027132749 oo1 boka Units Counts Display Overlay E Luminescent gt on X Ray gt Options ail Info sg w Luminescence Choose the type 300 of images to view An overlay of luminescent 600 data on X ray is shown in this example 400 200 Counts Color Scale Min 44 Max 817 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools 251 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 9 3 Click the button to remove the smoothing Figure 13 9 3D Multi Modality tools Process tab Tool Palette E3 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools 252 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 10 Perspective view and slice views displayed using different color
338. tion Results The quantification results can be saved with the image sequence and as a calibration database that is made available in the DLIT or FLIT 3D reconstruction tools in the Properties tab When you define the properties for performing a 3D reconstruction and a calibration database is specified the 3D reconstruction results will be displayed in calibrated units for cell numbers or molecule quantities in picomole units Figure 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 1 465 520 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
339. ton H Figure 4 31 Figure 4 31 Opening the Edit Sequence dialog box Sequence View Spectra TE 11120050624145507_SEQ Lolo fe Min 2140 Max 36446 Min 2949 i gt Mins 1122 Max 51182 95 Max 10348 76 Max 19545 Units Counts x E Use Saved Colors Options x Info R a l Single images in the Living Image Browser that can be added to the sequence i Edit Sequence Sequence Clicks TLT20050624145507_001 TLT20050624145507_002 TLT20050624145507_003 TLT20050624145507_004 TLT20050624145507 005 TLT20050624145507 006 Images in the active Move Up been removed Browser Images JJH20050630142125 001 JJH20050630142125_002 JJH20050630142125_003 JJH20050630142125 004 Retired Images Images that have from the active EAEI 4 In the Edit Sequence box that appears choose the image s to add or remove retire from the sequence Figure 4 31 94 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 4 Working With Optical Image Data 95 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 s
340. tor 1 4 Animation Setup Time Scale 4o E og v XD Frames Per Second 10 4 Total Duration secs 5 3 5 To capture the first key frame click the f button The first key frame is added to the key frame box 6 Adjust the position of the reconstruction in the 3D View using an image tool for example 47 or a For more details on the image tools see page 194 7 Click the fj button The second key frame is added to the key frame box Figure 11 41 Example key frames for a custom animation Pa 3DAnimation Preset Animations Presets Spin CCW on X Axis z Frame Factor Animation Setup Time Scale B 0 5 1 7 Key Frame 1 Key Frame 2 oo Solan iF Key Frame 1 Play Frames Per Second 10 db db Total Duration secs 5 Save Load Key Frame 3 Key Frame 2 Key Frame 4 232 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 233 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 233 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 ge
341. ts for the two types of ROIs are displayed in separate tabs of the ROI table Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 6 2 Drawing a 3D ROI 1 Load a CT image or a DLIT or FLIT sequence 2 Click the 3D ROI button in the ROI tools Figure 6 3 A red bounding box appears in the 3D View 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 m 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 ET 3s NG Subject Height 194mm Perspective 3 Adjust the position of the 3D ROI using the transform tools NOTE It may be helpful to view the CT image from different perspectives to check the 3D ROI position and size To turn and rotate the CT image press and hold the left mouse key then drag the mouse when the hand am appears a Click the 3D ROI Transform button and select the ROI from the drop down list The first 3D ROI created durin
342. ube This helps improve the pseudo color visualization of the image cube Copies the composite image to the system clipboard Click to export the composite image to a graphic file for example jpg dig Opens the Print dialog box 160 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 I HX20120419114703_SEQ bodka sci GD s
343. uence 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 36 Opening the sequence table LC IMIS Acqu isition Control Pane Lo x Imaging Mode Exposure Time Excitation Filter Emission Filter hito sec v Medan gt Medium vje v Standard One Mo v Field of View System Status X Rays will be produced en energized Idle Acquire Subject height 15o GF cm Sequence Setup Focus use subject height v Temperature AG Locked sition Control Pane x D Binning F Stop Excitation Filter Emission Filter Display Photographic Settings Subject Auto sec medum v E sea O Medium vla Standard One Mol Lal Field of View System Status X Rays will be produced when energized Acquire jae cm Imaging Wizard Subject height 1 50 Z cm Image Setup Focus use subject height v Temperature I Locked E Number of Segments 1 G Delay o0 E min Apply to All X Remover Aj Update Insert Add 3 Choose a subject and probe from the drop down lists Figure 3 37 Figure 3 37 Choose a subject and probe C Display Photographic Settings Subject Probes 7 O Seq 0 Bioluminescent Probes gt Unselect Probes Fluorescent Probes gt Bacteria CBGreen CBRed Firefl
344. uire Idle 13 4 cm Imaging Wizard Subject height 1 50 cm Sequence Setup f ONG Focus use subject height w Temperature NN Locked Initialize roi nog 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 Ag Fluorescence i imaging in the wizard rer 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 CT 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 bol
345. umetric 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 Stdev Counts Standard deviation of the count values for all voxels inside the 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 Volumetric Data Absorption Measurements Fixed 32 bit scale with values that are consistent between images 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 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
346. uorescent 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 CT Appendix B Preferences 277 Figure B 5 Acquisition preferences Camera Settings C Freferences Acquisition Optical Properties Suto Exposure Camera Settings Default Image Exposure Default Image Binning Photographic Auto Photographic Luminescent Luminescent 1 00 sec Il F
347. ur custom animation setup page 234 Save an animation setup page 233 Record an animation to a movie file page 233 Edit an animation setup page 233 Figure 11 38 Individual 3D views key frames in the preset animation Spin CW on Y Axis iG Fi 3DAnimation Hl Preset Animations Presets Spin CW on Y Axis v Frame Factor 1 Animation Setup Time Scale CJ Key Frame2 Key Frame 3 Key Frame 4 Key Frame 5 Keyframe 1 Keyframe 2 Play Frames Per Second 10 Total Duration secs 5 Load 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 viewthe animation composed of the key frames Keyframe 3 Keyframe 4 Keyframe 5 229 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources Table 11 14 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
348. urface 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 the animal surfaces derived from IVIS data Organ files must be segmented from MRI or CT 3D volumetric data in third party medical imaging analysis software 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 CT 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 cj E kale stomach iv uterus iv Organ At My Recent Documents Add Organ Files Save Organ Atlas My Network Places File name File
349. uring 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 17 Autosave prompt Fa E Living Image 4 3 64 bit Auto Save E3 a 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 61 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 18 See Table 3 4 on page 35 for details on the image window 41 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition Figure 3 18 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 3 TLT200492 17115344 Saree Pharescert Background Teste Tue Feb 17 DOA 03 34 57 Doperment 2 female LevelsHigh Emy 5 5 Famy 5 Shig Barnikummabon Bino MI8
350. us injection of a radiotracer As the radiotracer circulates through the body each organ 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 Option
351. utton 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 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 239 Living Image 4 3 1 User s Manual IVIS Spectrum CT Table 12 1 Quantification results continued Chapter 12 Quantification Database 240 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 Quantifica
352. vel 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 181 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 195 198 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 11 3D Reconstruction of Sources 199 Figure 11 10 FLIT 3D Reconstruction tools Analyze tab Images selected for reconstruction Type of image used in the reconstruction If no NTF data are available only Radiance is available LEEEEEEEE n A gt Optimization algorithm
353. ves 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 3 _ ROI Tools _ Spectral Unmixing and DyCE gt Surface Topography _ 3D Multi Modality Tools _ 3D Optical Tools DUT 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 02 Starting vsiz Copy Kappa best Nsurf best Select All Total surf sa Threshold a Export Results Kappa limits 0 50 4 00 Nsurface limits 200 00 200 00 pe Photon Density Maps sb Export Results Save Results Name DLIT 1 Delete Load Overwrite 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 is a useful way to check the quality of a 3D reconstruction The photon density is closely related to the measured radiance Photon density
354. w unang Composite V Show Labels V Individual Scale V Normalized Legend a Units 1 00 Image Ust i J AFEED 7 AFIS 0 50 J Photograph 0 0 660 700 740 780 Emission Wavelength nm Spectrum List seb xX Note Select Name Color Pick 1 V TissueAF Mime z r dkg 2 V AF680 E zf 3 V AF750 blue 4 Mins 0 00 ax 6 4888 image Adas Unghiress xy s Loparitiwec Scale Libel TasurAF Cose 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 components 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 c
355. ws 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 178 Living Image 4 3 1 User s Manual IVIS Spectrum CT 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 0 400 800 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 C Computed Spectru
356. ximum Intensity Projection MIP Gradient Illumination L gt 3D Optical Tools DLIT 3D Reconstruction Click a button to save or load a color opacity map 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 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 246 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 13 3D Multi Modality Tools 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 Aa Figure 13 5 Level of Detail slider W Display Volume Level Of Detail T erformante Quali Resolution 0 5x 1x 2x 3x Table 13 2 Example volume with 512 slices at 1x resolution Volume Resolution
357. y hRenilla Tritium Bead 5 XPM 2 LED ZnS Unselect Probes E Number of Segments 1 Delay 0 0 min x Remover 4 Update Insert Add Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter 3 Image Acquisition 58 4 Specify the imaging settings for the first image in the sequence See Appendix A on page 268 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 38 the IVIS Spectrum CT acquires only one photograph for the entire sequence If this option is not chosen the system acquires a photograph for each image in the sequence 5 Click the Add button Gadd The acquisition parameters appear in the sequence table Figure 3 38 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 mj in the sequence table b Select a directory enter a file name and click Save in the dialog box that appears Figure 3 38 Control panel and sequence table with image settings TC WIS Acquisition Control Panel Imaging Mode Exposure Time Binning FiStop Excitation Filter Emission Filter F Display Photographic Settings Subject Mouse Probes v O
358. y in a user specified area of the image that is considered background Subject ROI Identifies a subject animal in an image The software automatically associates a measurement and an average bkg ROI that are included in the same subject ROI Using this type of ROI is optional 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 121 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 Thre
359. zard see page 48 Acquire and load the sequence 2 Generate or load a surface using the Surface Topography tools See Chapter 10 on page 181 for more details 3 Inthe DLIT or FLIT 3D Reconstruction tools select the a Wavelengths or excitation point images to analyze m Tissue and source properties 4 Reconstruct sources See page 192 for detailed DLIT steps See page 198 for detailed FLIT steps 5 View source measurements see page 205 E pumaroon s2 Tas 20 7 r eorrer View D Wew Secre Unts HaGarke Photons Use Saved Colors Tool Palette L gt Image Adjust lel gt Corrections Filtering lal gt ROI Tools gt 1 gt Spectral Unmixing and DyCE Surface Topography E gt 3D Multi Modality Tools 7 DLIT 3D Reconstruction Analyze Properties Results Sequence 6120111027132749 SEQ Tissue Mouse Tissue Source Firefly Select Filters Filter Threshold 600 106 62 68 640 81 Tool Falata Donnections S Merg _ ROE Took Sppckral Liming and DyCE gt Sells Topography 3D Helki Hodality Toots J DLT 3D Reconehnectaan kaka Properties hendte Top Properties Howse Tiue Tirit Sot Era Frei Pivi Tease Proper bes Lier ki CMR Br gt eee Fi i 1 a0 ag 100 Wavelength fern Jad AP loo pe prp atja 190 Living Image 4 3 1 User s Manual IVIS Spectrum CT Chapter
360. zing the System and Checking Temperature on page 7 Overview of Image Acquisition on page 10 Overview of Living Image Tools and Functions on page 13 Managing User Accounts on page 20 Tracking System and User Activity on page 23 2 1 Starting the Living Image Software The Living Image software on the PC workstation that controls the IVIS Spectrum CT 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 version 64 bit Windows C Program Files Caliper Life Sciences Living Image NOTE All components of the IVIS Spectrum CT 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 Sp

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