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Living Image® Software - NYU Langone Medical Center

1. GALT 30 ietervteruetion gt SetEnd Undies Figure 8 11 Drawing an ROI using the free draw method 8 5 Subject ROIs A subject ROI identifies a subject animal in an image It provides a convenient way to automatically associate link a measurement and average background ROI for background corrected ROI measurements when there is significant autoluminescence or autofluorescence For more details on background corrected ROI measurements see page 64 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 4 Select Subject ROI from the Type drop down list 5 Click the B button and select 1 6 Position the subject ROI so that it includes the measurement ROI s and the associated average background ROI 8 6 Measuring Background Corrected Signal If a subject has significant autoluminescence or autofluorescence you can obtain a background corrected ROI measurement by subtracting an average background ROI from a measurement ROI The software computes Background corrected intensity signal Average signal in the measurement ROI Average signal in the average background ROI j Y Caliper Chapter 8 Working With ROI Tools 65 NOTE This is an optional background correction that is applied in addition to the el
2. Fy TLT30017151343517_ So Figure 13 4 Point source fitting results 13 4 Checking the Point Source Fitting Results 1 In the Results tab click Photon Density Maps The Photon Density Maps window appears Select an image from the drop down list p l m Proton Pantiiy ape e TR Perce ft Eg see l Erir Eiaa Figure 13 5 Point source photon density map 2 Select the image from the Image sources drop down list 3 Compare the simulated and measured photon densities 4 To rotate the surface and view it from a different angle move the thumb wheel to the left or right zi m Y Caliper Chapter 13 Point Source Fitting 125 Thumb wheel Photon Dansity Maps C Lewy Sre Hartrental Profile J aiii F Me a Phofon derathy photosaesen Phos cesaety phoboraimes Figure 13 6 Rotating the surface 5 To view the photon density profile at another location drag the cross hairs or click a location 13 5 Exporting Results To export all results 6 In the Results tab click Export results Gi Export Results 7 In the dialog box that appears select the destination folder for the results and click OK The results include a txt csv xsc source information and a xmh surface file To export user selected results 1 Right click the item of interest in the results list a
3. Sequence View Surface Source Animate Registration Registration Tools ejas me d r e Display Organs A 8 Opacity lo K Organ Atlas Organs LI rectum O salivary skin E Wepleen E stomach LI tongue L uterus O Bular Use Tab key to switch between transformation tools photons sec Use Y or 2 keys to restrict scaling to only one axis Gpurce Intensity Scaling On YZ p gt Spectral Unmixing Ubject Height 26 4 mm Perspective Figure 15 31 Displaying the transform tool 4 To adjust the x y or z position of the organ drag the transform tool 5 To return the transform tool to the default location click the Reset button 6 Press the Tab key to put the transform tool in scale mode A red cube M appears at each corner of the transform tool 7 To increase or decrease scale the size of the organ drag a red cube at a corner of the transform tool To restrict scaling to a particular axis press the X Y or Z key then drag a red cube 8 Press the Tab key again to put the transform tool in rotate mode A red green and blue circle appear around the surface 172 Living Image Software User s Manual The selected circle line is thicker Figure 15 32 Rotating the organ on the surface 9 To rotate the organ on the x y or z axis click the blue green or red circle and drag the mouse arrow in the direction of interest To return the digital orga
4. 2 To stop the playback click the Stop button m 2 3 To view a particular frame do either of the following e Move the top frame slider or enter a frame number in the box next to the frame slider e Click a location in the Maximum vs Time graph 1 To select a particular range of kinetic data move the start and end frames selection handles Alternately enter a frame number in the box next to each slider Only the selected frames will be played back or saved Kinetic data dcm can also be edited in the Image window For more details see page 91 48 Living Image Software User s Manual Kinetic Acquisition Control Panel Display Photograph 14 Framesisec Acquisition Settings Overlay I Dynamic Range mage i al i Min 111 Frame number 121 _ F t S Max 15366 12000 10000 S000 6000 Current frame number Enter anew number Excitation Filter or use the top slider to view another frame Emission Filter FL Lamp Level ie ae Photograph Light Level 40 7 3 fiero oa E O 00 1 Start frame in a user specified data range T E ac Scale wl Auto l Maximum Vs Time Minimum 1200 biad courts 111 Position of the displayed current frame 200 A axtari i Position of the start frame File Size 110 9 ME Al l i l Position of the end frame Time secs Time secs 17 05 A Save Figure 6 4 Acquisition window Viewing Options After acquisition has been stoppe
5. 5 In the Properties tab make a selection from the Tissue Properties and Source Spectrum drop down lists Figure 15 3 Muscle is usually the best choice of tissue properties for general in vivo reconstructions Tool Palette Fluor Calib WPOLANT_1 wt m j ern 1 Figure 15 14 FLIT 3D Reconstruction tools Properties tab 6 To view the tissue properties M4 Hefo H s for the tissue you selected make a selection from the Plot drop down 7 To include the number of fluorescent molecules source in the results select a luminescent calibration database For details on generating a luminescent calibration database see page 133 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 146 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 15 13 10 Click Reconstruct The reconstruction requires about 1 5 minutes depending on the parameter settings and the processor speed When the analysis is finished e The 3D View window displays the surface and the reconstructed sources e In the Tool Palette the Results tab displays the results data and the algorithm parameter values Figure
6. Caliper ifeSchences Chapter 11 Spectral Unmixing 109 Table 11 5 Spectral unmixing tools Results tab continued Item Description Weighting Mode The weighting method applied to the data Column Weighting Indicates if column wise weighting was used Mode Row Weighting Indicates if row wise weighting was used Mode Save Results Name The name of the selected spectral unmixing results Delete Removes the selected spectral unmixing results from the system Save Saves the spectral unmixing results 11 5 Spectral Unmixing Options In the spectral unmixing tools the Options tab shows the user modifiable parameters in the spectral unmixing algorithm Figure 11 9 It is recommended that you first perform spectral unmixing using the default settings Then if necessary change the option settings and reanalyze the data Tool Palette Spectral Unmixing Analyze Options Results Constraints Reset Values Init Fix HP LP Unimod 1 Auto ai Nja v NjA v NA v 2 Auto EC Nia v NjA v Na v Sort Force Denoise by PEA MediumiDeraulk Unimod Tolerance Cay 5 pl Pca Node Explained Variance Biplot Update Figure 11 9 Spectral unmixing options Table 11 6 Spectral unmixing options Option Description Constraints The constraints for unmixing the components Reset Values Returns all constraint settings to the default values Init The method for generating the initial guess of the spectrum for the
7. Figure 12 1 Analyses that require a surface 114 Living Image Software User s Manual 12 1 Generating a Surface 1 Load the image sequence for the reconstruction For example a sequence that was acquired for DLIT analysis 2 Inthe surface topography tools make a selection from the Object drop down list nude mouse fur mouse or phantom Select an orientation dorsal or ventral 4 Select a smoothing level Click Reconstruct The Tomography Analysis box appears By default the entire subject is selected for the reconstruction Tool Palette Surface Reconstruction Object Orientation Surface Smoothing P Singir Virw Surface Topography Analysis Level Restore Save Results Name SURFACE_1 t Delete Load B Greer aregon of interest enaga bo proceed _ Point Source Fitting The green rectangle selects the region to reconstruct TL Tae HA 0 Figure 12 2 Surface topography tools and tomography analysis box 6 If you want to reconstruct only a particular region of the subject resize the rectangle drag a green handle so that it includes only the area of interest 7 Click Next The purple data mask appears The mask is an overlay on the subject image that defines the area of interest for the surface topography reconstruction The mask should match the underlying photograph of the subject as closely as possible without including any area
8. Reverse Choose this option to reverse the min max colors of the selected color table Restore Defaults Click to apply the default settings feSciences YY Caliper d p Appendix B Preferences 243 B 5 Tissue Properties Taanil Palette gt ROU Tools gt Planar Spectral Insaging gt Surface Topig aphy gt Pint Goeurce Ting J DIT 2D Reconnection ish pe Properties Rep s Toews Properties Hooke Inher mandi index of retraction 1 4 Source kanna Pirefiy ka Prue Met Soc Specie Bree Liri Gah hra mai li Acquisition Theme Pincus Progeertiees Musde Beemer ceri Homers Ampek tate con lrurs Fre i LOO yi I on n H carmvedzztad nip Lt Tack Fluorescent i iy Source Spectrum j Pit Ma ao To fn 1 Worekazih pen TAL IT J0 Aei oatu a a Task gt Spectral Uniig Figure B 9 Set the default tissue properties preferences left for the Properties tab in the Planar Spectral Imaging DLIT or FLIT tools Table B 7 Tissue properties preferences Item Description Tissue Properties Choose a default tissue type that is most representative of the area of interest This tissue type will be used if a Subject Type is not selected in the Imaging Wizard and saved during acquisition Source Spectrum Choose the default luminescent source This Source Spectrum will be used if a Subject Type is not selected in the Imaging Wizard and saved during acquisition for D
9. lve Oecrley Window tape Awe GEO AR DOS eS OC a Fiica Fr Ll AR DOS LEO OO O awaa ra oe w kring Aliri Dokr Tais Cokarhia Tpi flac wel ted C hra C ogai Faite Labe Scales pa Gima g S Figure 7 21 Image Overlay window 5 To overlay all images click the 4 button The overlay appears The photograph is at the bottom of the stack and the last fluorescent or luminescent image in the list is at the top of the stack mapt Overlay Window Tapa AWW a alee Links Badass 3 Mabara AMAS l Pie lise El amean Peon ton AEn Ern ii p a E h Daar Da Cab trae Tepe fied aed 6 Pati Lebel GERE eke car Coke 5 C eerie Wate as asi l i 7 Figure 7 22 Generated overlay y Caliper a _ Chapter 7 Working With Data 93 ifeSchences Table 7 10 Image Overlay window Item Description Units Choose the type of units for displaying the fluorescent or luminescent image For more details on measurement units see page 251 Photograph A drop down list of the photographs in the image sequence Fluorescent or The sequence images Luminescent Images Ba Copies the overlay to the system clipboard Click to export the overlay to a graphic file Click to include all fluorescent or luminescent images in the overlay Click to remove all fluorescent or luminescent images from the photograph Image Adjust Tools for adjusting the appearance of the highlighted fluorescent or lumin
10. Simulates the red spectral shift of a spectrum produced by a signal that is located at a depth of 5 mm in tissue Note This option is only available if Show Advanced Options is selected in the general preferences For more details see page 232 Adding Spectra to the Plot You can add library spectra or a spectrum calculated for an ROI to the plot To display library spectra 1 Click the F button and select From Library A new row appears in the spectrum list 2 In the new row select a probe name from the drop down list Sequence view Spectra Unmixed Images Normalized Legend _ Red Shift p z Spectrum List a UWE 2 w a Tsw F 1 o0 Hormalzed Amplitude a SFAZ0Em Label Color TissueAaF D uez E arcscem 0 50 0 0 650 700 T50 s00 s50 Wavelength rrr Figure 11 7 Selecting a library spectrum to display To add spectra from an ROI 1 Create an ROI on an image and apply it to the sequence 1 Click the button and select From ROI A new row appears in the spectrum list 2 In the new row select an ROI name from the drop down list 108 Living Image Software User s Manual To remove a spectrum 1 In the spectrum list select the spectrum row that you want to remove 2 Click the button 11 4 Spectral Unmixing Parameters The Results tab in the Spectral Unmixing Tool Palette shows the optimized fit parameters used by the software to derive the spectral unmixing results Figure 11 8 Tool Pal
11. 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 Nsurf best The number of surface element data analyzed per wavelengths images Total surf samples The total number of surface element data analyzed for all wavelengths images Threshold angle The angle that the object surface normal makes with the optical axis The optical axis can be considered to be a line perpendicular to the stage The default setting for this limit is 70 for IVIS Spectrum or IVIS 200 data Uniform Surface Sampling TRUE the option is chosen and the surface data for each wavelength is sampled spatially uniformly on the signal area FALSE the option is not chosen and the N brighter surface elements are used as data in the reconstruction NNLS Weighted Fit TRUE the option is chosen and the DLIT or FLIT algorithm weights the wavelength data inversely proportional to data uncertainty in the NNLS reconstruction FALSE the option is not chosen Bkgd Fluorescence FLIT Image Threshold Tissue Properties The background fluorescence is derived from the data Background fluorescence can be due to non specific dye or tissue autofluorescence The percentage of the minimum radiance at each wavelength DLIT or source location FLIT is of the maximum radiance This defines the minimum intens
12. feSciences 7 6 Viewing Intensity Data amp Making Measurements The Image Information tools enable you to view intensity data and measure distance on an image You can view pixel data in different formats Image Information Description See Page x y coordinates and The x y pixel coordinates of the mouse pointer location in the 84 associated intensity image and the intensity counts or photons at that location Histogram Histogram of pixel intensities in an image 85 Line profile Plots a line graph of intensity data at each pixel along a user 86 specified horizontal or vertical line in the image Tan Palette lee Adjust Comectons Fterng C J Image Indernnation w rts cm s bange Binnig 8 With 12 6 erm Height 12 6 om imago X Y JASE IION cm linage Data JJ counts Cra ae TA CELES i geog maaan bitane 0 p gt ROI Tools Point Source Fitting lt gt DLT 30 Reconstruction GADLIT 3D Reconstruction Figure 7 12 Tool palette Image Information tools Table 7 5 Tool palette Image Information tools Description Click this button to display a histogram of pixel intensity For more details see page 85 fo o Click this button to display the distance measurement cursor in the image e Click this button to display a line profile For more details see page 86 Ter A window For more details see page 88 Click this button to draw and measure a rectangle on an image For
13. selected component Auto means this is automatically determined by the software Alternatively you can used a loaded spectrum as the initial guess 110 Living Image Software User s Manual Table 11 6 Spectral unmixing options continued Option Description Fix This option determines whether the spectrum is allowed to change If this option is chosen the spectrum of that component is not updated during unmixing HP Sets a high pass filter for the spectrum Signal below the HP cut off frequency is forced to zero Choose N A to turn off the high pass filter Otherwise the value represents the high pass cut off frequency This constraint can help isolate components that are physically mixed and difficult to distinguish LP Sets a low pass filter for the spectrum Signal above the LP cut off frequency is forced to zero Choose N A to turn off the low pass filter Otherwise the value represents the cut off frequency of the low pass cut off frequency This constraint can help isolate components that are physically mixed and difficult to distinguish Unimod Choose this option to apply the unimodality constraint Unimodality forces the spectrum to have only one peak one extremum however Small magnitude extrema are allowed if they are less than the Unimod Tolerance value This tolerance value limits the rising slope of the second spectral peak For example 5 tolerance means that the increase in magnitude of the neighboring nodes
14. E CS ed a lands tee pe aE 3 aN il Ciim es wu S a Crerar Pie sa nam toman nt bry eater ata one heme naay Tenens Tirang Pur nam Tiri E Beijo vise E eae Lhhininlciowe TA py mall be proche en a ii shen meryrrd m A g pE l y aie age 1 50 om Peay p ri Eaa EEL PAEA X ray reuse option is selected by default One X ray image is acquired for the sequence Figure 5 10 Control panel and sequence editor 36 Living Image Software User s Manual Acquire the Image Sequence 1 Confirm that the VIS Imaging System is initialized and the CCD temperature is locked For more details see page 17 2 When you are ready to acquire the images click Acquire Sequence in the control panel The Edit Image Labels box appears The information entered here appears in the image label Figure 5 13 ie ee ee ee ee a a Figure 5 11 Edit Image Labels box 3 In the Edit Image Labels box enter information about the image and click OK If you do not want to enter image information click Cancel 4 If this is the first image of the session you are prompted for an autosave location All images acquired during the session are automatically saved to this folder You can choose a different folder at any time select Acquisition Auto Save on the menu bar To select a folder for autosaved data click Yes in the prompt and choose a folder in the dialog box
15. Figure H 3 Light emission from a surface element passes through the lens entrance pupil and is recorded in the image The imaging system collects the light emitted from the surface element at an angle 0 measured with respect to the normal to the surface element into the solid angle dQ subtended by the entrance pupil The value of the surface radiance L 0 is directly related to the photon density p photons mm just inside the surface of the element H 3 Defining the Linear Relationship Between a Source and Photon Density The software divides the interior of the subject into a solid mesh of volume elements voxels Each voxel is considered to contain a point light source at its center The index i enumerates the set of voxels S is the value of the strength or flux in photons sec of the point source inside the i voxel The solid mesh defines a collection of point sources that approximate the actual source distribution The accuracy of the approximation is improved by increasing the density of the solid mesh The reconstruction method is based on the principle that there is an approximately linear relationship between the source strength in each voxel S and the photon density p at each surface element described by a Green s function G The photon density at the j surface element is the sum of the contributions from all the voxels p XG S 1 i 286 Living Image Software User s Manual The Green s function con
16. ROT Tonks gt Planer Spectral Imaging Surlavce Topography gt Point Source Fitting lt ULL JU Reconstruction Pavan oe Progenrtins firsa Tisus Jie Seea Piters Fit r O seo El o EI azo O E gay A LALIT 30 Reconstr Him _ Spectral Unmixing i gt Rot Tants ooo gt Planar spectral imagina E gt Paint Source Fitting Bruner DARE SD Source FaF Threshold x 0 7 0 5 0 5 US WS Figure 15 2 Analyze tab 4 In the Properties tab make a selection from the Tissue Properties and Source Spectrum drop down lists Figure 15 3 Muscle is usually the best choice of tissue properties for general in vivo reconstructions 144 Living Image Software User s Manual Toal Palette cl j Surface Topography F OLIT 30 Reconstruction BAN ie Progeer ties AT Tiaua Proper lies Made Source Sperbrum Firedhy F FA Bari airu w The selected plot type is displayed below Mormalzad Amplilude j Tiree lengh Bm gt GABLIT 30 Reconstruction gt 30 Tonk gt Spectral Urimixing Figure 15 3 Properties tab 5 To view the tissue properties M4 Mef W s for the tissue and source you selected make a selection from the Plot drop down 6 To compute the number of cells per source select a luminescent calibration database For details on generating a luminescent calibration database see page 133 7 In the Analyze tab click Start The Dat
17. Segment Colors available for the pencil tool Opacity Adjusts the opacity of the pencil tool markings Reset Removes all pencil tool markings Y Caliper eSclences Chapter 15 3D Reconstruction of Sources 149 Steps to Reconstruct Luminescent Sources Using GADLIT 5 Load an image sequence 6 Generate or load a surface using the Surface Topography tools For details on generating the surface see page 113 7 In the Tool Palette choose GADLIT 3D Reconstruction The Analyze tab shows the data that the algorithm automatically selects for the reconstruction Figure 15 2 For more details about the Threshold see page 146 Tuol Palette m al gt Surface Topography l 7 GALIT 30 Resconestriction Anaie Progeer Le Poevulls Seguame AS a ee uc ares Thrazhoti o gt 30 Tanks gt Spectral Urimixing Figure 15 8 Analyze tab 8 In the Properties tab make a selection from the Tissue Properties and Source Spectrum drop down lists Figure 15 3 Muscle is usually the best choice of tissue properties for general in vivo reconstructions 150 Living Image Software User s Manual Tow Pabethe gt Planar Spectral imaging o L gt DLT 40 Reconstruction Arani Fronention Reais Tssa Properties Miracle kum Fretky w The selected plot type is displayed below Want length rez Figure 15 9 Properties tab 9 To view the tissue properties U He H fo
18. The Living Image software applies spectral unmixing to distinguish the spectral signatures of different fluorescent or luminescent reporters and calculate the respective contribution of each on every pixel of an image Use spectral unmixing to e Extract the signal of one or more fluorophores from the tissue autofluorescence Images can be acquired using epi illumination excitation light above the stage or transillumination excitation light below the stage e Analyze luminescent or fluorescent images when more than one reporter is used in the same animal model 11 1 Image Sequence Requirements Use the Imaging Wizard to set up the image sequence that is required for spectral unmixing For more details on the wizard see page 25 If you do not use the Imaging Wizard to set up the image sequence it is recommended that the image sequence include images acquired using several filters that sample the emission or excitation spectra at multiple points across the entire range Make sure that the band gap between the excitation and emission filters is sufficiently large for example gt 35 nm so that the excitation light does not leak through the emission filter where it can be detected by the CCD 11 2 Performing Spectral Unmixing 1 Load the image sequence In the example in Figure 11 1 the fluorophore is Quantum Dots 800 Images were acquired using a 675 nm excitation filter and emission filters from 720 to 820 nm in 20 nm increments 1
19. The surface topography of the subject is determined by analyzing the displacement Ax or bending of the laser lines as they pass over the subject The displacement is defined as the difference between where the line should fall on the stage in the absence of the subject and where it appears in the image due to occlusion by the subject 284 Living Image Software User s Manual ai LAX Figure H 1 Parallel laser lines projected onto a subject Given knowledge of the angle 9 the height of the subject h can be determined by analyzing the displacement Ax of the laser lines as they pass over the object The parallel lines are projected onto the surface of the subject at an angle 0 The angle is known by instrument calibrations of the distance between the structured light projector and the optical axis D and the distance between the stage and the structured light projector Figure H 2 SL projector ey CCD i Y x p i A aa A ak 3 T Y a a Ts g 7 Y e A s x _ ii Figure H 2 Structured light projector and subject D and form two perpendicular sides of a triangle giving tan 8 D l Together Ax and h comprise a smaller version of this triangle The height A can be determined from h Ax tan Q by measuring the displacement Ax The software utilizes fast numerical methods to rapidly evaluate Ax over the entire image to determine the surface topography The surface topography determination is
20. ae re See ah ae Aah Da i jp e ee gee ee te teed Le prt hia mars Figure 4 13 Imaging Wizard Table 4 3 Imaging Wizard options Bioluminescence Description Page Open Filter Acquires an image at maximum sensitivity Planar Spectral Analyze the sequence to compute the average depth and total 139 photon flux of a luminescent point source in a region of interest ROI Caliper HeSclences Chapter 4 Luminescent or Fluorescent Imaging 15 Table 4 3 Imaging Wizard options continued Bioluminescence Description Page Spectral Unmixing Analyze the sequence to determine spectral signature of different 147 reporters in the same image and calculate the contribution of each reporter on each pixel in the image DLIT Apply the DLIT algorithm to the sequence to reconstruct the 3D 171 surface topography of the subject and the position geometry and strength of the luminescent sources Fluorescence Description Page Filter Pair Use the data to make fluorescence measurements Spectral Unmixing Analyze a sequence to extract the signal of one or more fluorophores 147 Filter Scan from the tissue autofluorescence Helps you determine the optimum excitation and emission filter for a probe FLIT Apply the FLIT algorithm to the sequence to reconstruct the 3D 180 Surface topography of the subject and the position geometry and Strength of the fluorescent sources For details on acquiring a sequence on the Lumina XR that include
21. gt Play Starts playing kinetic data m Stop Stops playing kinetic data Edit and Save Shows or hides the bottom sliders that enable you to select a range of options data and the Extract button that provides save options for the user selected image or data Accumulate If this option is chosen the software computes and displays the cumulative intensity signal Choose this option and playback the kinetic data to visualize accumulation as it happens Extract Click to select a save option for the current image or selected data Extract Current Image Displays the current image in a new image window The software prompts you to save the image when you close the image window Extract Accumulated The software computes the cumulative signal for each image Sum of Image the signal in all images up to and including the current image then displays the cumulative signal of the current image in a new image window The software prompts you to save the image when you close the image window Extract Kinetic Data Choose this option if you want to save a particular range of images Opens the Browse For Folder dialog box that enables you to select where to save the selected data Viewing Kinetic Data 1 Open the kinetic data 2 To start playing the kinetic data click the Play button If you want to start the playback at a particular image first move the top slider to the starting image then click the Play button To stop
22. 0 Sequence Setup Setup Focus use subject a ind Temperature gt D Locked OOO O miee Oe Figure 5 2 Lumina XR control panel 4 Select the Auto exposure time click the arrow Alternately manually set the exposure binning and F Stop For more details on the control panel settings see page 225 5 Make a selection from the Energy drop down list Table 5 1 Lumina XR energy options Energy Option Suitable For X Ray Energy Level Animal Living subject 35 Kv 100 uA filtered X rays Specimen Non living specimen 28 Kv 100 uA unfiltered X rays NOTE A Caliper field service engineer can customize the default X ray energy levels Table 5 1 to settings from 5 0 40 Kv and from 1 100 uA with or without the low energy X ray absorbing Al filter 6 Set the Field of View Make a selection from the Field of View drop down list For more details on the field of view see page 227 NOTE To view the subject s inside the chamber before image acquisition take a photographic image uncheck the luminescent or fluorescent option choose the Photographic and Auto options and click Acquire 7 Set the Focus e Select use subject height from the Focus drop down list and use the arrows or the keyboard arrows to specify a subject height cm NOTE The subject height for X ray images is restricted to 2 8 cm or less The subject height is critical to ensure proper optical and X ray overlay The subject height is used
23. App to a Pj Photon Density bps EIE Teel Palette z m E Wivealangth U gts of view C Lag Secale h e a c fatal airis fhe pia Cee i Fm a a deren j Figure 15 19 Photon density maps 3 Select a wavelength from the drop down list y7 Caliper eSclences Chapter 15 3D Reconstruction of Sources 159 The photon density profiles at the crosshairs location are displayed In a good reconstruction the simulated red photon density curves closely resemble the measured blue photon density curves 620 nm wavelength selected Photon Density Maps Wavelength 620 Angle of view C Log Scale zai d 4 mS l 05 if i photons mm3 l photons rom 3 Horizonial Profile a Sunulated Vertical Profile a Situated a m Wleasured aa a Wwleasured ag Photon density photonsimm 3 Ag Photon density photons ter s 3 0 3 0 2 0 1 0 00 10 5 2 Position rrr Position irar Position 5 Position Figure 15 20 Simulated and measured photon density plots 4 To view the photon density profile at another location on the animal surface drag the cross hairs or click a point on the photon density map Table 15 7 Photon Density Maps window Item Description Image sources A list of images used in the reconstruction Select all images ora 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
24. Panel Tools gt Well Plate Quantification for Tools gt Image Overlay for Tools gt Colorize Tools gt Transillumination Overview Tools gt Image Math for Acquisition gt Background Measure Dark Charge Acquisition Background Add or Replace Dark Charge Acquisition gt Background Measure and Replace Dark Charge Acquisition Background View Available Dark Charge Acquisition gt Background Clear Available Dark Charge Acquisition gt Background Auto Background Setup Acquisition gt Fluorescent Background gt Measure Fluorescent Background Acquisition gt Fluorescent Background Add or Replace Fluorescent Background Acquisition Fluorescent Background Measure and Replace Fluorescent Background Displays the Activity window at the bottom of the main application window The Activity window shows a log of the system activity Displays the Image Information box that shows the label set and image acquisition information for the active data Displays the ROI Properties dialog box Displays the ROI Measurements table Opens the Image Layout window that enables you to paste an image of the active data in the window Displays the control panel Opens the Well Plate Quantification window Opens the Image Overlay window for the active data Opens the Colorized View tab for the active sequence Note This menu item is only available if S
25. e Apply flat field correction to the image data e Specify pixel binning e Smooth the pixel signal Caor Satake Lint E s reivatins oder Tabir Pare A AAGE Phata Sed jushment J m _ Logarithmi Siak L gt Corrections Filtering gt m ge Informati n a2 iar d ap a O Manual Image Information Tools page 83 Display x y coordinates and intensity data ata user selected location on the image Display a histogram of pixel intensities in an image Plot the intensity y axis at each pixel x axis along a user specified line in the image Measure distance in an image Kinetic sequence ROI Tools page 101 M easure counts or photons in a user specified region of interest ROI and compute measurement statistics for example average min max standard deviation Measure efficiency radiant efficiency or NTF efficiency in the ROI and compute measurement Statistics for fluorescent images only Figure 2 4 Tools that are available for a single image luminescent fluorescent X ray or a kinetic sequence 12 Living Image Software User s Manual Image Adjust Tools page 99 e Tune the photograph brightness gamma similar to contrast or Opacity e Set the image display color scale minimum and maximum e Select a color table for image display Planar Spectral Imaging page 139 Determines the average depth and total photon flux of a luminescent po
26. see page 257 NOTE Transillumination is only available on the IVIS Spectrum Imaging System I Start the Living Image software double click the Fy icon on the desktop 2 Initialize the IVIS Imaging System and confirm or wait for the CCD temperature to lock For more details see page 17 3 In the control panel put a check mark next to Fluorescent and Transillumination WS Acquisition Control Panel Peldot View E a4 Jem Subject height 1 50 em Focus use subject height Gorri Locked Figure 4 9 Acquisition control panel 4 Select an excitation and emission filter from the drop down lists For more information about the standard filter sets see Table F 1 page 260 5 Click Setup If you are prompted to acquire a subject photograph click Yes 6 In the Transillumination Setup box that appears Figure 4 10 choose the location for transillumination and image acquisition click a W square 12 Living Image Software User s Manual VS Acquisition Control Panel Liu gy Pe Exe Cire Hiria A O A Transillumtnation Sctup Single Location Mode PAA Ae Mohan To oe Dart ucar fatima L Mek Grid Paints To Subat fork Types 1S P Poel cof view wf gyi Salas l Ter matio Cancel Subset height 130 on Focus ue qubject height Temperature A Locked Single location mode acquires one image at the location marked by a green square ff tities et ee
27. the sequence information appears in the sequence editor Figure 4 16 YP Caliper feSciences Chapter 4 Luminescent or Fluorescent Imaging 17 FJ Tk af kig Far I fi iial KTh ppi ete F Tht af d kij ira j j laa Lie Se G Fai Fifi Pitia Pier Par a 3 Pap Ce Ep irr gt oiir That option pabacts the beet garitan and ernmaon fers For eai huoria prii The Airini gaai nt ther aberh or they guria of Imaging Wizard Bioluminescence options Imaging Wizard Fluorescence options Figure 4 15 Choose the type of image sequence NOTE The imaging options available in the Imaging Wizard depend on the IVIS Imaging System and the installed filter set a E C i a a immi Piga Eg a i Rsi T E E der ca et L Eri LFI m 1 els Peki i Hai Saez Tacs E iai lee Sh E Blac Sa a i ce mas Sequence Editor Pieper i E Figure 4 16 Control panel and sequence editor during acquisition Each row in the sequence editor specifies the acquisition parameters for one image in the sequence For more details on the sequence editor see Table 4 5 page 23 Acquire the Image Sequence 1 Confirm that the IVIS Imaging System is initialized and the CCD temperature is locked For more details see page 17 2 When you are ready to acquire the images click Acquire Sequence in the control panel The Edit Image Labels box appears 18 Living Image Software User s Manual The information entered here appears in the
28. tools 141 142 point source fitting 167 169 export results 171 manage results 172 results 170 172 sequence requirements 167 tools 167 173 176 177 preferences 235 300 Living Image Software User s Manual print images 95 97 pseudocolor image 249 0 quantification database 179 183 create 180 182 manage results 183 samples 179 R radiance photon 253 radiance units 252 read bias 255 reconstruct 3D fluorescent sources 198 200 reconstruct 3D luminescent sources 188 194 reconstruct particular pixels 194 reduced Chi2 201 ROI 101 automatically draw 107 109 background corrected signal 110 112 delete 124 edit dimensions 120 free draw 109 managing 115 manually draw 106 measurement ROI free draw 109 measurement ROIs 106 109 Measurements table 103 move 119 move or edit label 123 quick guide 102 ROI line 122 save 123 subject 110 tools 104 105 ROI kinetic quick guide 113 114 ROI Measurements table 126 128 configure 129 130 copy or export 131 ROI properties 115 119 ROI types average background 102 110 measurement 102 subject 102 S save data manually 41 ROI 123 kinetic data 66 65 scientific image data 249 segment 39 sequence requirements DLIT or GADLIT 188 FLIT 198 planar spectral imaging 139 point source fitting 167 spectral unmixing 147 smoothing 82 248 software help 20 source depth 206 source spectrum 201 Spectra window 152 154 spectral imaging See pl
29. 121 Point Source Fitting 24 4 tk aeK SERGE Re SEH EER ESR SE KH DES 122 Checking the Point Source Fitting Results 0 124 Exporting Results 1 a a a a a 125 Managing Results 26h Che SEK KEEEDRE DSR sadara RRS ESD ES 8 126 Point Source Fitting Tools 2 ee ee 127 The point source fitting algorithm is a tool for advanced users that can be used to estimate the optical properties of tissue the location and power of a point source or the fluorescent yield of fluorophores 13 1 Image Sequence Requirements The software analyzes the images in a sequence acquired in one of the following imaging modes e Luminescence e Transillumination fluorescence bottom illuminated fluorescence e Epi illumination fluorescence top illuminated fluorescence e Transmission The point source fitting algorithm requires an image sequence that includes one or more images and a structured light image 13 2 Displaying the Point Source Fitting Tools The default Tool Palette does not include the point source fitting tools These tools are available if the advanced options are selected in the user preferences 1 Select Edit Preferences on the menu bar 2 Inthe dialog box that appears put a check mark next to Show Advanced Options and click OK Figure 13 1 The point source fitting tools appear in the Tool Palette Figure 13 1 For more details on the tools see page page 127 122 Living Image Software User s Manual Pre
30. 133 9 1 Creating a New Image Using Image Math e 133 9 2 Subtracting Tissue Autofluorescence 2 a a e a 136 10 Planar Spectral Image Analysis 2 000000 0s 139 10 1 Image Sequence Requirements 2 a a 139 10 2 Planar Spectral Image Analysis 1 2 139 10 3 Viewing Graphical Results 2 2 a 142 10 4 Managing Planar Spectral Imaging Results 2 2 2 0 0 0002 eee ee ee 144 11 Spectral Unmixing 1 2 a 147 11 1 Image Sequence Requirements 2 k a a 147 11 2 Performing Spectral Unmixing 2 2 ee 147 LGS Specia WidOW g 4us eae te een hee ee bee ee ee ee ee eee ie a 152 11 4 Spectral Unmixing Parameters 2 154 11 5 Spectral Unmixing Options 2 155 12 Reconstructing a 3D Surface 0 00002 2 eee eee ees 159 12 l Generar a SursCe xe en ee ee ee ee eR EERE ERS ee ae 160 12 2 Managing Surfaces sasra aa tt Eaa nee e aa a a eaa a a a 164 12 3 Export or Import a Surface 2 164 13 Point Source Fitting 2 a es 167 13 1 Image Sequence Requirements s 2 2 yoe eien t osaan 167 13 2 Displaying the Point Source Fitting Tools 2 2 0 2 ee ee 167 13 3 Point Source Fitting as s sa seara eke e Set ee Gee eee eed eeu he eewe eva 3 168 Y Caliper Contents iii ifeScihences 13 4 Checking the Point Source Fitting Results a oa aaa a a 2 2 00 2000 0000048 170 13 5 Exporting Results lt s ss sese sudaca amad i r ead
31. 15 16 154 Living Image Software User s Manual e The 3D Tools appear in the Tool Palette For more details on the 3D Tools see page 165 175 For details on managing results for example save load or delete see page 157 Bo Windy Hei Pe bo oii Aut ua He A 5 WY Unie Radare Effcwery w C Apply to al aj OKI00B0A07145405_SEQ F a tend cverote Figure 15 15 FLIT results 3D View window and Results tab For details on the 3D View toolbar see Table 15 3 page 145 15 3 3D Reconstruction Results The Results tab displays information about the photon density voxels and algorithm parameters DLIT or FLIT Results NOTE For more details on DLIT see Appendix H page 279 Sometimes adjusting the DLIT algorithm parameters improves the fit of the simulated photon density to the measured photon density data Figure 15 16 Example DLIT 3D reconstruction results ifeSchences Chapter 15 3D Reconstruction of Sources 155 Table 15 5 DLIT or FLIT 3D reconstruction results ltem Final voxel size mm Number of voxels Reduced Chi2 Description The voxel size length of a side mm that produces the optimum solution to the DLIT or FLIT analysis The number of voxels that describe the light source s A measure of the difference between the computed and measured photon density maps at the optimum solution A smaller y2 value indicates a better quality of fit
32. 6 5 Helenie hetae he Gaa ular Septet Al Retreat Figure 15 6 Adjusting the Threshold 148 Living Image Software User s Manual To select particular regions for reconstruction 1 Open the Data Preview window as shown in Figure 15 6 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 a appears 4 To automatically select all pixels in a source right click with the region with the pencil tool Alternately 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 If the pencil tool markings are applied to the image only the marked pixels are included in the analysis a Data al jure bre rit Thread Tak Piercy Tabanlar Treks i Grow Ci Erme Panina ae Laga capati Ma w Dpi Use these tools to select particular image data to include in the analysis Figure 15 7 Selecting regions to include in the reconstruction Table 15 4 Region Selection Tools Item Description Draw Choose this option to display the pencil tool 4g 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
33. Chery Coad eee WE ets E Sequence View window Check the image min and max in the color scale to determine whether the signal of interest is above the noise level and below CCD saturation Figure 4 19 Sequence View window amp Tool Palette Double click an image in the sequence to open it in a separate image window NOTE The Spectra window is available if the acquisition included multiple wavelengths The Spectra window provides a convenient way to view probe spectra from the factory installed library and ROIs For more details see page 152 Table 4 4 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 For more details on measurement units see page 247 Use Saved If this option is chosen data are displayed using a user specified color palette For Colors 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 20 Living Image Software User s Manual Table 4 4 Sequence View window continued Item Description Layout Choose a display option for the images in a sequence Default Dynamic or Film Strip For example here is Film Strip mode Options Sort by Options for ordering images in the sequence window This option only applies to images that were ope
34. Chrerkay dees 6 3 teH Figure 8 30 Copy all rows in the ROI Measurements table to the system clipboard 86 Living Image Software User s Manual This page intentionally blank Y Caliper feSciences Q Image Math Creating a New Image Using Image Math 2 508 87 Subtracting Tissue Autofluorescence 2 2 0 ee eee ee ee 90 The Living Image software provides tools that enable you to mathematically combine two images to create a new image The primary use of image math is to subtract tissue autofluorescence background from signal Living Image Tool Use This Tool To Image Math M athematically combine add multiply subtract or divide two 87 user specified images Image Math Remove autofluorescence from a fluorescent image 90 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 98 9 1 Creating a New Image Using Image Math 1 Load an image sequence 2 Select Tools Image Math for lt name gt _SEQ on the menu bar Fi L pa EEJ o o Hi a N F haf a Quartier bo LT E lopi ho ll o fbn lee SPS oy ae Pa re ee i i 4 Mr TOO ee 7 ae Pont let lek Bade foe OO TLE iad Laa i ea TEF H GP Sn U hioi a TEE HE eS G Tm ew il oe VLR pi HEA el piad Piuma eT ee MENEN AT H ian aai aa Pia i a a STT ee ST ee a ae he oe 2 el E ape
35. 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 176 Living Image Software User s Manual Table 15 12 3D animation tools continued Item Description Preset Key Frame Determines how many key frames are used to generate one revolution in a Factor 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 FPS Frames displayed per second in the animation sequence a Creates a new key frame from the current 3D view oA Updates the selected key frame to the current 3D view Deletes a selected or all key frames from the key frame box a Moves a selected key frame up in the key frame box x Moves the selected key frame down in the key frame box Total Duration The total time of the animation sequence Play Click to view the animation sequence defined by the current key frames and animation parameters Record Displays a dialog box that enables you to save the current animation to a movie mov mp4 or avi Animation Setup Load Displays a
36. Editing the ROI Line 1 Double click the ROI that you want to edit The ROI Properties box appears Figure 8 20 ROI Properties T fx ROI ROL 2 w ROI Label ROI Subj ROI Info Image Number TLT20050624145507_006 w ROI Bka 1 k Lock Position xci pis 105 00000 Yc pix 163 00000 Angle ideg 0 0000 H Select color Brightness slider Lack Size Widtht pix 16 00000 Height pix 15 00000 Ll HLT Cross hairs in the custom color field TIULE EEEHEE EEHEEHE BEEHEE Tite ie ten Figure 8 20 ROI Properties dialog box and Select Color box a 2 To edit the ROI line thickness enter a new value in the Line Size box Alternately click the arrows 3 To change the ROI line color Click the Browse button a b The Select Color box appears T To select a basic color for the ROI line click a basic color swatch and click OK To define a custom color drag the crosshairs in the custom color field adjust the brightness slider and click Add to Custom Colors e To select a custom color for the ROI line click a custom color swatch and click OK y Caliper r ho hile Chapter 8 Working With ROI Tools 77 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 dn drag
37. FLPA DOL SEE rbk iana L Aarne hgn E Well Mate Tee i e emendes C Cis Sek priina k vaa i cote il A For kh ELIDIR 14101005 001 Figure 14 2 Well Plate Quantification window 4 Select the well plate dimensions from the Well Plate Type drop down list The first image in the sequence opens and a grid ROI appears on the image E a0 004 Figure 14 3 Well plate image with grid ROI 5 Adjust the grid ROI to closely fit the plate wells YP Caliper eSclences Chapter 14 3D Quantification Database 135 6 In the well plate table select the sample wells and click Set Figure 14 4 Clicking a row or column header selects the entire row or column Hell Plate Quantification Window P Hall Plate Quantification Window for Sequence DAH a _ So Chek MAGH oo oe Fhaoraphore Type CE Cpe malande LD Cale For Sequence D ida L4101005_400 Chk LAN O00 COL Plrophore Trpe Ce Dye mabnbe C Cade pi aa Type 7 i aa Type 7 MFhimiranmani _ ape be Soge Mano rere Sample Welk 30 BA Ose Ly Appt be Sequtnen Wied Hate enna Pees rati Wied Hias nels es rai Tat paAHSA pnd antar dua vais in aak Tat paAHSA bnd anta ltt vis in calle B A Por Cide BLODOSOA 4102005 00 Canard Pop Click BLD 14109 006 004 Figure 14 4 Select the sample wells and enter the number of cells or molecules 7 Enter the dilution values in the table cells 8 Choose the Apply to Sequence option 9 Choose the Backgroun
38. Figure C 3 This produces a smaller image that contains one fourth the pixels compared to an image at binning 1 However due to summing the average signal in each pixel is four times higher than at binning 1 At binning 4 16 pixels are summed prior to read out Binning significantly affects the sensitivity of the IVIS Imaging System Binning at higher levels for example gt 4 improves the signal to noise ratio for read noise an electronic noise introduced into the pixel measurement at readout If four pixels are summed before readout the average signal in the summed pixel super pixel is four times higher than at binning 1 The read noise for the super pixel is about the same as it was for the individual pixels Therefore the signal to noise ratio for the read noise component of the image noise is improved by a factor of four Read noise is often the dominant source of noise in in vivo images so a high binning level is a very effective way to improve the signal to noise ratio Unfortunately binning reduces the spatial resolution in an image For example at binning 2 a super pixel is twice as wide as a pixel at binning 1 This results in a factor of two loss in image spatial resolution However for in vivo imaging the added sensitivity is usually more important than the spatial resolution Further since in vivo signals are often diffuse due to scattering in tissue little is gained by increasing spatial resolution For more backgro
39. NOTE You cannot overwrite a factory loaded configuration You can modify a factory loaded configuration and save it to a new name To delete a custom table configuration 1 Select the configuration from the User Lists drop down and click Delete NOTE Factory loaded table configurations cannot be deleted YP Caliper feSclences Chapter 8 Working With ROI Tools 85 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 select a folder and enter a name for the file txt then click Save To copy the table to the system clipboard Copy selected rows Select the rows of interest and click Copy Alternatively select the rows then right click the table and choose Copy on the shortcut menu All rows Click Select All and then click Copy Alternately press Ctrl A then right click the table and choose Copy on the shortcut menu ROI Measures imege Laver fetal Count Gang lournts Stes Cou pan Ceurks ILI SSL aS AL Lerereay Beet 2 JAHE 4 ote Love Dee TLT 20050624 L OT 1 Chrerkay 23H 6 Siete le Pde aH LT el SS act Leari En PE ge ot SALTEA tinea AAH Ceverlay ORY fe S eH ge G P a 3 over ay Sokak A EER stbcebeh iil Cilie eTil TLT 200500241 45507_003 th Say R2theeO0 3 225401 2 745e 01 TL eet Sei ie Leani l jjnelh 4 06 BS teil Ser ILI SSL aS rani Lerner nar UPAH 1 eH Gee LL ett TLT20050624145507_005
40. 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 Saving ROIs page 77 Auto ROI Parameters that specify how the auto ROI tool draws an ROI Note These are Parameters advanced options that are only available if Show Advanced Options is selected in the general preferences Threshold If the Auto All or Auto 1 method is selected the Threshold specifies the minimum per cent of peak pixel intensity that a pixel must have to be included in an ROI identified by the software Note After ROIs are drawn on an image if you modify the Threshold move the Slider or enter a new value the software automatically updates the ROIs Lower Specifies a multiple 1 to 10 of the color scale minimum that sets the lower Limit threshold for identifying an ROI For example if the lower limit 2 and the color scale minimum 1000 counts then the auto ROI tool will only draw an ROI on areas of 2000 counts or greater This helps create ROIs only in the visible range Minimum Sets the minimum size of an ROI measured in pixels For example
41. SE J Links E hird Piima ikea Hie oboe Sera z main Pear Figure 6 2 Kinetic Acquisition window 6 Click the Record button to start acquisition After acquisition begins the button changes to a Stop button a To stop acquisition click the Stop button a 2 The maximum vs time graph appears when kinetic acquisition begins and plots the maximum intensity signal in each frame The graph provides a convenient way to look for signal trends or select particular frames for viewing SS pr p H Kinetic Acquisition Control Pare di fe Disapharys Phiteg agh Ui irama Aipha tliey lund ag ar eigas j4 b ae sa M 11 Plax 1 Eee Enpre Tia i h T DOSE Catena Pier Fae Filer ipen Fl Larg Lai e Click a point in the graph to view Photoorach Uk Levak the corresponding image 40 7 frame OEEEBEELI e Put the mouse pointer over the graph to view a tooltip that Shows the frame number and time Maximum Vo Time e Right click the graph to view the Nhl Akl hs e available shortcut menu of mite graph display options Fi a Tune ak POF Figure 6 3 Maximum vs time graph YY Caliper ife Sciences Chapter 6 Kinetic Imaging 45 Table 6 1 Kinetic acquisition settings Item Description Fe pura ae Le Lerner ior ka H Cverlay Leise Ha E8 i Epopee Time imas E a hiie 4 w Erpe a ILPA Goes oo wr Etg Fiber Drion Pier pe
42. Surface If an experiment includes luminescent and fluorescent reporters DLIT or GADLIT and FLIT reconstructions can be displayed on one surface NOTE If the DLIT GADLIT and FLIT image sequences are acquired during the same session the generated surfaces are nearly identical 1 Load a DLIT or GADLIT reconstruction and a FLIT reconstruction 2 Choose one of the reconstructions click the By button and select Copy source voxels If copying a GADLIT source select Copy source surface 3 In the other reconstruction click the pa button and choose Paste source voxels or Paste source surface 15 7 Exporting a 3D Scene The items in the 3D View comprise a 3D scene For example the 3D scene in Figure 15 26 includes a surface and voxels The 3D scene can be exported to DICOM format and viewed in third party software a Sapana ie E IO Wew Spedra suja e mee o D Figure 15 26 3D scene 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 15 8 164 Living Image Software User s Manual 3D Scene Exporter Save DICOM as Single Frame DICOMs Slice Orientation Transaxial Slice vw C Export voxels using original resolution Parameters Total slices 256 s Slice spacing tmm 0 3970 Approx s
43. Y Caliper ifeSchences Appendix J Menu Commands Tool Bar amp Shortcuts 295 Table J 1 Menu bar commands and toolbar buttons continued Menu Bar Command Toolbar Description Button Acquisition Fluorescent Background gt View Available Fluorescent Background Acquisition gt Fluorescent Background Clear Available Fluorescent Background Window Close Window Close All Window Cascade Window Tile Window 1 dmage or Sequence name gt Window 2 lt image or Sequence name gt Window etc Window gt Other Windows Browser Window Help User Guide Help gt About Living Image Help License information Help Plug in Information Opens a dialog box that displays the fluorescent background measurements for the system If a fluorescent background is selected the Sub Fluor Bkg option appears in the control panel Choose the Sub Fluor Bkg option to subtract the user specified background measurement from the image data Opens a dialog box that enables you to remove the fluorescent background measurements from the system Closes the active image window Closes all image windows Organizes the open image windows in a cascade arrangement See page 99 Organizes the open image windows in a tiled arrangement See page 99 A list of the open image windows Click a window in the list to make it the active window indicated by a check mark If the Liv
44. Yet pix 4ngle deg Lock Size Width is Height pix 20 00000 Line Size Line Color Done x Drop down list of subject ROIs in the image Enter label information here for the subject ROI selected above ROI Properties Info tab Shape Contour Lock Position sci pie 112 00000 vt 4ngle deg 0 0000 Lock Size width pi Heightt pix z0 00000 Done ck BkgROI SubjROI Info Information about the ROI selected from the drop down list above Figure 8 17 ROI Properties Subj tab and Info tab The items in the ROI Properties box depend on the type of ROI selected in the image For more details see Table 8 3 page 72 Table 8 3 ROI Properties dialog box Item Description ROI A drop down list of ROIs in the active image or image sequence To select an ROI double click the ROI in the image or make a selection from the drop down list ROI Label Click to edit the selected ROI label name Image Number A drop down list of open images BKG ROI tab The Bkg ROI tab shows a drop down list shows all average background ROIs in the click number selected above that can be linked to a user specified measurement ROI or subject ROI selected from the drop down list at the top of the dialog box 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 avera
45. 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 74 Living Image Software User s Manual Table 7 1 Living Image browser continued Item Description Load as Group Enables you to select particular images that you want to view as a sequence The images may be acquired during different sessions To select adjacent images in the browser press and hold the Shift key while you click the first and last file in the selection To select non adjacent images in the browser PC users Press and hold the Ctrl key while you click the images in the browser Macintosh users Press and hold the Cmd key apple key while you click the images in the browser Note The Load as Group option is only available when two or more images non kinetic are selected in the browser Load Opens the selected image or image sequence Remove Removes a user selected image sequence s from the browser Close Closes the Living Image browser Opening Data from the Menu or Toolbar NOTE To open a recently viewed file select File Recent Files on the menu bar 1 Click the Open button G amp on the toolbar Alternately select File Open on the menu bar 2 In the Open box that appears double click the file of interest Al
46. a two dimensional array of numbers Each element of the array pixel is associated with a number that is proportional to the light intensity on the element A charge coupled device CCD camera used for scientific imaging is essentially an array of photo sensitive pixels and each pixel collects photons during an image exposure The subsequent electronic readout provides a photon intensity number associated with each pixel In a bright area of the image more photons are detected and the photon intensity number is greater than the number in a dim area of the image The image data can be visualized in different ways including pseudocolor images generated by the Living Image software contour plots or isometric displays Graphic Image Data A graphic image is a two dimensional array of pixels with a color assigned to each pixel There are several schemes for digitally storing the images For example a common scheme assigns a red green blue RGB color code to each pixel The RGB code defines how much of each color to apply in order to create the final pixel color Color photographs or color screenshots are examples of RGB images An RBG image is also a two dimensional array of numbers but unlike a scientific image the numbers are only color codes and are not related to light intensity A graphic image can be exported to a graphic display application Pseudocolor Images An image can be generated from scientific image data by assigning a c
47. and maximum court 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 Double click the color swatch to open a color palette that enables you to select a new color for the figure legend Label The name of the image selected in the image list To edit the name double click the name in this box Right click the label name to show a short cut menu of edit commands for example Cut Copy Paste Copies the composite image to the system clipboard 106 Living Image Software User s Manual Table 11 3 Composite image window continued Item Description Opens the Print dialog box 11 3 Spectra Window The Spectra window plots the normalized spectra of the unmixed results E 111 20060406164950_SEQ E 5 Ed Sequence View Spectra Unmixed Images Composite Normalized Legend _ Red Shift r Spectrum List a UWI 2 m Tiesu F 5 too Normalized Amplitude On Type Mame Label Color Lint TissucnF P Lime 2 uzez E List of the spectra in the results Add remove a check mark to show hide Spectrum Plot T60 TsO 00 Wavelength rrn Figure 11 6 Spectra window Table 11 4 Spectra window Item D
48. and select Auto All to automatically draw ROIs in the image using the auto ROI parameters Click and select Auto 1 to automatically draw one ROI at a user selected location using the auto ROI parameters For more details on using the auto ROI features see page 61 Click to display the ROI Measurements table or compute intensity signal in an ROI 2 e Click to display a drop down list of options to delete an ROI s in the active image For more details see page 78 Note These commands do not delete the ROIs that are saved to the system listed in the M enu Name drop down list Apply to Sequence Choose this option to apply the selected ROI to all images in a sequence ifeSchences y Caliper a Chapter 8 Working With ROI Tools 59 Table 8 2 ROI tools continued Item Description Type Choose the ROI type from the drop down list Measurement Measures the signal intensity in an area of an image Average Bkg Measures the average signal intensity in a user specified area of the image that is considered background Subject ROI Identifies a subject animal in an image The software automatically associates a measurement and an average bkg ROI that are included in the same Subject ROI Using this type of ROI is optional Save ROIs Name 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
49. appears and displays the selected image 3 To save a snapshot of the current image click the Export Graphics button in the image window In the dialog box that appears select a destination folder enter a file name select a file type and click Save 54 Living Image Software User s Manual This page intentionally blank gr Y Caliper r Working With Data Opening Data oaoa 44K Owe RRR eR REE ee KE CO HO 71 About the Image Window amp Tool Palette 2 50 58 258 75 Viewing Image Information 0 eee ee a a 77 Adjusting Image Appearance 2 eee ee 79 Correcting Image Data 2 ee 81 Viewing Intensity Data amp Making Measurements 2684 83 Creating a Transillumination Overview 2 eee ee ee ee 90 Overlaying Multiple Images 2 2 ee ee ee 91 Rendering Intensity Data in Color 0 00 eee ee ee es 93 Exporting or Printing Images 22k dae ee a a ew H amp S 95 Editing an Image Sequence 2 0 eee ee ee ee ee 97 Creating an Image Sequence from Individual Images 98 7 1 Opening Data You can open load data from the e Living Image browser see below e Toolbar or menu bar page 74 Multiple data sets can be open at the same time NOTE To open a recently viewed file select File Recent Files on the menu bar Loading Data From the Living Image Browser The Living Image Browser provides a convenient way to browse and previe
50. cannot exceed 5 Sort Choose this option to automatically sort the unmixed spectra in ascending order of their center wavelength Force Choose this option to force the first component to non zero throughout the image Denoise by PCA Determines how much of the data will be filtered by principal component analysis Stronger denoising means less principal components will be used in the data and more details will be lost Stronger denoising also may slow down the unmixing Unimod Tolerance The threshold for the unimodality constraint It is the percentage of overshoot allowed for the second spectral peak PCA Mode Standard Principle component analysis performed on the original data Correlation Principle component analysis performed on the correlation matrix of the original data Covariance Principle component analysis performed on the covariance matrix of the original data n Explained Variance Click to display the PCA variance plot Figure 11 10 Biplot Click to display the biplot graph Figure 11 11 Update Click to redo the spectral unmixing results with updated constraints YY Caliper ifeSchences Chapter 11 Spectral Unmixing 111 PCA Explained Variance The PCA Explained Variance histogram shows the part of variance y axis that can be explained by a number of principal components x axis A TTA Se C Seapets ew Spala UFtaces red Tai BA Hipki FCA Erian Figure 11 10 PCA explained variance hi
51. dialog box that enables you to export the active view as a graphic file wy Takes a snapshot that is displayed with the data in the Living Image Browser For more details on the browser see page 71 Cn ae mrke Pie ine fees Pit i Wi a m G Erei A a Ea Fe LH Enpe tS Soe WO Loree y ee RS dd Sita Bett 102 Soe TD Lae the ce re DHS od aoe Living Image browser smo Laadi dei 1 pet be int POT 4 Oo Tir Ihi E i psia N Lidyana dey a U m ar IEG LPC is oe tim Tiki Er if Sot M LOL doe iy 2 Li pa ha IEG DCDC ja eiim Iii Dip j ipia 2 Lee iey a LT p hae IED ICIC a le Sy Ihi Eip 19 ipebi PS PL does ey a LP p ha i acie Bimm ie Eee Prva Label Set A lt Ekot Brome ris isst Fema Che Lacuna C Eero nd ete r egg Cat a A L j a i a cho Snapshot 76 Living Image Software User s Manual Table 7 2 Image window continued Item Description Display A list of image types available for display for example overlay For more details single image the different types of image displays see Appendix A page 205 Note If the acquisition included more than two imaging modes for example luminescent x ray and photograph additional drop down lists appear so you can choose any two images to overlay Display C Luminescent on BY Photograph hi To change the foreground or background click the button or select from the drop down list Color Scale Provides a reference for the pixel intensities in a lumi
52. 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 Viewing a Preset Animation Preset animations are factory loaded animation setups They include predefined key frames which are used to generate the animation Table 15 13 Preset animations Name Choose This Animation Setup to Spin CW Rotate the 3D reconstruction clockwise Spin CCW Rotate the 3D reconstruction counterclockwise Zoom In Magnify the 3D reconstruction Fade In Increase opacity from 0 100 Fade Out Decrease opacity from 100 0 1 Open the reconstruction results 2 Select properties to display in the 3D View window for example organs sources surface or photon density maps 3 In the 3D tools click the Animate tab Y Caliper Yi 3D Tools Surface Source Registration Animate Preset Animations Presets Spin CW on 4 Axis hz Frame Factor 1 Snimation Setup Time Scale E 7 x 7 m Play 7 T mi D o Figure 15 37 3D animation tools Chapter 15 3D Reconstruction of Sources 177 Select a preset animation For more details on the animation tools see Table 15 12 page 175 4 If necessary clear the key frame box click the button and select Delete All 5 To view a preset animation make a selection from the Presets drop down list See Table 15 13 for a d
53. e 10 15 Width cm Figure D 4 Illumination profiles at different FOVs Measurements were taken at the center of the FOV on the IVIS Imaging System 100 Series Displaying fluorescent image data in terms of efficiency eliminates the variable excitation light from the measurement and enables a more quantitative comparison of fluorescent signals When you select efficiency for the image data Figure D 2 the software normalizes the fluorescent emission image to a reference image and computes Efficiency Radiance of the subject I umination intensity 254 Living Image Software User s Manual Prior to instrument delivery Caliper Life Sciences generates a reference image of the excitation light intensity no emission filter incident on a highly reflective white plate for each excitation filter at every FOV and lamp power The data are stored in the Living Image folder Image efficiency data does not have units The efficiency number for each pixel represents the fraction of fluorescent photons relative to each incident excitation photon and is typically in the range of 10 2 to 10 When ROI measurements are made the total efficiency within the ROI is the efficiency per pixel integrated over the ROI area so the resulting units of total efficiency is area or cm2 D 3 Flat Fielding Flat fielding refers to the uniformity of light collected across the field of view FOV A lens usually collects more light from the center of the FOV than at the
54. edges The Living Image software provides a correction algorithm to compensate for the variation in the collection efficiency of the lens This enables uniform quantitation of ROI measurements across the entire FOV To apply the correction algorithm choose the Flat Field Correction option in the Corrections Filtering tools The algorithm multiplies each pixel by a predetermined scale factor The scale factor for each pixel depends on its distance from the center of the image The scale factor near the center of the field of view is one but can be up to two or three near the corners on the VIS Imaging System 100 Series The IVIS Imaging System 200 Series has a larger lens with a smaller flat field correction You may notice an increase in noise near the edges and corners of the FOV when flat field correction is applied this is normal D 4 Cosmic Ray Corrections Cosmic rays are extraterrestrial high energy particles that register a false signal on a CCD detector Cosmic rays as well as other sources of ionizing radiation cause infrequent interactions a few per minute on the CCD These interactions result in large signals that are usually isolated to a single pixel making them easy to correct The Living Image software searches for isolated high amplitude hot pixels and replaces them with a collective average of surrounding pixels The Cosmic Correction option should always be selected for in vivo image data because hot pixels can signific
55. edit a parameter in the sequence editor 1 Double click the cell that you want to edit ry WE cgin Contrel Panel a Peete Pape lie i j Pp ae zii Pe Exon Fine sy amala juan a apa oe E imago aard cat iad C Deena Phonsgrantie batte Gjat Maaa 8 CG gre e m ou Hio E LS D hio z LS i z 1 bao ho im 5 GA ad z Serie 154 om Subect hehe gt m i i i ain mr aa e Pona dba ahja a ee Tencer sure TD scctes iii Forai Figure 4 23 Control panel and sequence editor era _ EA Io ae 2 Enter a new value in the cell or make a selection from the drop down list To apply the new value to all of the cells in the same column click Apply to an 3 Click outside the cell to lose focus To edit a parameter in the control panel 1 In the sequence editor select the row that you want to modify 2 In the control panel choose new parameter values and or imaging mode 3 Click in the sequence editor Inserting Images in a Sequence Method 1 1 In the sequence editor select the row next to where you want to insert the image 2 Set the imaging mode and parameters in the control panel 3 To insert the new image above the selected row click Gj insert Method 2 Select the row s of interest and right click the sequence editor to view a shortcut menu of edit commands y Caliper imagna wizard css E CI Dspiy Photograche settings Mije ope U
56. ee Fae Poo Pr bhe Bhgi 2 GALT 20 Ripi Figure 8 7 Automatically drawing measurement ROIs detected by the software 3 Click the Measure button in the ROI tools to show the ROI Measurements table 62 Living Image Software User s Manual RO deacons S ree Imaz Number Image Laye Total Count Avg Counts Stdev Coun Min Courts Max Counts DEEN DF J l J A eer a TLT20050624145507 a Serer r ia E s090403 TLTANnSne 24145507 AH F Ta Araian MT20050624145507 a rrin d 1 3198403 j EE TL ZUe 2414250 Oe 3 o AL SIHH H e TLT20050624145507 _006 Curvborrsined Selene Lites cous Figure 8 8 ROI Measurements table 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 80 4 Double click a thumbnail to view the ROI measurements on the image Figure 8 9 iE t iit g K inte tern lpp ber E MESTIE a per i iea fpertre Panar Fira eos ee te Tapera Fai tam Riy j ELT 1 es eal ew Te a T ee i e auaum ma Pae mE en Figure 8 9 ROI measurements on image To automatically draw an ROI at a user specified location 1 Open an image 2 Click an ROI shape button Circle 2 Square m or Contour and select A
57. gt 30 Tonks gt Spectral Unmising Bad Photon Density fit The optical properties or source spectrum may have been chosen erroneously For example for mice M uscle optical property is appropriate while XPM 2 is only appropriate for the Caliper phantom 182 Living Image Software User s Manual YP Caliper feSciences Appendix A IVIS Acquisition Control Panel 229 Appendix A IVIS Acquisition Control Panel Control Panel 229 Manually Setting the FOCUS 4 6c se eawweeve ewe Siaueaee eveuaee 233 A 1 Control Panel The control panel provides the image acquisition functions Figure A 1 Luminescence imaging settings Fluorescence Imaging settings Photographic Imaging settings Structured light imaging settings X ray imaging settings To acquire an image using auto exposure click the 4 arrow and select IVIS Acquisition Control Panel BE Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter Field of view System Status Idle Acquire Galen Subject height cm Sequence Setup Figure A 1 IVIS acquisition control panel auto exposure selected 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 Item Description Luminescent Choo
58. image label Figure 4 19 ES a i a et a Figure 4 17 Edit Image Labels box 3 In the Edit Image Labels box enter information about the image and click OK If you do not want to enter image information click Cancel 4 If this is the first image of the session you are prompted to enable the autosave function Living Image 4 0 Do vou want bo enable auto saving of acquired data For this session This can be changed anytime From the Acquisition menu Figure 4 18 Autosave prompt 5 To enable autosave click Yes in the prompt and choose a folder in the dialog box that appears If autosave is enabled all images acquired during the session are automatically saved to a user selected folder You can choose a different folder at any time select Acquisition Auto Save on the menu bar Image acquisition proceeds The Sequence View window appears and displays the images as they are acquired The Tool Palette appears when acquisition is completed Figure 4 19 6 To stop acquisition click the Stop in the control panel To pause acquisition click Pause in the control panel y7 Caliper feSclences Chapter 4 Luminescent or Fluorescent Imaging 19 Plo Bh View beb Agiis Window Heb gt U H wt A g K rcar w C Appir te all si nTM zB SEO OOOO O Sequence View Spectre nts Cour Seguint TLE AME F jentia O i aaa beads Sout Ciak Wi 200 Beta Il SEE Comment Dia Label Teo bier ete or Gs Aad AA di
59. items in the control panel depend on the particular IVIS Imaging System and the imaging mode selected luminescent or fluorescent Image Setup or Sequence Setup mode For more details on the control panel see Appendix A page 229 The IVIS Imaging System is ready for imaging after the system 1s initialized and the operating demand temperature of the CCD camera is reached locked 3 4 About the IVIS Acquisition Control Panel amp Auto Exposure Feature The control panel Figure 3 5 provides the image acquisition functions For details on the imaging parameters in the control panel see Appendix A page 229 The auto exposure setting is useful in situations where the signal strength is unknown or varies widely for example during a time course study When you choose auto exposure Figure 3 5 the system acquires an image at maximum Sensitivity then calculates the required settings to achieve as closely as possible an image with a user specified target max count If the resulting image has too little signal or saturated pixels the software adjusts the parameters and takes another image In most cases the default auto exposure settings provide a good luminescent or fluorescent image However you can modify the auto exposure preferences to meet your needs For more details see page 239 YP Caliper Mccann Chapter 3 Getting Started 19 To acquire an image using auto exposure click the arrow and select Auto Luminescence
60. limited to the topside of the object facing the lens H 2 Converting Light Emission to a Photon Density Map The input data to the FLIT algorithm for 3D reconstruction of fluorescent light sources includes e A surface that defines the surface of the subject e A sequence of images acquired at different transillumination source positions using the same excitation and emission filter at each position Use the Imaging Wizard to acquire the images ifeSciences Caliper Appendix H DLIT amp FLIT Reconstruction of Sources 285 The input data to the DLIT algorithm for a 3D reconstruction of luminescent light sources includes e A surface that defines the surface of the subject e A sequence of two or more images of the light emission from the surface of the subject acquired at different filter bandwidths Table A 1 Use the Imaging Wizard to acquire the images Table A 1 IVIS System filters for luminescence amp fluorescence tomography IVIS Imaging System Filters Bandwidth nm 200 Series 6 emission filters 550 670 nm 20 Spectrum 10 excitation filters 415 760 nm 20 18 emission filters 490 850 nm The IVIS Imaging System 200 Series and the IVIS Spectrum are absolutely calibrated so that the electron counts on each CCD pixel can be mapped back to the surface of the object to produce an absolute value of the surface radiance photon s cm steradian from each imaged surface element Figure H 3 Entrance Pupil
61. luminescent image can significantly improve the signal to noise ratio The loss of spatial resolution at high binning is often acceptable for in vivo images where light emission is diffuse For more details on binning see Appendix C page 246 Recommended binning 1 4 for imaging of cells or tissue sections 4 8 for in vivo Imaging of subjects and 8 16 for in vivo imaging of subjects with very dim sources Sets the size of the camera lens aperture The aperture size controls the amount of light detected and the depth of field A larger f stop number corresponds to a smaller aperture size and results in lower sensitivity because less light is collected for the image However a smaller aperture usually results in better image sharpness and depth of field A photographic image is taken with a small aperture f 8 or f 16 to produce the Sharpest image and a luminescent image is taken with a large aperture f 1 to maximize sensitivity For more details on f stop see Appendix C page 245 A drop down list of fluorescence excitation filters For fluorescent imaging choose the appropriate filter for your application For luminescent imaging Block is selected by default If you select Open no filter is present For systems equipped with spectral imaging capability choose the appropriate emission filter for your application Note The excitation filter selection automatically sets the emission filter position A drop down list of fluorescenc
62. measurements 128 correction filtering tools binning 82 cosmic correction 82 dark background subtraction 82 flat field correction 82 smoothing 82 cosmic correction 82 cosmic ray corrections 254 counts 251 crop box 89 D dark background subtraction 82 dark charge 256 dark current 255 data graphic image 249 scientific image 249 Data Preview window 192 194 298 Living Image Software User s Manual detection efficiency 245 detection sensitivity adjusting the lens aperture 245 exposure time 246 field of view 246 DICOM file size limit 65 diffusion model 276 display units counts 251 efficiency 253 photons 252 DLIT sequence requirements 188 DLIT results 200 201 manage 203 DLIT FLIT troubleshooting 227 drift correction 255 E edit image label 78 edit image sequence 97 98 efficiency 253 262 265 266 electronic background dark charge 256 dark current 255 drift 255 read bias 255 EM gain 62 export image data 42 point source fitting results 171 surface 164 165 export image data 42 export images 95 97 exposure time 246 F f stop 245 246 fluorescent imaging 265 field of view 246 filter bandpass 263 fluorescent 264 filter spectra 263 flat field correction 82 flat fielding 254 FLIT sequence requirements 198 FLIT results 200 201 manage 203 fluorescence adaptive background subtraction 272 reconstruct 3D sources 198 200 fluorescence efficiency 265 266 fluorescence imaging components 261 263 flu
63. on the imaging system camera M anual Allows the user to choose a binning value 1 2 4 or 16 Auto Save Specifies the folder where images are automatically saved Click the button to select a folder Restore Defaults Click to apply the default settings B 4 Theme Preferences General Lear Mequstion Themis Ophea Properhes iaga Yew AD wie olor Falete linearer Eai Pereyra Piporescert vrdliewuHee r Rewerce C Le saved coder piette while lading datasets Reston Gefais Bacher cued amp Tesh Coku REI Cii Makgro K Lurrarescent B Teat Color a Fiaweswrert a eeina k Caneel Apy Figure B 7 Image view preferences Table B 5 Image view preferences Item Description Color Palette Use these controls to select a color table for luminescent and fluorescent image data Choose the Reverse option to reverse the min max colors of the selected color table Y Caliper feSciences Appendix B Preferences 241 Table B 5 Image view preferences continued Item Description Use saved color palette If this option is chosen data are displayed using a user specified while loading datasets color palette For example after you load data specify a color table in the Image Adjust tools and save the data The user specified color table is automatically applied whenever the data are loaded Background amp Text Color Sets the colors for the background and text the image window To cha
64. open the Image Layout window select View gt Image Layout Window on the menu bar 2 To paste the active image into the Image Layout window click the x button To resize the image drag a handle W at a corner of the image 4 To reposition the image in the window drag the image 96 Living Image Software User s Manual A kmagi Laysui Winda Db Bk amp e amp eos h a Figure 7 25 Image Layout window Table 7 12 Image Layout window Item Description C Clears the Image Layout window Note If you do not clear the layout click the button before you close the Image Layout window the same window contents are displayed the next time the window is opened Opens a dialog box that enables you to save the Image Layout window contents to a graphic file Pastes the active image in the Image Layout window Copies the contents of the Image Layout window to the system clipboard Pastes the contents of the system clipboard to the Image Layout window Rectangle drawing tool Ellipse drawing tool Pointer tool 70 0 p 0 Arrow and line drawing tool AA 2 A E x Select an the item in the Image Layout window To move the item to the front CTSA or back in the window choose an option from the drop down list E bsi i w Deletes the selected image y Caliper 4 es iii Chapter 7 Working With Data 97 Table 7 12 Image Layout window continued Item Description A drop down list o
65. selected image parameters warrant a dark charge measurement large binning and long exposure time If a dark charge image is not required the read bias will be used If a dark charge is recommended the software provides the option of using the read bias measurement instead Since the read bias is by far the largest component of background using a read bias measurement instead of a dark charge measurement is often acceptable If read bias is used instead of a dark charge background the read bias image is stored with the image data rather than the usual background information 256 Living Image Software User s Manual Dark Charge If the amount of dark charge associated with an image is negligible read bias subtraction is an adequate substitute for dark charge background subtraction Dark charge increases with exposure time and is more significant at higher levels of binning A good rule of thumb is that dark charge is negligible if t B2 lt 1000 where T is the exposure time seconds and B is the binning factor Under these conditions dark charge contributes less than 0 1 counts pixel and may be ignored Dark charge refers to all types of electronic background including dark current and read bias Dark charge is a function of the exposure time binning level and camera temperature A dark charge measurement should be taken within 48 hours of image acquisition and the system should remain stable between dark charge measurement and im
66. signal can be subtracted from ROI measurements For more information on ROI measurements see Chapter 8 page 110 Note that the background light emission is not uniform over the entire animal In Figure E 2 images of control animals mice show a somewhat higher background component originating from the abdominal and thoracic regions Therefore care must be taken when selecting a representative background area Photograph Luminesce Photograph Luminescent nt overlay overlay Figure E 2 Background light emission Background light emission from a female white furred Swiss Webster left and a female nude Nu nu mouse right Usually only very low signals at the highest level of sensitivity require this type of background subtraction For more information on how best to handle these types of measurements please contact Caliper technical support 260 Living Image Software User s Manual This page intentionally blank Y Caliper Appendix F Fluorescent Imaging Description and Theory of Operation 0 a a a a a 261 yoo ela Ga ee Gee ee E E A E E EE E E E E eG 263 Working with Fluorescent Samples a a ees 264 Image Data Display aoaaa aa a a 265 Fluorescent Background a 267 Subtracting Instrument Fluorescent Background 271 Adaptive Background Subtraction 2 056 eee ee ee es 272 Subtracting Tissue Autofluorescence Using Background Filters 273 F 1 Description a
67. single image photograph and read bias Save Accumulated Image Saves the accumulated signal for the selected frames tiff Save Kinetic Data Saves all selected photographic luminescent or fluorescent images frames and the read bias image dcm The signal is not accumulated Done Closes the Kinetic Acquisition window Y Caliper ifeSciences Chapter 6 Kinetic Imaging 47 Accumulated Signal The Accumulate option enables you to view increasing signals in real time If you plan to accumulate signals it is recommended that you perform a test acquisition to optimize settings so that the photographic image luminescent or fluorescent signal is not saturated To perform a test acquisition 1 Confirm that the Accumulate option is selected Do not select the Auto color scale option 2 Start the acquisition click the Record button lt 3 Ifthe photographic image is saturated stop the acquisition click the 2 button and reduce the photograph light level CAUTION Extended acquisition of saturated images can shorten the life of the EMCCD and should be avoided 4 Restart the acquisition If necessary repeat step 3 and step 4 6 2 Viewing amp Editing Data in the Kinetic Acquisition Window After stopping acquisition you can view the data in the Kinetic Acquisition window 1 To start the playback click the Play button After playback starts the button changes to a Stop button m 2
68. 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 Click Next in the wizard In this screen you will select the subject type and signals components to unmix Spectral Unmbdng Wizard Choose the components to unmix Tip Cheese ths numer of components ba urenibe Pick thes significant backgroud horas lire Cheers ad Ue pokes kara lua Ue Labbe l E E rect feted I yiu bra uiar bbout th pobe or R is ett im th rary chee Linirn magng subject v Choose a subject type from the drop down list Barchyrarmd Sigrak NEEE ESEA Choose one or both types of background O Fani yal signals Prob Ini onimatsii Ifthe probe does not automatically appear in this list select the probe s If you are not sure about the probe that was used choose Unknown Match Probe Labets Lhe Cashar Huer al coea eris bo iA Fd Figure 11 3 Choose the component to unmix Select at least two but no more than four components to unmix Table 11 2 Spectral unmixing wizard choose the components to unmix Item Description Imaging Subject A drop down list of subject types 104 Living Image Software User s Manu
69. splits into four separate bundles that deliver filtered light to four reflectors in the ceiling of the imaging chamber Figure F 1 The reflectors provide a diffuse and relatively uniform illumination of the sample stage Analyzing image data in terms of efficiency corrects for nonuniformity in the illumination profile When the efficiency mode is selected the measured fluorescent image is normalized to a reference illumination image For more details on efficiency see page 253 The emission filter wheel at the top of the imaging chamber collects the fluorescent emission from the target fluorophore and focuses it into the CCD camera All IVIS Imaging Systems require that one filter position on each wheel always be open for luminescent imaging Y Caliper ifeSciences Appendix F Fluorescent Imaging 263 IVIS Imaging System Number of Emission Filter Wheel Number of Available Positions Fluorescence Filters 200 Series 24 two levels each with 12 positions 22 60 mm diameter Lumina 8 7 100 or 50 6 5 75 mm diameter F 2 Filter Spectra High quality filters are essential for obtaining good signal to background levels contrast in fluorescence measurements particularly in highly sensitive instruments such as the IVIS Imaging Systems Figure F 4 shows typical excitation and emission fluorophore spectra along with idealized excitation and emission filter transmission curves The excitation and emission filters are called bandpass fi
70. start the software 1 PC Users Click the Windows Start menu button and select All Programs gt Living Image Alternatively click the Living Image software icon Fg on the desktop Macintosh Users Click the Living Image icon Fy on the desktop or run the software from the application folder The main window appears Figure 3 1 16 Living Image Software User s Manual ig Par i eel FETTEN Figure 3 1 Living Image main window 2 Select a user ID from the drop down list or enter a new User ID up to three letters and click OK The control panel appears if the workstation controls the IVIS Imaging System Figure 3 2 For more details on the control panel see Appendix A page 229 M enu bar for more details see Appendix J page 293 Toolbar T orcii Gee fad eed N Iirimaa hE origen A ard ac wami Poet eee Activity window Figure 3 2 Living Image main window and acquisition control panel NOTE The Living Image software on the PC workstation that controls the IVIS Imaging System includes both the acquisition and analysis features The Living Image software on other workstations includes only the analysis features Macintosh users have access to the analysis features only E i Y Cali er Chapter 3 Getting Started 17 3 2 Initializing the IVIS Imaging System The imaging system must be initialized each time the Living Image software 1s started or if the power has be
71. that appears Living Image 4 0 Do you want bo enable auto saving of acquired data For this session g S This can be changed anytime From the Acquisition menu Figure 5 12 Autosave prompt appears if this is the first acquisition of the session Image acquisition proceeds The Sequence View window appears and displays the images as they are acquired The Tool Palette appears when acquisition is completed Figure 5 13 5 To stop acquisition click the Stop in the control panel To pause acquisition click Pause in the control panel YY Caliper 4 Pe oe Chapter 5 X Ray Imaging 37 Pie Eat ata ee equi Wicca Hisi E ye amp Lo k Unn beiribh O p ol 4 0 20090623147613_S10 G sequence view Tool palette Linde Se ee Ol C e Seemed Coders HL P0000K75147613_007 Uns Bader ieee e eit erli map P ea edd Tu Jun 2 a aa Gy Liebig Layo Leey S Lae ee erin Image label Tere Be A a li Chart Comet NE 2 Avi Epi Anma Murti coira Epia 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 Figure 5 13 Image sequence and Tool Palette Double click an image in the sequence to open it in a separate image window Table 5 4 Image window Sequence view Item Description Units Select the measurement units for the image display from this drop down list The available units depend on the type of image
72. the menu bar 3 In the dialog box that appears click Add Organ Files Figure 15 35 174 Living Image Software User s Manual P Import Organ Atlas Select Shan Mesh Senne Mires Co Pcie oe Lite Urpan Filer elect Shan Piast Serer abn fired Cin edifices Figure 15 35 Import Organ Atlas dialog box 4 In the next dialog box that appears select all of the files iv dxf stl that you want to include in the atlas one file per organ and click Open 5 In the Select Skin Mesh drop down list select the skin organ file which must include skin in the file name 6 Click Generate Mesh Coefficients Enter a name for the atlas and click Save Organ Atlas The organ atlas atlas is created and is added to the Organ Atlas drop down list in the 3D tools Registration tab y Caliper Per ae Chapter 15 3D Reconstruction of Sources 175 15 12 3D Tools Animate The Living Image software can create an animation from a sequence of 3D views or key frames For example an animation can depict a rotating 3D scene Figure 15 36 The animation series of key frames can be recorded to a movie file mov mp4 or avi I0 Tools Surface Cre Depar ations Dias d Presets Sota a a We ia Fr i Faler Parner feet Keyframe 1 Keyframe 2 Keyframe 3 Keyframe 4 Keyframe 5 EEA om The L box shows the key frames in the current animation setup Click a key frame in Frama Per Seam l
73. the tissue autofluorescence without exciting the fluorophore To reduce autofluorescence signal in the primary image data use the image math tool to subtract the background filter image from the primary excitation filter image For more details on tissue autofluorescence see Appendix F page 269 The software computes Background corrected signal A B x k where A primary image acquired using the excitation filter B background image acquired using the background filter k primary signal background signal The background signal is obtained from a measurement ROI that is located in an area where no fluorophore signal is present The scale factor k accounts for different levels of tissue autofluorescence due to different excitation wavelengths and filter transmission characteristics After you acquire an image sequence that includes a primary and background image use the image math tool to subtract tissue autofluorescence For more details on acquiring an image sequence see Chapter 3 page 23 Y Caliper feSciences Chapter 9 Image Math 91 To subtract tissue autofluorescence 1 Load the image sequence that includes the primary and background fluorescent images LT AHO TOAST SEO ED Sequence view I Units Photons Use Sawad Colores TLT2005 1221160239 TLT2005 1221160320 Wims 1 2629 b Wir 2 2468 Tine 247610 l Tite 4 4309 Figure 9 3 Image sequence 2 Open either the primary or background image
74. to determine the X ray resizing coefficient Select a subject height which suits the region of interest or Y Caliper ii Chapter 5 X Ray Imaging 29 e Select Manual focus from the Focus drop down list For more details on manual focusing see page 229 OO If you want to acquire a photograph set the Photograph image settings a Put a check mark next to Photograph b Enter an exposure time or choose the Auto option c Confirm the binning and f stop defaults or enter new values 9 If necessary click mageSetup in the control panel to operate in single image mode NOTE In single image mode the Sequence Setup button appears in the control panel Click this button to set up Sequence acquisition For more details on setting up a Sequence see page 32 10 When you are ready to acquire the image click Acquire The Edit Image Labels box appears ns E fl agp lel lage Sel mi ee r Ei ie JERR The information you enter here appears 2 eee in the image label Figure 5 6 Comt Tipe ai o a ee ee a E T E A L Figure 5 3 Edit Image Labels box 11 In the Edit Image Labels box enter information about the image and click OK Figure 5 3 If you do not want to enter image information click Cancel Image acquisition proceeds During acquisition the Acquire button becomes a Stop button To cancel the acquisition click Stop When acquisition is complete the image windo
75. uses luminescent image data for surface elements that are less than the angle limit The default angle limit setting is 70 for the IVIS Imaging System 200 Series or the IVIS Spectrum For IVIS Spectrum or 200 Series data if there is significant signal on the side of the subject a larger angle of 70 85 can be used Spatial Filter Filters out the noisy data at the mouse edges A setting of 0 1 means that the analysis includes 90 of the data from the center of mass to the edges y Caliper Chapter 13 Point Source Fitting 129 ife Sciences Table 13 2 Params tab Item Description Parameter starting Note Selecting a tissue Properties tab automatically updates MuaEm values MusEm MuaEx and MusEx in the Params tab x y OFZ Source coordinates d F yield Power Fluorescence yield strength of illumination or luminescence source MuaEm Absorption coefficient at the emission wavelength MusEm Reduced scattering coefficient at the emission wavelength M uaEx Absorption coefficient at the excitation wavelength M usEx Reduced scattering coefficient at the excitation wavelength Restore Defaults Resets the model type algorithm starting parameters and algorithm options to the default values Mask A drop down list of ROIs in the selected image Select an ROI to compute only the source in the ROI Statistics Weighting Choose this option to apply a statistical weighting technique to help reduce the error associated with high radiance meas
76. vV Vv Vv Vv Vv Structure V v aa X ray V Kinetics V NOTE For details on your IVIS Imaging System please see the imaging system hardware manual Ee GE b bp W iiH t D a Unki caurte_ C ipei iy ad Double click an image in the Sequence View to open it in a separate window Figure 2 3 Example image sequence Overlay images luminescent image on photograph 8 Living Image Software User s Manual Table 2 2 Image types Imaging Mode Description Example Fi Photograph A short exposure of the subject illuminated by the lights located in the ceiling of the imaging chamber The photographic image is displayed as a grayscale image Luminescent A longer exposure of the subject taken in darkness to capture low level luminescence 9 emission The luminescent image is displayed in l l pseudocolor that represents intensity For more Luminescent image details on luminescent image data see Appendix D page 249 Overlay Luminescent image on photograph _ Fluorescent An exposure of the subject illuminated by filtered light The target fluorophore emission is captured and focused on the CCD camera gt p Fluorescent image data can be displayed in units of counts or photons absolute calibrated orin Fluorescent image terms of efficiency calibrated normalized For more details on fluorescence image data see Appendix F page 261 Overlay Fluorescent imag
77. we arrows or e Select use subject height from the Focus drop down list and use the the keyboard arrows to specify a subject height cm or e Select Manual focus from the Focus drop down list For more details on manual focusing see page 229 If necessary click mageSetup in the control panel to operate in single image mode NOTE In single image mode the Sequence Setup button appears in the control panel Click this button to set up sequence acquisition 8 To acquire an overlay image coregistered images for display after acquisition put a check mark next to Overlay NOTE To view the subject s inside the chamber before image acquisition take a photograph Uncheck the Luminescent option choose the Photograph and Auto options and click Acquire 9 When you are ready to capture the image click Acquire _ The information entered here appears inthe image label Figure 4 4 Ree ee ee ee ee eil Figure 4 2 Edit Image Labels box 10 In the Edit Image Labels box that appears Figure 4 2 enter information about the image and click OK NOTE You can enter image label information at any time during or after acquisition If you do not want to enter image information click Cancel 11 If this is the first image of the session you are prompted to enable the autosave function Y Calip er feSciences Chapter 4 Luminescent or Fluorescent Imaging 7 Living Image
78. you to export the active image data to DICOM format dcm 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 Opens a dialog box that enables you to save the voxels from the active data in Open Inventor format iv 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 Displays the Print box Displays the Print Preview box that shows what will be printed Shows recently opened data sets Note The number of files displayed can be set in the Preferences box Select Edit Preferences and click the Customize tab Closes the Living Image software Copies the active image window to the system clipboard Opens the Edit Image Labels dialog box that enables you to edit the label set information for the active data Opens the Preferences box Choose this option to display the toolbar Choose this option to display the status bar at the bottom of the main window Choose this option to display the Tool Palette 294 Living Image Software User s Manual Table J 1 Menu bar commands and toolbar buttons continued Menu Bar Command Toolbar Description Button View Activity Window View Image Information View ROI Properties View ROI Measurements View Image Layout Window View Acquisition Control
79. 0 Planar Spectral Image Analysis 97 Fic Edt View Tools Acquistion Window Hil SAHM BB MP niade frac vO ipy toa A Plat af Linear Fit R tul g Lang Moam Fik POM 2 Sa Oe es ee Figure 10 5 Viewing the linear fit graph Plot of Intensity Vs Lambda Intensi Lambda Flot for ROI 1 Normalized Flux a u Wavelength run Figure 10 6 Intensity graph The intensity graph displays a graph of the measured intensity in the selected ROI at each wavelength in the analysis The intensity is normalized to the selected source spectrum and the filter transmission properties To export graph data 1 Click the Export Data button fg 2 In the dialog box that appears select a directory for the data and enter a file name csv The data can be opened in a spreadsheet application such as Microsoft Excel 98 Living Image Software User s Manual 10 4 Managing Planar Spectral Imaging Results Go to the Results tab to select results that you want to view or manage Tual Palette z 5 Anikhga Progeerbies iea Spectral fesulbse Urara RO Depth mm Toke Phuc eheys i ROLI SI QL Lael i 159a ROZ JAHO O01 1 09e0 He Z lt Mot Linear FE Mot Intensiby Save Results Ame Spim l i gt Surface Topography D Point Source iting L gt GADLIT 30 Reconstruction Figure 10 7 Planar spectral imaging tools Results tab To save results 1 Select the results of interest
80. 02 Living Image Software User s Manual ae jE tt A g MP ca se C Apeh ba al TL 2004040461 64950_S60 a SS E a Lirios rh Ua Cats O use Savea Cer JE Excitation wavelength Emission wavelengths of the sequence G5 iz F F E F Figure 11 1 Sequence for spectral unmixing 2 In spectral unmixing tools click the Analyze tab and put a check mark next to the emission wavelengths that you want to include in the analysis 3 Click Start Unmixing The Spectral Unmixing Wizard appears and shows the purple data mask that specifies the analysis area By default the data mask includes the entire subject Special Ummidia Wizard w Special Ummi Wizard Select the data mask Select the data mask Deks Firkete C Photograph ok EE Dram Hik Enae L ENE P Default data mask applied by the software Data mask manually applied to a user selected area Figure 11 2 Selecting the data mask For more details on the data mask options see Table 11 1 W Caliper Chapter 11 Spectral Unmixing 103 4 If you do not want to analyze the entire subject draw a data mask on a user selected area using the data mask options Table 11 1 Data mask options Option Description Photograph If this option is chosen the software automatically draws the data mask so that it includes the entire the photograph MI Threshold If necessary use the threshold slider or arrows to adjust the mask
81. 14 1 Preparing amp Imaging the Samples 1 Prepare a well plate 4 x 6 6 x 4 8 x 12 or 12 x 8 well format that contains a dilution series of luminescent cells or fluorescent dye at four or more concentrations 2 Include at least four background wells that contain diluent only 3 Place the well plate on the IVIS stage positioning it so that it is centered and squared in the field of view 4 Acquire the images Bioluminescent samples Acquire one Open filter image of the well plate Fluorescent samples Acquire reflectance illumination Filter Scan images using the appropriate excitation and emission bandpass filters The well plate in Figure 14 1 contains a dilution series of a sample at four concentrations The image sequence is a filter scan set of images with the excitation filter centered at 465 nm for all the images and emission filter images centered at 520 nm 540 nm 560 nm and 580 nm a ToS Vi Dair Lint Redken Bificercy U Sawer Colors DEn e sn 7 3 c W i T 1 PT Emis EPfickercy pbbeciin ter E pki Figure 14 1 Well plate data 134 Living Image Software User s Manual 14 2 Creating a Quantification Database 1 Load the well plate image sequence 2 Select Tools Well Plate Quantification for lt name gt _SEQ on the menu bar The Well Plate Quantification window appears 3 Choose the Dye molecules or Cells option Well Pirr Ouantificatian Window For Seene
82. 2 Right click the measurement ROI and select Set BkG ROI to Bkg X on the shortcut menu that appears i hd Di me TOS id Ted eg e Se i Te Jahe fT Sp rra mi bii Lag Sell Ee op Method 3 a Right click a background ROI and select Properties on the shortcut menu b In the ROI Properties box that appears click the Bkg ROI tab and put a check mark next to Use as BKG for future ROIs in ee ee ft eee eel EE ina i OG a Pita PDE ih D Levee oe 0 Cb eee 8 7 Kinetic ROIs Kinetic ROIs help you track signal sources on an unanesthetized mobile subject The software automatically creates a separate ROI in each frame based on the user specified auto ROI settings As aresult kinetic ROIs are continuously displayed during kinetic data playback You can draw a kinetic ROI using any of the methods or shapes in Table 8 1 page 56 j Y Caliper Chapter 8 Working With ROI Tools 67 NOTE Large kinetic data sets may require more time to create plot and measure the ROIs because the software first applies corrections to a frame specified in the Corrections Filtering Tool Palette then draws the ROIs in the frame The process can be aborted at any time These steps provide a quick guide on how to apply a measurement ROI to kinetic data For more details about measurement ROIs see page 60 1 Open the kinetic data and click ROI Tools in the Tool Palette 2 In the ROI tools s
83. 2 sr uW cm 2 Transillumination fluorescence Fluorescence emission radiance per incident excitation power p sec cm2 sr mW Efficiency Fluorescent emission yield normalized to the incident excitation intensity epifluorescence radiance of the subject illumination intensity NTF Efficiency Fluorescent emission image normalized to the transmission image transillumination measured with the same emission filter and open excitation filter fluorescence 82 Living Image Software User s Manual Table 8 4 ROI Measurements table continued Item Description Image Attributes Make a selection from the drop down list to specify the click number image file information to include in the table Click attributes include label name settings and camera settings None Excludes image attributes from the table All Possible Values Includes all of the image attributes for example label name settings and camera settings in the table All Populated Values Includes only the image attributes with values in the table Living Image Includes all Living Image Universal label name settings in the table Universal ROI Dimensions M ake 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 c
84. 4 0 Do vou want bo enable auto saving of acquired data For this session wf This can be changed anytime From the Acquisition menu Figure 4 3 Autosave prompt 12 To enable autosave click Yes in the prompt and choose a folder in the dialog box that appears If autosave is enabled all images acquired during the session are automatically saved to a user selected folder You can choose a different folder at any time select Acquisition Auto Save on the menu bar Image acquisition proceeds During acquisition the control panel Acquire button becomes a Stop button To cancel the acquisition click Stop in the control panel The image window appears when acquisition is completed Figure 4 4 oe ee aia amp T K Ltr ore Tool palette 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 Figure 4 4 Overlay luminescent image on photograph in the image window Table 4 1 Image window Item Description Units Select the measurement units for the image display from this drop down list The available units depend on the type of image data For more details on measurement units see page 247 Display A list of image types available for display for example overlay For more details on the different types of image displays see Table 2 2 page 8 Note If the acquisition included more than two imaging modes for exam
85. 45910403 Cutleria Senter Lane Saat _ Figure 8 4 ROI Measurements table 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 80 58 Living Image Software User s Manual 8 3 ROI Tools Table 8 2 provides brief explanations for the ROI tools Tool Palette ype Measurement ROT Save ROIs ame ROL 2 K54 Delete Auto ROI Parameters hreshold Lower Limit Minimum Size Preview Restore Defaults gt Planar Spectral Imaging gt Surface Topography gt Point Source Fitting gt BLIT 3D Reconstruction gt GADBLIT 30 Reconstruction gt Spectral Unmixing Figure 8 5 ROI tools Table 8 2 ROI tools Description The ROI tools that appear in the Tool Palette depend on the type of ROI selected from the Type drop down list and whether an image or sequence Is active T Click to select the number of circle ROIs to add to the active image Click to select the number of square ROIs to add to the active image gt BE 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 feo Click
86. 49907_006 cer Waka Counts w pe Chelan k sy E w A al PB uns cm iii linning F Wih Lb on Hist 1b ci Image X Y Jase ey om image Dates 2 coua pae lE a Ta lamem B n OJI hance JP om Crop box position amp dimensions a4 Mamar Spectral Ternir o Solace Topography Pomel Simmer Filli gt BLIT 3D Reconstruction GADLIT 30 Reconstruction L Figure 7 17 Using a crop box to make measurements 2 When the mouse pointer changes to a draw a rectangle on the area of interest 3 To change the size or position of the crop box drag a handle at a corner or side of the box 4 To delete the crop box from the image click the fj button Table 7 9 Crop box position amp dimensions pen x y coordinates at the upper left corner of the box x y coordinates of lower right corner of the box Box width and height Distance Length of the diagonal from the upper left to lower right corner of the box 90 Living Image Software User s Manual 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 specified location in an image To apply a tag 1 Right click a location in the image 2 Select Insert Tag on the short cut menu Figure 7 18 Insert a tag on an image left move the tag label right To move a tag 1 Position the mouse pointer over the tag 2 When the hand tool appears
87. 8 Figure 4 10 Opening the Transillumination Setup dialog box Table 4 2 Transillumination Setup box Item Description Move Motors to Selected Transillumination motors will move to the grid location selected in Spot the Transillumination Setup dialog box Mask Grid points To When setting up a transillumination sequence choose this option to Subject automatically select only the grid locations within the subject boundaries Grid locations outside the subject are masked 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 Select a grid type from the drop down list 15x23 11x23 5x10 or 8x12 well plate Xenogen Sparse Mask 6x8x1cm 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 7 Confirm that the Lamp Level is set to High 8 Make a selection
88. 90813193532 Retired Images Retire gt lt lt Reactivate Images that have Move Up been removed from the active sequence Move Down Figure 7 26 Opening the Edit Sequence dialog box 4 In the Edit Sequence box that appears choose the image s to add or remove retire from the sequence Figure 7 26 98 Living Image Software User s Manual 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 To restore a retired image to the sequence select the retired image and click Reactivate Nn 6 To reorder the sequence select an image and click Move Up or Move Down NOTE The Move Up and Move Down buttons are only available when the sequence view window displays images in the default sort order If the TimeStamp or UserID sort order is selected the images cannot be reordered 7 When you are finished editing the sequence click Close The updated image sequence is displayed 7 12 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 For more details on browsing see page 71 NOTE Browse for individual images which may or may not be part of a sequence not image sequences Individual images highlighted blue in this exa
89. Choose this option to display the photon density using a log scale Simulated The photon density computed from DLIT GADLIT or FLIT source solutions which best fit the measured photon density M easured The photon density determined from the image measurements of Surface radiance Horizontal Profile The photon density line profile at the horizontal plane through the Subject at the crosshairs location Vertical Profile The photon density line profile at the vertical plane through the subject at the crosshairs location Position mm Horizontal Profile The y axis position of the crosshairs horizontal line Vertical Profile The x axis position of the crosshairs vertical line 160 Living Image Software User s Manual 15 5 Measuring Sources Here is a convenient way to measure the source total flux or total florescence yield or if calibrated the abundance in cells or picomoles The volume and center of mass are also reported in the 3D Tools Source tab Determine Source Center of Mass 1 Click the ot toolbar button and then drawing a box around the source 2 Click Center of mass in the 3D tools Source tab Figure 15 21 The integrated source intensity volume and center of mass are displayed in the Source tab The coronal sagittal and transaxial planes intersect at the center of mass of the selected source Figure 15 22 To select asource click the Measure Source button then draw a box around the source H TL
90. Color Selections Width 4 Restore Defaults Folder Locations Window Size amp Position Height 591 65 2 Mosk Recently Used Dataset History Apply Individual Color Scale For Sequences Display ROT Label 4s Measurement Show Transillumination Locations ud Photons Total Flux we Show Advanced Options P Counts Total Count w apa Figure B 1 Preferences dialog box 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 236 Living Image Software User s Manual B 1 General Preferences Prelercmics General Leet Peco Thats Tea Pr caer lies Start Up Derauks Chine Atheky Werden aee J Erara Dork Toni Pakis Saver Settings C Save Aost correched image Window ier Color Selecion width 9 55 Fobier Locations vindos Sipe k Position Height 3 65 Mot Recently Used Datasst Hitoy 5 Apply Mirua Co Seabee bor Serea Dipli POL Lebel as Mesar enart Shree Trabar Locaia Pietas Ta Flux F Shoe Adae Oplia Counts Total Court Figure B 2 General preferences 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 it a distance greater th
91. DLIT or FLIT algorithm see Appendix H page 279 3D Reconstruction Description Algorithm Diffuse Tomography DLIT DLIT provides a complete 3D reconstruction of the 143 luminescent source distribution within the subject DLIT places no constraints on the geometry or spatial variation of the source strength throughout the volume DLIT is a more versatile algorithm than GADLIT and is well suited for analyzing complex and spatially extended luminescent sources The 3D recontracting is presented as voxels If a luminescent calibration database is available the number of cells per source can be determined in addition to source Intensity photons sec Note DLIT is the recommended algorithm for 3D reconstruction of luminescent sources Genetic Algorithm Diffuse GADLIT is based on a simpler model than DLIT The source 143 Tomography GADLIT distribution is assumed to consist of one or more isolated regions of quasi uniform source strength The 3D reconstruction is presented as a Surface surrounding each source region GADLIT is well suited for analyzing simple and compact luminescent sources Note GADLIT is only available if the Show Advanced Options is selected in the user preferences See page 236 Fluorescent Tomography FLIT provides a complete 3D reconstruction of the 152 FLIT fluorescent source distribution within the subject The 3D reconstruction is presented as voxels If a fluorescent calibration database is ava
92. EQUENCES se sss cede eehaehabeeagkeeexeeeeeaaehaess 10 2 4 Image Display amp Analysis o oaoa a 11 3 GEHING Sl rted s es rirrd daadaa rianaire naa 15 3 1 Starting the Living Image Software o oo aa ee 15 3 2 Initializing the IVIS Imaging System aoaaa 2 ee ee ee 17 3 3 Checking the System Temperature nooo 17 3 4 About the VIS Acquisition Control Panel amp Auto Exposure Feature 18 3 5 Tracking System amp User Activity 2 2 ee 19 4 Luminescent or Fluorescent Imaging 0 0 08 08 2 8 ee eens 21 4 1 Acquire a Luminescent Image ooo a ee 21 4 2 Acquire a Fluorescent Image With Epi IIlumination 0 24 4 3 Acquire a Fluorescent Image With Transillumination 2 000000 08 2I 4 4 Acquire a Sequence Using the Imaging Wizard oaoa a L 30 4 5 Manually Set Up an Image Sequence onono aa a 37 4 6 Manually Saving Image Data aoaaa a 41 4 7 Exporting Image Data 2 aa 42 5 X Ray Imaging aaao a a REE Ee PE eee ee ee eG 43 5 1 Acquire an X Ray Image anaa aaa 43 5 2 Acquire an Image Sequence That Includes X Ray Images aoaaa aa 48 5 3 Measuring Relative Density aoaaa 55 6 Kinetic Imaging aoaaa e 59 6 1 Kinetic Acquisition ooo 59 6 2 Viewing amp Editing Data in the Kinetic Acquisition Window aoaaa 000 63 6 3 Saving Kinetic Data aaa aaa 65 oA Playing Kinetic Data os lt sa 22544 464444044 ceo eb nana 66 7 Workin
93. I I IS Acquisition Control Panel imaging settings Imaging Mode Exposure Tine Binning F Stop Excitation Filter Emission Filter M bedie e fhi Aee oen f Fluorescence E imaging settings pg puto HA e x T Photographic a imaging settings lv Ej Field of view Ic r Structured light X Rays will be produced imaging settings de when energized Service fio crm me Acquire Subject height 0 00 cm pe Sequence Setup Focus fuse subject height Temperature ll roc Initialize Figure 3 5 IVIS acquisition control panel System Status f X ray imaging settings NOTE The options available in the IVIS acquisition control panel depend on the selected imaging mode the imaging system and the installed filter wheel or lens option 3 5 Tracking System amp User Activity The Activity window shows the imaging system activities The software creates and saves a log of the system activities related to data acquisition for example the type and number of acquired images fluorescent lamp usage X ray tube accumulated usage and kinetic camera usage This information may be useful for Caliper field service engineers to understand the imaging system behavior over time or for troubleshooting You ll find the activity log at C Program Files Living Image e ee i Airtareater berg ap 6b e 10 7008 Li j r el jprm E mgar Ms Poured ned eied iiss H p Poa a Pheter heng La Fip aed Shaye motor Ac
94. L cel aire starting yaue S Linon Suefars Sapir RLE Dagi Ae bin nt PSF Peter Deere ar fe Eager Bees Sue Feel Mares LT Ghar r LTT JD Aachen _ 2 Taos o Spectral iriki Figure 15 5 DLIT reconstruction results Table 15 3 3D view toolbar Tool Image Tools Gop fae Description A drop down list of tools for viewing and working with the surface or DLIT results 0 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 toward the bottom of the window To zoom out right click and drag the Qo 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 P ahs E Displays a bounding box around the subject 146 Living Image Software User s Manual Table 15 3 3D view toolbar continued Tool Description ii Displays a grid under the subject it r 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 15 34 page 173 es Select this tool from the drop down list to display the perspective view Rotates the 3D reconstruction resul
95. LIT sequences Plot Tissue Properties Choose this option to display a graph of the absorption coefficient ua effective attenuation coefficient uor and reduced scattering coefficient u or usp Source Spectrum Choose this option to display the source Spectrum for DLIT reconstructions 244 Living Image Software User s Manual This page intentionally blank y Caliper ad p Appendix C Detection Sensitivity 245 feSciences Appendix C Detection Sensitivity CCD Detection Efficiency 2 2 ee a 245 BHI s 6 we GEE RA Ee Eee Ee Eee EEG Ow ee ee 246 Sess l e 24 64 KG RE RES OSEKEAE KEE RGD EE SHS EERE EHS 248 The parameters that control the number of photons collected signal and the image background noise determine the sensitivity of low light imaging To maximize sensitivity the goal is to increase signal and decrease background Several factors affect the number of photons collected including the lens f stop image magnification size and detection efficiency quantum efficiency of the CCD transport efficiency of the imaging optics and the image exposure time C 1 CCD Detection Efficiency IVIS Imaging Systems use a back thinned back illuminated CCD cooled to 90 to 105 C depending on the system This type of CCD provides high quantum efficiency of over 80 across the visible and near infrared part of the spectrum Figure C 1 shows detection efficiencies for several commonly used photon det
96. Lrg irinli it verlyy Chery 6 eH beet ANHE AHG EIR AO didit atiii 2 Mile SAT SHED Yao Lie exe 2 MMe Sexe II d N AHO iHi ai Latte aiie 057800 Dern Ser TOWE AARNE TANG BOOTS Iie 2 a EN F aas LAOH BIHE Aei iiie IT ae LIHO Didi Deri SHieit pee pemr z Bnl Droad Copy StA Pik Wi Conii Saeco Meer eens Tepes T imga irb AD oT T Cordis Esperi co Figure 8 27 ROI Measurements table 3 To include image information in the ROI table make a selection from the Image Attributes drop down list 4 To include ROI dimensions in the table select units Pixels or cm from the ROI Dimensions drop down list To create amp save a custom table configuration 1 In the ROI Measurements table click Configure The Configure Measurements box appears Fj Configure Measurements Leer Lote Cudbhoniend d uae S Mabie Air blabi There Arabe Comin Selected hems Total Counts fog Cee Click to reorder the available items in Arabesk Liver 1D Aang Arira Barnier Ariens Strain Gdey Fouad Pin Caimi Place Cindi Eol Mior nal cd Pes Hires onc Asay Derk Change ars Bang Eier Haag Phoorescemt Blog Courts Bang Radana piir Zeina Teipit debe pete Heaghiy poets Lever D Sequence If Date ened Tie Lanna Laii Peki of Vier ascending or descending alphabetical order Cii Correcbesd Items column headers currently in the t
97. OI measurements for all images click the arrow next to Current Frame and select All Frames then click the Refresh button Kinetic ROI measurements are displayed in a separate tab Ba DE m l a ei amp a y mre air tm at Freem L Rcd Fesmzmmi MPa e eia El Hot Inne eh CS PT a EA eo Ter ogee E la ee ee ies We Ps i ki hes Tir isis hib M dee ee Soe A li f nem LAT te Pee Jee eS E Laney a Figure 8 13 Kinetic image window and ROI measurements table Plotting Kinetic ROI Measurements The kinetic ROI plot provides a convenient way to view and compare kinetic ROI measurements across user selected image frames from the same or different kinetic data sets 1 Open one or more kinetic data sets 2 Draw kinetic ROIs on the data sets in which you want to measure and compare ROIs 3 In the ROI tools click the ROI Measurements button W The ROI measurements table appears YY Caliper ad p i Chapter 8 Working With ROI Tools 69 feSciences AD Re apa ee Erer UO Pier ere Piat trebe PC Heta renanis Phe Aaii D Meares Fi eeeunemernt iret Core w RN Bearer ints S elf Total Counts Vi Tine CY A Ss BT I Lig Total Commis FP A G I 2 Figure 8 14 Plot Kinetic ROI Measurements tab 4 Click the Plot Kinetic ROI Measurements tab Make a selection from the Measurement Unit and ROI Measurement drop down lists Select a data set and an ROI Click Plot ROI Measurements To add other ROI data to t
98. ROI for more details see Drawing an ROI Using the Free Draw Method page 63 e ROI size dimensions or position e Subject ROI ID information There are two ways to move an ROI on an image e Drag the ROI to a new location e Edit the settings in the ROI Properties box An ROI cannot be moved if it was created using the auto ROI tool or if the ROI position is locked To drag an ROI 1 Put the mouse pointer over the ROI so that it becomes a arrow 2 Drag the ROI 3 Release the mouse button when the ROI is properly positioned To move an ROI using the ROI Properties dialog box 1 Double click the ROI in the image The ROI Properties box appears and displays the position and dimensions of the selected ROI 74 Living Image Software User s Manual ROI Properties A fx ROI EKG 1 ki ROI Label BKG 1 Subj ROI Use as BEG For Future ROIs in O TLT 200506241 45507_006 O Entire sequence L Lock Position viel poe 120 47356 E l Position of the ROI Yet pie 109 36069 E selected in the image Angle deg 0 0000 E L Lock Size widtht pix 17 49013 E Height pix 17 04815 E Line Size Line Color Done Figure 8 18 ROI Properties dialog box 2 To set ROI position enter new Xc pix and Yc pix values in the ROI Properties box 3 To rotate the ROI clockwise enter the degrees in the Angle deg box and click outside the box 4 To lock the current ROI position choose the L
99. RR GS 66 Menado ROIS a ket bw eee beet HEE EHEC EE KERA EH HREMR ODI 69 Managing the ROI Measurements Table a ee eee 80 8 1 About ROIs A region of interest ROI is a user specified area in an image Figure 8 1 The ROI tools enable you to create three types of ROIs measurement average background or subject ROI Table 8 1 During a session the Living Image software records information about the ROIs you create and computes statistical data for the ROI measurements The ROI Measurements table displays the data and provides a convenient way to review or export ROI information Figure 8 1 For more details see Managing the ROI Measurements Table page 80 a Fe Et i A re K Ln Cha vege Mian all J Ti Fy ThE r ee ie gps ee iis yi em hs ita 7 Ore Bale i Spee TGS LT ae Wy ear hee eet Ce ee ee hE ROI Measurements table t Pe q i fas s o TEE b 7 j tal i T J J i t E ee h a f r IE z Ibr 3 me e Pe qa Hu a Figure 8 1 Example measurement ROIs and ROI measurements table 56 Living Image Software User s Manual Table 8 1 Types of ROIs Type of ROI Description Available ROI Drawing Methods Available Shapes Measurement ROI Measures the signal intensity in an area of an image e Manual e Automatic e Free draw Circle Square grid or contour Average Background ROI Measures the average signal Intensi
100. Registration 214 220 Source tab 213 214 Surface tab 211 212 3Dsurface export or import 164 165 generate 160 161 manage 164 view perspective 162 163 A accumulate signal 62 63 acquire image sequence using Imaging Wizard 30 34 image sequence with X ray images 48 55 kinetic data 59 single fluorescent image epi illumination 24 26 single fluorescent image transillumination 27 30 single luminescent image 21 24 single X ray image 43 48 activity window 19 adaptive fluorescent background subtraction 272 adjusting image appearance 79 animation 221 226 custom 224 225 edit an animation setup 226 preset 222 animation tools 221 226 auto exposure feature 18 autofluorescence 110 267 miscellaneous material 268 270 See tissue autofluorescence subtract using background filters 273 274 well plate 267 268 Index 297 autoluminescence 110 automatically draw ROIs 107 109 average background ROI 102 110 B background adaptive fluorescent background subtraction 272 fluorescent 267 272 light on sample 256 258 tissue autofluorescence 273 274 background light from sample 258 259 on sample 256 258 background corrected signal 110 112 band gap 264 bandpass filter 263 binning 82 246 248 browse image data 71 74 browser 73 C Caliper Corporation technical support 3 cascade images 99 color table 249 colorize data 93 95 composite image 133 135 control panel 18 229 232 conventions 2 copy ROI
101. Registration Animate Display Subject Surface A O Opacity J 24 Display Photon Density Map Apply Measured Wavelengths 580 Intensity Jez S Color Table Reverse k C Log Scale r att Height 26 4 mm Perepeetie Figure 15 28 Surface tools and example DLIT reconstruction with photon density maps Table 15 9 Surface tools Item Description Display Subject Choose this option to display the surface in the 3D View window Surface 166 Living Image Software User s Manual Table 15 9 Surface tools continued Item Description Drawing styles for the surface Point cloud TAN Wire frame Wire frame amp ih ih Surface face surface face Shading styles for the surface teal Surface face Smooth surface fy Reflect surface Reflect smooth face ST face Surface face Click to open the color palette from which you can select a display color for the a Surface and the cross section views Opacity Adjusts the surface opacity Display Photon Choose this option to display the photon density on the surface Density Map Apply Choose measured or simulated photon density maps for display Wavelengths Choose the data to display in the photon density map DLIT Images FLIT Color Table Color scheme for the photon density map Reverse Choose this option to apply the colors of the selected color table in reverse order For exampl
102. See GSi SS ii EELT DA ae 55l mi 240 75 2S aL 4 S 1240 7S 200 59 Rakad Eltickerry r SARL 2009 pyireciomler paas OS PATY C d LPSE ZMU 2b ay Oe TS TH 73 Coir Scale Fin 1 73e1 Pa J42 Figure 7 19 Transillumination overview 7 8 Overlaying Multiple Images The image overlay tool provides a convenient way to view multiple reporters in one image You can use the image overlay tool to display multiple luminescence or fluorescence images on one photographic image To coregister multiple images 1 Acquire an image sequence using the appropriate filters for each reporter Alternately create a sequence from images acquired during different sessions For more details see page 98 2 Open the image sequence A ARW 20050826124002_ SEO Ey Sequence view Units Efficiency C Use Saved Colors hins 2 546 5 Mur 4 698 4 Figure 7 20 Image sequence 92 Living Image Software User s Manual 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 gt 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
103. Sequence Editor Initialize Click to initialize the IVIS Imaging System For more details on initializing the system see page 17 Table A 2 Additional controls for the IVIS Imaging System 200 Series or IVIS Spectrum Item Description Alignment grid Choose this option to activate a laser generated alignment grid on the Stage when the imaging chamber door is opened The alignment grid is set to the size of the selected FOV The grid automatically turns off after two minutes If subject alignment is not completed in two minutes place a check mark next to Enable Alignment Grid to turn on the grid Note The horizontal cross hair of the alignment grid is offset appropriately to take into account the height entered in the Subject height box Focus Scan Mid Image Choose this option in the Focus drop down list to set the focal plane at the maximum dorso ventral height of the subject at the middle of the animal This focusing method uses the laser to scan horizontally across the middle of the subject to determine the maximum Subject height along this line This option is well suited for animal imaging because the peak height is clearly identified as the maximum height on the dorsal side along the mid plane of the animal Note This focusing method is not recommended for microplates or when using a high magnification field of view FOV A 4 0 cm In these situations Manual or Subject Size focus methods are recommended Transilluminatio
104. Shortcuts ae A A eh Figure J 1 Living Image toolbar Table J 1 Menu bar commands and toolbar buttons Menu Bar Command Toolbar Description Button File gt Open File gt Browse File gt Save File gt Save As File Import gt 3D Surface File gt Import gt 3D Voxels File gt Import Organ Atlas File Export gt Image Sequence as DICOM File gt Export 3D Surface File gt Export gt 3D Voxels File gt Export 3D Scene as DICOM File gt Print File gt Print Preview File gt Recent Files File Exit Edit gt Copy Edit gt Image Labels Edit Preferences View Tool Bar View Status Bar View gt Tool Palette Lai E Displays the Open box so that you can select and open an image data file 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 Saves overwrites the AnalyzedClicklnfo text file to update the analysis parameters but the original image data files are not altered Displays the Browse For Folder box so that you can specify a folder in which to save the image data The original data is not overwritten Opens a dialog box that enables you to import a surface Opens a dialog box that enables you to import a source volume Opens a dialog box that enables you to import an organ atlas atlas Opens the Browse for Folder dialog box that enables
105. Splm_ lt name gt from the Name drop down list 2 Click Save The planar spectral imaging results are saved with the image To view results 1 Select the results of interest from the Name drop down list 2 Click Load To delete results 1 Select the results that you want to delete from the Name drop down list 2 Click Delete To copy selected results 1 Right click the results row of interest select Copy Selected from the shortcut menu that appears The selected results are copied to the system clipboard To copy all results 1 In the Results tab right click the results table and select Copy All from the shortcut menu that appears All of the results table is copied to the system clipboard y Caliper Ber ie Chapter 10 Planar Spectral Image Analysis 99 To export results 1 Right click the results table and select Export Results from the shortcut menu that appears In the dialog box that appears choose a folder for the results enter a file name txt and click Save 100 Living Image Software User s Manual This page intentionally blank y Caliper 1 1 Spectral Unmixing Image Sequence Requirements 2 a ee ee ee ee ee a 101 Performing Spectral Unmixing lt 2646 4 se twee Se ew 101 Spectra Window gt iss eS wk ER RE SHRED EE REDE atasan ai 106 Spectral Unmixing Parameters 2 0 ee eee ee ee ee 108 Spectral Unmixing Options eee ee 109
106. T200006274145507_S00 Aia y 3D Tools Sequence Yew Sppira Surface SOUFCE Registration Animate i E I baa gt ga Fi Bs F C Display Source Surface 4 R Display voxels Threshold 6 807 Opacity J E Voxel size Display vowels as Color Table F Reverse T C Log Scale eaae f Total Flux 349610 photonsisec volume 6 427 mm 3 Host Organ Unknown Se Center of Mass 4 9 70 2 79 2 pere The source intensity integrated within the om BA selection box is reported in the Source tab Figure 15 21 Selecting source voxels in the 3D View window Measure Source Depth 1 Select the source see Figure 15 21 2 Click the ma button The distance from the center of mass to the surface is measured in the three planes Figure 15 22 e Coronal and transaxial planes display the shortest distance from the center of mass to the surface e The sagittal plane displays the distance from the center of mass to the bottom of the subject 3 To display slice planes through the center of mass click the ae button For more details on planes see page 161 ie TE Y Caliper Chapter 15 3D Reconstruction of Sources 161 Sequence View 3D View Spectra eas m t e l S tate cone At ae i te a an TE Me oP photons sec ee ee Source Intensity Subject Height 26 4 mm Perspective F Figure 15 22 Slice planes through the source center of mass amp distance mea
107. Telephone 1 877 LabChip 877 522 2447 Toll Free in the United States 1 508 435 9761 E mail tech support caliperLS com Fax 1 508 435 0950 Address Caliper Life Sciences 68 Elm Street Hopkinton MA 01748 USA 4 Living Image Software User s Manual This page intentionally blank y7 Caliper feSciences 2 Overview of Imaging amp Image Analysis Workflow Overview megma MOUDE s rasare rtea r rdg we OEE Ee EGR ar 7 About Image Sequences Image Display amp Analysis This chapter provides a brief overview of imaging and image analysis 2 1 Workflow Overview The Living Image software provides image acquisition viewing and analysis functions for IVIS Imaging Systems Figure 2 1 shows the steps to acquire an image Figure 2 2 shows an example sequence acquisition workflow Start the Living Image software rj amp initialize the IVIS Imaging System Inthe control panel e Select the imaging modes for example luminescent amp photograph Set the imaging parameters When you are ready to acquire the images Enter image label click Acquire information optional E e ee a e a is Gh a ie miu aam tap ol fp emi e T EFi F fon harm Pr 2 i ait amp EE a The image window amp Tool Palette appear when acquisition is finished Figure 2 1 Steps to acquire a luminescent or fluorescent image 6 Living Image Software User s Manual Start the L
108. The Tool Palette shows the position and length of the cursor File Fre Teme ink OA Ol Wiles fo Cae 4 Il ihn Ei cs K Lita Gih lt Ap be all Tool Palette las fF E E e om a w A TL 2003624145507 00i T er diaple Geirliy y a Wikis 12 cm heg df on Lu or Image X Y PS 6 SF om Image Deta counts i a Smem LB oe Measurement cursor position i n amp length r 9 p w 7 Jre ny Cetate J _ Planar Spectral Imaging E A e rr A i Figure 7 16 Measurement cursor The Tool Palette displays the measurement cursor position and length 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 wiles button Table 7 8 Measurement cursor position amp length Item Description geene Pixel x y coordinates of position A on the cursor Pixel x y coordinates of position B on the cursor Length of the cursor from A to B number of pixels vertical distance from A to B number of pixels Distance Lenoth of the cursor from A to B number of pixels YP Caliper feSciences Chapter 7 Working With Data 89 To measure distance using the crop box 1 Open an image and in the Image Information tools click the Image Crop button i_ ae mm A g RP rte cones 1 Apok te al ILT200906241
109. W Ca li 7 Living Imagee Software User s Manual Version 4 0 2002 2009 Caliper Corporation All rights reserved PN 126545 Caliper Life Sciences 68 Elm Street Hopkinton MA 01748 USA 1 877 522 2447 US 1 508 435 9500 Fax 1 508 435 3439 E mail tech support caliperls com www caliperls com Discovery in the Living Organism IVIS Imaging System Living Image DLIT and FLIT are either registered trademarks or trademarks of Caliper Life Sciences Inc The names of companies and products mentioned herein may be the trademarks of their respective owners Apple Macintosh and QuickTime are registered trademarks of Apple Computer Inc Microsoft PowerPoint and Windows are either registered trademarks or trademarks of Microsoft Corporation in the United States and or other countries Adobe and Illustrator are either registered trademarks or trademarks of Adobe Systems Incorporated in the United States and or other countries Y Caliper ifeScihences Contents I WeEICOMG cress nanena nrnna RSENS AEETI 1 1 1 What s New In Living Image 4 0 Software oaoa aa 1 1 2 About This Manual sce wee ee Pee REDE De He Eee eee EO 2 1 3 Contacting Caliper Technical Support a aoa aoa e 3 2 Overview of Imaging amp Image Analysis a noaoono 6 0 08 2 eee eee ee 5 2 1 Workflow Overview lt oaoa ce Gd Ce ebave Abe CEH HEE EEE SE EX 5 ZZ Maes INIOUES 2 sk ena eee Bh eee eR eee Eee eee eee eee Ee ae ee GE 7 2 3 Abou tlmage S
110. White furred animals were No increase or decrease in background emission levels shaved prior to imaging Test 3 Alfalfa known to be An alfalfa free diet reduced background emission phosphorescent was eliminated from slightly but not significantly the animal diet The sources of autoluminescence are not yet fully understood No external sources have been proven to cause natural light emissions so it is possible that a chemiluminescent process associated with metabolic activity in living animals is the source of animal background This is supported by the observation that the level of background light drops significantly in euthanized animals In Figure E 2 the background light emission is clearly visible in the images of a control white furred mouse and a nude mouse The images are five minute high sensitivity high binning exposures The average emission from a white furred mouse and a nude mouse is approximately 1600 photons s cm sr and 1000 photons s cm sr respectively Since these values are well above the lower limit of detection of the IVIS Imaging System 100 feSciences i E Y Caliper Appendix E Luminescent Background Sources amp Corrections 259 photons s cm 7 sr the background light emission from the mouse determines the limit of detection An approximation of this background determined by making similar measurements on either control animals or regions of the subject animal that do not contain the primary
111. Ww FL Lita Lee Piaget Light Lev 07 Coko Satie Sa Minar Piirre File Size 10 9 E A save Dore Overlay Dynamic Range Exposure Time msecs Binning F Stop Select the type of data to acquire luminescent or fluorescent from this drop down list Choose the Overlay option to acquire photographs Choose this option to acquire photographs 14 bit If this option is chosen the signal intensities range from O to 16383 counts per pixel 16 bit If this option is chosen the signal intensities range from O to 65535 counts per pixel Note The14 bit dynamic range enables faster imaging The exposure time for the luminescent image Shorter exposure times enable faster frame rates longer exposure times provide greater sensitivity The14 bit dynamic range enables faster imaging by attaining a higher frame rate at the cost of a smaller dynamic range Controls the pixel size on the CCD camera Increasing the binning Increases the pixel size sensitivity and frame rate but reduces spatial resolution Using larger binning for a luminescent or fluorescent image can significantly improve the signal to noise ratio The loss of spatial resolution at high binning is often acceptable for in vivo images where light emission is diffuse For more details on binning see Appendix C page 242 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 i
112. a 289 I 2 Tracking Infusion in the Maximum vs Time Graph 2 0020002 G 291 I 3 Closing the Infusion Pump Control Panel 2 aa a 291 Appendix J Menu Commands Tool Bar amp Shortcuts 06 293 Y Caliper ifeSchences 1 Welcome What s New In Living Image 4 0 Software 1 a ee ee POO This Manual 6 s s ee hea eRe SHEE EDS HESS eH HH Ome HOH 2 Contacting Caliper Technical Support 2 0 ee eee es 3 The Living Image software controls image acquisition on IVIS imaging systems and provides tools for optimizing image display and analyzing images 1 1 What s New In Living Image 4 0 Software The major new features in the Living Image software include New Feature or Updated Tool Page X Ray Imaging Acquire X ray images on the IVIS Lumina XR imaging system Co register an X ray image 43 with a luminescence fluorescence or photographic image Determine relative density in an x ray image using ROIs 55 3D Surfaces Select a particular region of the subject to reconstruct 160 Export a 3D surface or a user selected portion of the surface in DICOM format Multi frame 164 export option supports 3D CT reconstruction in third party software Expanded export and import capabilities for reconstructed 3D surfaces XM H DXF STL IV 164 3D Sources Living Image software automatically selects the data to include in the reconstruction based 188 on the Threshold value Select particular
113. a 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 146 Tool Pakette a IL MOs06 74145507 SEO C Sepeernce view E a veew Spectra Gata Prewiews abire en Peter Sia Sourte Arc F Image Label F Medien Fiter Restore Threshold bate Achushment C Soleck All carmel Reconstruct Figure 15 4 Data Preview window right 8 Click Reconstruct YP Cali er feSciences Chapter 15 3D Reconstruction of Sources 145 The reconstruction requires about 1 5 minutes depending on the parameter settings and the processor speed When the analysis is finished e The 3D View window displays the animal surface and the reconstructed sources e In the Tool Palette the Results tab displays the results data and the algorithm parameter values e The 3D Tools appear after a reconstruction is generated or loaded For more details on the 3D Tools see page 165 175 For details on managing results for example save load or delete see page 157 3D View toolbar i En m m EN ee he Wilt oun s C Apply be al TLT20050624145507_SE0 Jea Toot Paietse d Tipi V e aD View gered a aa m os ROI Temas E E s Briat Tiparipiy Peat Sur HU DL S30 Ber ro E Til art deh Siu Thr iiki caregle PE
114. a displayed in terms of radiant efficiency When efficiency is selected the fluorescent image data is normalized divided by a stored calibrated reference image of the excitation light intensity incident on a highly reflective white plate The resulting image data is without units typically in the range of 10 to 10 NOTE On each IVIS Imaging System a reference image of the excitation light intensity is measured for each excitation filter at every FOV and lamp power The reference images are measured and stored in the Living Image folder prior to instrument delivery Y Caliper feSciences Appendix F Fluorescent Imaging 267 F 5 Fluorescent Background Autofluorescence Autofluorescence is a fluorescent signal that originates from substances other than the fluorophore of interest and is a source of background Almost every substance emits some level of autofluorescence Autofluorescence may be generated by the system optics plastic materials such as microplates and by animal tissue Filter leakage which may also occur is another source of background light The optical components of the VIS Imaging Systems are carefully chosen to minimize autofluorescence Pure fused silica is used for all transmissive optics and fiber optics to reduce autofluorescence However trace background emissions exist and set a lower limit for fluorescence detection To distinguish real signals from background emission it is important to recognize t
115. able Available items column headers that can be added to the table Figure 8 28 Configure Measurements dialog box 84 Living Image Software User s Manual 2 Do either of the following Select a configuration that you want to modify from the User Lists drop down OR Select Customized Unsaved from the User Lists drop down to create a new configuration To add an item to the table click an item in the Available Item list and then click Add To remove an item from the table select the item that you want to remove in the Selected Items list and click Remove Configure Measurements User Lists Customized Unsavec Name My ROI Table Available Items Analysis Comment Analysis User ID 4ngle Animal Model Animal Mumber Animal Strain Area cme vg Dark Charge Counts Avg Efficiency 44g Fluorescent Bkg Counts vg Radiance pfsicmZysr Binning Cell Line Comment Comment 1 Comment2 Add gt Delete Selected Items Total Counts Avg Counts Stdey Counts Min Counts Max Counts ROI Pixels Areatccd Pixels mel picels Yel pixels Widthi pixels Heighti pixels Items in the ROI Measurements table Figure 8 29 Configure Measurements dialog box 5 To reorder an item in the Selected Items list select the item and click Move Up or Move Down The columns in the ROI Measurements table are updated 6 To save the table configuration enter a name in the Name box and click Save
116. ae i i n2 Lt Fs a y l humiae off ai Chapter 4 Luminescent or Fluorescent Imaging 25 meid Hi w Probes F mop ol Cote Daan ra M pey i Beck i Ye w en Copy ida i Grei AJ L BRAF LID ho oo mt Delate romli Eizh fea hs S Replace fowls Bixi fetal hio 1S0 Pirie foa Figure 4 24 Sequence editor edit commands in the shortcut menu Table 4 6 Sequence editor shortcut menu edit commands Command Description Copy row s Select All Delete row s Replace Row s Paste Row s Copies the selected row s to the system clipboard Selects all rows in the sequence editor Deletes the selected row s from the sequence editor Replaces the row s selected in the sequence editor with the rows in the system clipboard Note The Replace function is only available when the number of rows in the system clipboard is the same as the number of rows selected in the sequence editor 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 editor to view a shortcut menu of edit commands Figure 4 24 4 6 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 acqu
117. aeda 233 Appendix B Preferences 2 2 we ee et 235 B 1 General Preferences 2 2 d kaeka ntria oraaa a 236 B User Pictctenccs a eww eee ek ee eee eR ee eee eee eee ee ee 238 Boo ACGUISIOM ony osu sd oe bates OG ba eu beet wee bee doe oe ES oa ee 239 BA TWheme e82 ne85 6228S eR POPE eRe eee eee eee ee se eee 240 Bo Tissue Properties sss s6 ek bKo BYR KBHS OER Eee eee edhe RE EEE BEE SA 243 Appendix C Detection Sensitivity 0 002 2 eee eee ees 245 C l CCD Detection BIMciency gt s e asaw bes ewes base Dede eee ae dee as ba 245 C2 BINNIE 244446424 Sb FE GER RA REDE tetas ranieri ranei 246 Coo SMOOUINS eu ead exeeoauwt PERSE eS wee es eee ee SEG we eee es 248 Appendix D Image Data Display amp Measurement 0 000 8 ee wae 249 DA tases Dat see ke ake eeyst ea de eee eee eh Sete eee ee ee eee eee 249 D 2 Quantifying Image Data oaoa a a 251 D3 Plat PiclditG 42i64e46255048 ee 8 oe RE He REESE He eee Emo KRG eee EES 254 iv Living Image Software User s Manual D 4 Cosmic Ray Corrections aooo a 254 Appendix E Luminescent Background Sources amp Corrections 255 E 1 Electronic Background i 644 4444444 a Ghd we tauri EEE HS BEE ESS eG 25 E 2 Background Light On the Sample 0 202 000 eee eee 256 E 3 Background Light From the Sample 0 0 00 eee ee ee ee ee 258 Appendix F Fluorescent Imaging 2 6 ee ee ee ee 261 F 1 Description and Theory of Operati
118. age acquisition If the power to the system or camera controller a component of some IVIS Imaging Systems has been cycled or if the camera temperature has changed a new dark charge measurement should be taken The dark charge is measured with the camera shutter closed and is usually performed automatically overnight by the Living Image software The software acquires a series of zero time exposures to determine the bias offset and read noise followed by three dark exposures The dark charge measurement usually takes more than three times as long to complete as the equivalent luminescent exposure E 2 Background Light On the Sample An underlying assumption for in vivo imaging is that all of the light detected during a luminescent image exposure is emitted by the sample This is not accurate if there is an external light source illuminating the sample Any reflected light will be detected and is indistinguishable from emission from the sample The best way to deal with external light is to physically eliminate it There are two potential sources of external light a light leak through a crack or other mechanical imperfection in the imaging chamber or a source of external illumination IVIS Imaging Systems are designed to be extremely light tight and are thoroughly checked for light leaks before and after installation Light leaks are unlikely unless mechanical damage has occurred To ensure that there are no light leaks in the imaging chamber c
119. age acquisition to ensure that photons incident on the CCD camera the camera settings are property adjusted Radiance photons A calibrated measurement of the photon Luminescent measurements emission from the subject Radiant Efficiency Epi fluorescence A fluorescence Fluorescent measurements emission radiance per incident excitation power fluorescence Transillumination fluorescence Fluorescence emission radiance per incident excitation power Efficiency epi Fluorescent emission normalized to the Epi fluorescent measurements fluorescence incident excitation intensity radiance of the subject illumination intensity NTF Efficiency Fluorescent emission image normalized Transillumination fluorescent to the transmission image measured measurements with the same emission filter and open excitation filter Counts When image data is displayed in counts the image pixel contents are displayed as the numerical output of the charge digitizer on the charge coupled device CCD Figure D 2 The counts measurement also known as analog digitizer units ADU or relative luminescence units RLU is proportional to the number of photons detected in a pixel Counts are uncalibrated units that represent the raw amplitude of the signal detected by the CCD camera A signal measured in counts is related to the photons incident on the CCD camera The signal varies depending on the camera settings for example integration time bi
120. age browser w AmA eee 7 i IBE a ae hii Ihi Eji Sacha PS 2 IHE LPC os iim Iii Eri if Sota M LL IHG ee a r a eiir Ili Cope j ipea TS a L IEG IO r Opar Ihi Eip 148 faecal Pe LLS 4 Hike Bete viaa Choker Prarie ated ah A oe Te iec Grimace Lacuna C Eerme d ete eee pig 2 Cea ae ee j a e cho Snapshot Color scale Provides a reference for the pixel intensities in a luminescent or fluorescent image Pixels less than the color scale minimum do not appear in the image Pixels greater than the color scale maximum are displayed in the maximum color Image label Information about the image that the software automatically records and user specified information entered in the Edit Image Label dialog box 5 2 Acquire an Image Sequence That Includes X Ray Images To acquire an image sequence first specify the acquisition parameters for each image in the sequence editor Figure 5 7 The Imaging Wizard provides a convenient way to do this for some imaging applications The wizard guides you through a series of steps prompting you for the information that the software needs to set up the sequence in the sequence editor If you don t use the wizard you can set up a sequence manually for more details see page 21 Sequence Setup Using the Imaging Wizard 1 Click Sequence Setup in the control panel Figure 5 7 The sequence editor appears 2 If necessary click the Remove button Xrm and select All to clear the sequenc
121. age information select File Save As on the menu bar 7 4 Adjusting Image Appearance Use the image adjust tools to adjust the appearance of an image Figure 7 9 NOTE Not all tools are available for all image display modes Pes e l ee ra eh Dm G a ec es ip al ea mi he J Image Adjust tools Lite Figure 7 9 Tool palette Image Adjust tools 80 Living Image Software User s Manual Table 7 3 Image Adjust tools Description T Click this button to incrementally zoom out on the image reduces the image dimensions in the image window Note The zoom tools are also available in the shortcut menu when you right click the image Ctrl click for M acintosh users Click this button to incrementally zoom in on the image incrementally magnifies the image in the image window Click this button to magnify the area inside a rectangle that you draw using a click and drag operation Sets the dimensions of the magnified area equal to image window dimensions m 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 81 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 informa
122. ages obtained at two or more wavelength filters from 560 660 nm To reconstruct fluorescent sources the software requires a structured light and fluorescent images obtained using the same excitation and emission filters at different transillumination source positions on the IVIS Spectrum To localize and quantify the light sources in a subject the software e Reconstructs the subject surface topography surface from structured light images The surface is defined by a set of connected polygons or surface elements e Maps the surface radiance photons s cm2 steradian to the photon density photons mm3 just beneath the surface of each element of the surface e Divides the interior of the subject into a solid surface of volume elements or voxels Each voxel is considered to contain a point light source at its center that contributes to the photon density at each surface element e Defines equations that relate the source strength of each voxel to the photon density at each surface element e Determines the optimum approximate solution to the system of linear equations to reconstruct the source strength in each voxel H 1 Determining Surface Topography The software determines the surface topography from a structured light image Parallel laser lines are projected onto the subject to produce a structured light image Figure H 1 If the Structure option is chosen in the Control panel a structured light image is automatically acquired
123. al Table 11 2 Spectral unmixing wizard choose the components to unmix continued Item Description Background Signals Any undesired fluorescence that the camera detects for example autofluorescence from the animal food or instrument background Probe Information Specify the probes a Click to add a probe 4 Click to remove the selected probe M atch Probe Labels If the probe names are specified the software attempts to automatically match the unmixed spectra with the specified probe names Note A correct match is not guaranteed due to the complexity of the in vivo spectra and filter sections especially when only part of the emission excitation spectrum is sampled Use Constraints Choose this option to apply the recommended constraints when performing spectral unmixing For more details on the constraints see page 109 To disable the constraints uncheck this option Alternately in the Options tab of the Spectral Unmixing tools click Reset Values and then click Update Number of components The total number of components background and probe signals to unmix selected for unmixing 6 Click Finish when you are done choosing the components to unmix The unmixed images and results are displayed Figure 11 4 The results include a signal distribution map of each unmixed result and a composite image that includes all of the fluorescent signals each displayed in a different color Gor En lel Ha L a k7 Lr Cee s A
124. ale from black to red If Reverse is chosen the source intensity photons sec from low to high is represented using the color scale from red to black Log Scale Choose this option to apply a logarithmic scale to the color table Quantification For uncalibrated sources the total flux measured for the sources selected DLIT using the Measure Source tool For calibrated sources this unit will be in cell units For details on using this tool see page 160 Quantification For uncalibrated sources the fluorescence yield measured for the voxels FLIT selected using the Measure Source tool ire Fluorescence yield is expressed in units of pmol M cm here for uncalibrated sources For calibrated sources this unit will be in either cells or pmol For details using this tool see page 160 Volume Volume of the selected source mm3 Center of Mass The weighted average x y and z coordinates of the selected voxels where DLIT GADLIT or the weights are the flux of each highlighted voxel FLIT 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 173 Click to compute the center of mass for the source selected with the Measure Source tool ee For details using this tool se
125. an its width Window Size Specifies the dimensions of the main application window Width Height Sets the dimensions of the image window Restore Defaults Click to apply the default settings Apply Individual Color Choose this option to apply a separate color scale to each thumbnail of Scale for Sequences a sequence If this option is not chosen all of the thumbnails are displayed using the same color scale Show Transillumination Choose this option to display a cross hair at each transillumination Locations location when you load transillumination data When you mouse over 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 If this option is chosen the Options e Tool Palette includes the Point Source Fitting and the GADLIT tools e Image window includes the Colorized View tab Show Activity Window A drop down list of options for when to display the activity log Figure on B 3 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 th
126. anar spectral imaging spectral unmixing 147 157 options 155 156 parameters 154 155 PCA biplot 157 PCA explained variance 157 results 150 sequence requirements 147 starting the software 15 starting the system See system initialization steradian 253 subject ROI 102 110 surface 159 165 export or import 164 165 generate 160 161 manage 164 view perspective 162 163 syringe injection system 289 291 system initialization 17 optics autofluorescence 271 temperature 17 system activity 19 T tag an image 90 technical support 3 temperature 17 threshold angle 201 tile images 99 tissue autofluorescence 273 274 eliminate by spectral unmixing 147 157 subtracting with background filters 136 138 tissue properties 201 tool palette 77 3D tools 211 226 correcting filtering 81 82 point source fitting 167 173 176 177 ROI tools 104 Y Caliper feSchences toolbar 293 transillumination overview 90 troubleshooting DLIT FLIT 227 U uniform surface sampling 201 units See display units user activity 19 user preferences 235 V ventral imaging chamber 59 voxel 285 vsize starting 201 W well plate autofluorescence 267 268 X X ray acquire image 43 48 acquire sequence 48 55 Index 301 302 Living Image Software User s Manual
127. and a Optimize the image display using the color scale Min and Max sliders in the Image Adjust tools b Draw a measurement ROI on an area of the animal that represents background signal area where no fluorophore signal is present N da W i LN w 1 You only need to draw the ROI on one of the images The software copies the ROI to the other image A TLT AOS 4S DOi is Pha e Sheer ni l ifo image Mn OM Max i a piece aii Cok Ba Min 1 2503 Max 2 47610 Figure 9 4 Draw measurement ROI on an area that represents background signal 92 Living Image Software User s Manual Select Tools Image Math for lt name gt _SEQ on the menu bar 4 In the Image Math window that appears select the primary image in box A Select the background image in box B For more details on items in the Image Math window see Table 9 1 page 89 5 Select the math function A B k in the Result drop down list 4lmoage Moth Window fil Sequence TLT 20061 220101746_SE0 FL TELETHON 221 1 TETH 1221 12 L TAIR TES TL TF 1 Coker ica Leids hor fi ad Ful C Auto Fiesi Cala beas Limni f Ful gt Aug Mine Hewat 2 Eo k 1 00 Compute k bom Al F eth Photo from A w r Drip Redull For Hebbur Figure 9 5 Select a math function and view the mathematical result 6 Click and select the ROI created in step 2 from the drop down list The background corrected signal is displ
128. and source spectra are specified in the Preferences box For more details see Appendix B page 243 288 Living Image Software User s Manual This page intentionally blank Y Caliper _ Appendix IVIS Syringe Injection System Controlling the Infusion PUuMp ona a eee ee ee 289 Tracking Infusion in the Maximum vs Time Graph aaa a a a 291 Closing the Infusion Pump Control Panel aoaaa aa a 2 80 291 The IVIS Syringe Injection system is designed for use with the IVIS Kinetic Imaging System You can control the infusion pump in the Living Image software or manually For more details on the setup and manual control of the infusion pump see the V S Syringe Injection System instructions from Caliper or the PHD 22 2000 Syringe Pump Series User s manual from Harvard Apparatus Both are included on the Living Image installation CDROM The IVIS Syringe Injection system can be used during kinetic or still image acquisition however subjects must remain immobile 1 1 Controlling the Infusion Pump After the IVIS Kinetic Imaging System is initialized and locked you can access the infusion pump controls 1 Select Acquisition gt Infusion Pump Setup on the menu bar The Infusion Pump control panel appears above the IVIS acquisition control panel ma OS ae Eee a Figure I 1 Display the Infusion Pump control panel NOTE If you are going to acquire kinetic data open the infusion pump control panel before you o
129. and the Image Information tools choose Cm or Pixels from the Units drop down list 2 Put the mouse pointer over a location of in the image The x y coordinates and intensity data are displayed in the Tool Palette D E fe i e op ein Ih g y ran ir ia a iin Liew ee iE aiis i i ee x y coordinates amp taba ENE intensity dataatthe pointer location ae Ama j wy a falar Oy iem UT M imaina x oe T L ECIENT Figure 7 13 x y coordinates and intensity data at the mouse pointer location Ww Caliper Chapter 7 Working With Data 85 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 la Tood Falette gt Corrections Fiberi w ots Cm irene Erri F Wih 12 6 err Height 12 6 cr f Se aia CO 4 Hnlacian H imire Full Mnp r G He tee OS ee RID H hb TTEA eC verry Figure 7 14 Viewing a histogram of pixel intensities 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 t
130. anersoms Copy II Select fl Darii el A Pii takes 0 Moh yf Select the type of data to include in the table For more details see Table 8 4 page 81 E RO Measurements ROI Measurements Grid ROI Measurements Click He20070420121444 001 ROL ROL4 Measurement Total Counts w EM Refresh 2 a 4 4 07 fe 05 2 479e 05 6 015e 05 2 1 576e 06 4 350e 05 2 249e 05 1 411e 05 Grid ROIs 3 3 08 e 05 9 262e 04 9 039e 04 9 342e 04 Customized Selections Measurements Types Image Attributes ROI Dimensions Select All Figure 8 25 Opening the ROI Measurements table Column headers in the table include ROI information ROI measurements and dimensions and information about the image recorded at acquisition W Caliper a Chapter 8 Working With ROI Tools 81 ifeSchences P ROI Measurements EJ ox ROIL Measurements Kinetic ROI Measurements Kinetic ROI Plot Data click DA20081107162612 v ROL IROL a All Frames EM Refresh Image Number Rol Image Layer Total Flux pf Avg Radian Stdey Radia Min Radianc Max Radiam ROI Pixels 4Areatcced Fii i x H a z e a 4 2 0 m Customized Selections Measurements Types _ Image Attributes _ ROT Dimensions Copy Select All Photons All Possible Values Pixels Configure Export Close Figure 8 26 ROI Measurements table kinetic ROIs Table 8 4 ROI Measurements table Item De
131. anged anytime From the Acquisition menu Figure 4 7 Autosave prompt 13 To enable autosave click Yes in the prompt and choose a folder in the dialog box that appears If autosave is enabled all images acquired during the session are automatically saved to a user selected folder You can choose a different folder at any time select Acquisition Auto Save on the menu bar Image acquisition proceeds During acquisition the control panel Acquire button becomes a Stop button To cancel the acquisition click Stop in the control panel The image window appears when acquisition is completed Figure 4 8 Pe he ce oe ie E oe et t Aa p ae cores symp te fiaim Cre a jape iii he LLP LE eed Teper Deed Coupee Wa Eri a mat rra Tool palette Cmm FES o a Hl ty Um tarp rnn mir ___ Check the image min and nam max in the color scale to determine whether the signal of interest is above aa the noise level and below CCD saturation Figure 4 8 Overlay fluorescent image on photograph in the image window For more details on the items in the image window see Table 4 1 page 7 Y Caliper a Chapter 4 Luminescent or Fluorescent Imaging 11 4 3 Acquire a Fluorescent Image With Transillumination Transillumination provides an excitation light source located below the stage FLIT reconstruction of fluorescent sources analyzes a transilluminated image sequence For more information about fluorescent imaging
132. ans and surface first use the transformation tool to manually register the surface and atlas organs then click a registration tool to automatically fit the organs For more details on manual registration see below 4 If necessary adjust the opacity of the organs using the slider or enter a number in the box The organs are easier to view if you uncheck Skin in the Organs list To clear all organs from the surface click the Deselect All button Ra To hide a particular organ remove the check mark next to the organ name To display a specific organ s choose the organ name To display all organs on the surface click the Select All button If you manually change the location orientation or scale of an organ s you can click Reset button to restore the default size and position of the selected organs To manually adjust the scale or location of organs 1 Load reconstruction results and confirm that the surface is in the perspective view click the toolbar button in the 3D View window or press the R key In the 3D registration tools choose the Display Organs option and select an organ atlas The organs in the selected atlas appear on the surface In Figure 15 31 only Skin is selected Click the Transform tool button amp y7 Caliper ieGelances Chapter 15 3D Reconstruction of Sources 171 The transform tool appears Tool Palette Eal A 1L720050624145507_ SEO Took
133. antly affect an ROI measurement Cosmic ray correction is not recommended when imaging very small objects such as individual cells An individual cell may only light up one or two pixels and can sometimes be misinterpreted as a cosmic ray In this case clear the Cosmic Correction option in the Corrections Filtering tools to avoid filtering out single cell images Y Caliper ifeSchences Appendix E Luminescent Background Sources amp Corrections Electronic Background 246i i ss sssri we ee Re HE De ee Ee eS 255 Background Light On the Sample 2 0 2 eee eee eee 256 Background Light From the Sample 2 2 0 08 2 eee eae 258 The background sources of light from luminescent images are inherently very low This appendix discusses sources of background and how to manage them Due to the extreme sensitivity of the IVIS Imaging System residual electronic background dark current and luminescent emission from live animals autoluminescence are measurable and must be taken into account For information on fluorescent background see Appendix F page 267 E 1 Electronic Background The cooled CCD camera used in an IVIS Imaging System has electronic background that must be accurately measured and subtracted from the image data before the light intensity is quantified Raw data that is not corrected for electronic background results in erroneous ROI measurements Incorrect background subtraction may also result in serious error
134. aptive background subtraction e Identify the fluorescent subject in the original image using the photo mask e The software automatically fits the instrument background to the whole image using the pixels outside of the subject e The software subtracts the fitted instrument background from the original image In most situations such adaptive software correction works as effectively as the traditional method except the following cases e The subject is dark making it is difficult to mask the subject using the photo for example experiments that use black well plates e The subject occupies most of the FOV for example high magnification or multiple mice in the FOV As a result there is not enough information outside the subject that can be used to help fit the background feSciences y Caliper ad p Appendix F Fluorescent Imaging 273 F 8 Subtracting Tissue Autofluorescence Using Background Filters High levels of tissue autofluorescence can limit the sensitivity of detection of exogenous fluorophores particularly in the visible wavelength range from 400 to 700 nm Even in the near infrared range there is still a low level of autofluorescence Therefore it is desirable to be able to subtract the tissue autofluorescence from a fluorescent measurement The IVIS Imaging Systems implement a subtraction method based on the use of blue shifted background filters that emit light at a shorter wavelength see Table 9 2 page 136 The
135. ar spectral image analysis For more details on the Imaging Wizard see page 25 At a minimum the sequence must include a photographic and luminescent image at the first wavelength and a luminescent image at a second wavelength 560 660 10 2 Planar Spectral Image Analysis 1 Load the image sequence that you want to analyze Pe Ce Wew Took AORA Wi Pele ef we Ba BB MP lune con w C Apply to al TLI20050824145507 sa Tool Palette omaan O gt ADI Tooli E Planar Spectral birgai Dy anahot Progertios Renis Seence Fee E Gi Sequence view Soara Liebe ount w C Use Served Coors Timka p r Tarih Priit Pikari Filler eee San Ren cn Pon Inageset cc Mo active ROL selected 3m sre gt Surface Topography Point Source Fitting DALIT 30 Reconstruction _ GADLIT 30 Reconsbruction Specbral Linenining Figure 10 1 Sequence for planar spectral image analysis 2 In the Tool Palette click Planar Spectral Imaging 3 In the Analyze tab select the emission filter wavelengths for the analysis Figure 10 2 It is recommended that you do not include a wavelength in the analysis if the signal is less than or equal to the autoluminescent background If autoluminescent background 94 Living Image Software User s Manual is a concern you can create a background ROI and link it to the measurement ROI prior to planar spectral analysis For more details see Measuring Ba
136. at Field Correction Select this check box to apply a lens correction factor to the image data For more details on flat field correction see Appendix D page 254 Note In photons mode flat field correction is a mandatory default In counts mode the check box can be cleared Cosmic Correction Select this check box to correct image data for cosmic rays or other lonizing radiation that interact with the CCD For more details on cosmic correction see Appendix D page 254 Adaptive FL Opens the Photo Mask Setup box that enables you to set the photo mask Background for adaptive fluorescent background subtraction For more details on Subtraction adaptive fluorescent background subtraction see Appendix F page 272 Binning Specifies the number of pixels in the image data that are grouped together to form a larger pixel called soft binning Binning changes the pixel size in the image Figure 7 11 For more details on binning see Appendix C page 246 Smoothing Computes the average signal of the specified number of pixels and replaces the original signal with the average signal Figure 7 11 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 Binning at acquisition 8 no smoothing Binning 2 smoothing 5x5 Figure 7 11 Example of binning and smoothing image data Caliper Yr p Chapter 7 Working With Data 83
137. ayed 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 To save the new image 1 Click the Save button fj Alternately select File Save on the menu bar 2 In the dialog box that appears select a directory and click Save A folder of data is saved to the selected location AnalyzedClickInfo txt ClickInfo txt luminescent and photographic TIF images To export the new image to a graphic file 1 Click the Export button mg 2 In the dialog box that appears select a directory enter a file name and select the file type from the Save as type drop down list 3 Click Save Y Caliper 1 0 Planar Spectral Image Analysis Image Sequence Requirements aaao a a a 93 Planar Spectral Image Analysis 0 2 eee ee ee 93 Planar Spectral Image Analysis 2 eee ee 93 Viewing Graphical Results 1 a 96 Managing Planar Spectral Imaging Results 2 858285 98 The Living Image software applies planar spectral image analysis to a sequence to determine the average depth and total photon flux of a luminescent point source in a user specified region of interest For more information on planar spectral image analysis see Appendix G page 271 10 1 Image Sequence Requirements Use the Imaging Wizard to setup the image sequence required for plan
138. background within the region of interest In the corrected image the RMS error is used to quantify the background The signal to background ratio of the original fluorescent image DsRed filter is 6 5 The ratio increases to 150 in the corrected image an improvement factor of 23 This improvement reduces the minimum number of cells necessary for detection from 1 5x 105 to 6 7x 103 Primary Background Corrected excitation filter excitation filter 7 200 au Tr 3 5 jen TEA oF 41x10 ia iE 417x105 2 00 100 S gt 5 ni a 50 50 Signal Background 6 3 Scale Factor 0 47 Signal Background 100 Min detectable cells 1 6x10 l Min detectable cells 1 0x10 Figure F 17 Example of the autofluorescent subtraction technique using a background excitation filter a primary excitation filter DsRed b blue shifted background excitation filter DsRed Bko and c corrected data The corrected image was obtained by subtracting the scaled background filter image multiplied by 0 47 from the primary filter image The 6 week old female Nu nu mouse was injected subcutaneously with 1x 106 HeLa luc PKH26 cells in the left flank 274 Living Image Software User s Manual Bkg Ex Em Filter Filter Filter 2 1 0 y Excitation in I S E Emission 0 8 h i Autofluorescence E 12 D 06 aH Be No o E 04 i i i ll i 400 450 500 550 600 650 700 750 Wavelength nm Figure F 18 Spec
139. ce the mouse pointer over the ROI When the pointer becomes a f click the ROI b Place the mouse pointer over an ROI handle W so that it becomes a Drag the handle to resize the ROI You can also change the ROI position or size using the adjustment controls in the ROI Properties box see Moving an ROI page 119 and Editing ROI Dimensions page 120 7 Click the Measure button The ROI intensity measurements appear in the X ray image and the ROI measurements table appears For more details on the table see Managing the ROI Measurements Table page 126 For information on how to save ROIs see page 123 Pis Bii Ys e ue i H oo ad al E Ee 3 Ky ints dj 7 Apple be al Lines Louth Dee Loy w l E ita a Tend Piglets 4 x Simeeait o G tovedtons F reng ooo O l imap Inormatian l J ROI Tosia oO O E d o Y w D Appir bo ae Tyee Mepa pma E m eae Hoti Pi HG I CSA ba e save Pii Figure 5 17 Measurement ROIs on X ray image 42 Living Image Software User s Manual This page intentionally blank Y Caliper 6 Kinetic Imaging Kinetic Acquisition sooo a a a ROS RH ED 43 Viewing amp Editing Data in the Kinetic Acquisition Window 47 Saving Kinetic Data lt 44 5 684 eee EHE DERE EERE ESSE so 49 Playing Kinetie Data sce ec ce kee ewe eee ES RHR wres nisd oops 50 The IVIS Kinetic Imaging System is ready to acquire kinetic data after the system 1s initialized a
140. ce to display on the animal surface View the parameters for the selected source s below Table 15 6 GADLIT 3D reconstruction results Item Description Sources A list of the reconstructed sources Choose all none or a particular source from this list for display The total number of The number of sources in the reconstruction sources Source strength The sum of the luminescent source intensities photon s Source location The location of the source center of mass Source volume The total volume mm of a Source Min Radiance The minimum radiance at each wavelength used in the reconstruction Index of Refraction The internal medium index of refraction that is associated with the user selected tissue Tissue Properties The tissue properties for modeling the photon propagation Source Spectrum The emission spectrum of the type of luminescent or fluorescent source Photon Density Maps Click to view the photon density maps For more details see page 158 Export Results Opens a dialog box that enables you to save the results cSv Save Results Name The default name for the active DLIT or FLIT results Select results from this drop down list Delete Click to delete the selected results Load Click to load the selected results Save Click to save the active results y Caliper d p Chapter 15 3D Reconstruction of Sources 157 feSciences Table 15 6 GADLIT 3D reconstruction results conti
141. ckground Corrected Signal page 64 Tool Palette gt RUI Tools J Planar Sperchral nagar inre Prepees tte p AGAMA MLS Se pos LaF Emission filter wavelengths selected for the analysis Jmagerct lt lt Me aches Pol eetected 25 ROL Lint are Faint ae gt Point Source Fitting gt IT 3D Reconstruction gt GADLIT 3D Reconstruction Figure 10 2 Planar spectral imaging tools Analyze tab For more details on the Analyze tab see page 95 4 In the ROI List drop down select All or a particular ROI for the analysis If there is no measurement ROI draw an ROI that includes the area for analysis For more details on drawing ROIs see page 56 You only need to draw the ROI s on one image in the sequence The software copies the ROI s to all other images of the sequence during the analysis The ROI should include as much of the light emission from a single source as possible Aj TL 20050624145507_006 AGI Foo Fatette Ta a image Adjust i bige Infornrateor gt RDI T dh Planar Spectral Imaging Areas Papais Resuls AOL 1 12146006 cd f 5 94 bee Figure 10 3 Selecting ROIs for planar spectral image analysis W Caliper p Chapter 10 Planar Spectral Image Analysis 95 ifeSciences 5 Choose the tissue properties a In the Properties tab make a
142. clickinfo txt file i Displays a dialog box that enables you to save the information in the sequence editor 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 editor Subject If a subject and probe are specified optional the software uses the information to automatically set parameters in the Surface Topography DLIT FLIT Spectral Unmixing and Planar Spectral Imaging tools If a subject or probe is not selected here the default parameters appear in the tool palette Number of Segments The sequence specified in the sequence editor is called a segment Choose this option to set the number of segments to acquire and the time delay between segments This is useful for acquiring data for kinetic analysis Delay Specifies a time delay between each segment acquisition Apply to All Applies the selected cell value to all cells in the same column T Deletes the selected row from the sequence editor A Update Updates the selected row in the sequence editor with the acquisition parameters in the control panel Inserts a row above the currently selected row using the Insert information from the control panel Adds a new row at the end of the sequence setup list Add 24 Living Image Software User s Manual Editing Image Parameters You can edit parameters in the sequence editor or in the control panel To
143. creasing tissue density When acquiring X ray images for bone density evaluation it is best if the subject does not fill the entire field of view since the X ray absorption measurements are referenced to the exposed scintillator plate To determine relative bone density 1 Load an X ray image For more details on opening image data see page 71 2 Confirm that the X ray absorption correction default is applied Figure 5 15 3 Inthe ROI tools select Measurement ROI from the Type drop down list Figure 5 16 Pe E Weve en Wie He ase UHU wm A g E lune core 2 apy te al ini Garig w phy X Ray ar aho ig j Tool Feita gt Dera Actas l gt mectiesa itaring l gt Die Winika 7 Adit Took COo t x L appi io Sequences Pye Peer creer Bd Pare per ty nal Subsect maa rr Abo RO Faan aami a0 Figure 5 16 X ray image with absorption correction YY Caliper ad Per ne Chapter 5 X Ray Imaging 41 4 To select the ROI shape a Click the Circle or Square O button b On the drop down list that appears select the number of ROIs that you want to add to the image The ROI s and intensity label s appear on the image If you are working with a Sequence open an image to show the ROI intensity 5 Adjust the ROI position a Place the mouse pointer over the ROI When the pointer becomes a click the ROI b Drag the ROI 6 Adjust the ROI dimensions a Pla
144. ct Bul bo mora ada ROL a Diii AOE Dikta Al Peas rrr Figure 8 15 Opening the ROI Properties dialog box y Caliper ksd Chapter 8 Working With ROI Tools 71 S ILT20050624145507_006 j ROI Properties ER ROI selected ae 3 in the image fecal Fis ROI Label GL Wao sR Inte Use as BG For fure Gls ini Luria ee 0 ATi 145507 0E r I Erie sequence AOI 191 07 36406 L Lork Pesiion Xel pix 119 35238 RO 2 5 340405 Sacy PETT angle deg 0 0000 Ces Lock Sie Widhi pis 34 15235 Haighti pi 22 00508 Background ROI selected in the image Lire Size F vn cote M C oome Label of the ROI selected in the image Double ROI A I I click to edit RGI Label ROL 1 S TLI20050624145507 006 ROI Properties beg ROL Subj BH Imap Phuru Lumisen TLT 2005062414507 _006 Selected image REIL B e Po L 1 07 38 06 RO 2 5 346 ved piv 10 cee 3 L m angie dea uous Lock Size Wich pizi m m Height pixi l1 ao l Measurement ROI selected in the image 2 E vecer M C Figure 8 16 ROI Properties Bkg ROI tab The items in the ROI Properties box depend on the type of ROI selected in the image For more details see Table 8 3 page 72 72 Living Image Software User s Manual Subj ROI tab ROI Properties ROI ROI 1 v ROT Label ROT 1 Bkg ROI SubjROI Info Image Number TLTZO0S5062414550 O06 Subject 1 wi Lock Position ct pix
145. cted Figure F 16 If background subtraction is needed remove the fluorescent subject from the imaging chamber and measure the fluorescent background select Acquisition Fluorescent Background Measure Fluorescent Background on the menu bar In the Living Image software the Sub Fluor Bkg check box appears on the Control panel after a background has been acquired You can toggle the background subtraction on and off using this check box 272 Living Image Software User s Manual The fluorescence background also contains the read bias and dark charge Dark charge subtraction is disabled if the Sub Fluor Bkg option is checked iin A Ri Tim 100 inno Figure F 16 Comparison of dark charge bias subtraction left and fluorescent background Subtraction right The autofluorescence from the nose cone and filter leakage have been minimized in the image on the right by using Sub Fluor Bkg option F 7 Adaptive Background Subtraction Adaptive background subtraction is a simple way to reduce the instrument fluorescent background by fitting and removing the background using the existing image for example the left image in Figure F 16 Unlike the method described in section F 6 Subtracting Instrument Fluorescent Background where you acquire an actual instrument fluorescent background image by removing the fluorescent subject from the imaging chamber to correct the background the new method uses software correction To perform ad
146. d right click the image to access a shortcut menu of viewing options Table 6 2 Kinetic view options Item Description Zoom Area To magnify a particular area draw a box around the area that you want to zoom in on right click the area and select Zoom Area on the shortcut menu Zoom In Incrementally magnifies the view Zoom Out Incrementally reduces the magnification Reset Zoom Returns the image to the default display magnification Pan View Enables you to view a different area of a magnified image To view another area of the image choose this option then click and hold the pointer while you move the mouse over the image Crop Area To crop the image draw a rectangle over the area of interest in the image then right click the area in the box and select Crop Area on the shortcut menu Draw Grid Displays a grid over the frame Draw Scale Displays a scale along the x and y axis of the frame Insert Tag Displays a tag with x y pixel information at a user selected location of the image To insert a tag right click a location in the image and choose Insert Tag on the shortcut menu Remove Tag Removes a user selected tag from the image y Caliper a Chapter 6 Kinetic Imaging 49 ifeSchences Table 6 2 Kinetic view options continued Item Description Remove All Tags Removes all tags from the frame Display Color Bar Choose this option to display the color scale Display Color Min Max Choose this option to dis
147. d Wells option 10 In the well plate table select the background wells and click Set Well Plate Quantification Window for Sequence DL GOH igas Sng Chk CLA 4OLis oon we Fharcphore Trp OB erie Tea oye moter C Cale MAS urement oe i A F Dectugroured Weds ste Lv Apk bo Segura Welllste cuien s Benks Dat padihan bad rnha duhan walle ii Selle Fer Cheke ELASH 01005001 guare Figure 14 5 Set the background wells 136 Living Image Software User s Manual 11 Click Quantify The results are displayed B Well Flite Quantification Windew fa IBIR A Well Flite Quantification Window KIBIR Fer Sequence BLADH 4101005 Sep Okk ELSA IOLOS OL For Sequence BLAD 14 O100S SE Dkk ELS IOLOS OL ow Pippen yE Pigoropeore yest Ce Dye meues C Cels BH wel Plate Type 5 Oye molecules C Cels Pie a ira Pee aar Hit Wel Piste Type wka Wiek iD AA i Hakarl Wiek iD AA i Apply bo Sequence Apply bo Daquence Wel Piste Quantification lots Wal Plie umiksi Plesults Litt liots ood T Custebene WOLUANT I Erisin Emission Extinchon Cost Cross Section air Linder Fii rer im Ct Mi a nC of nd jga Tol BAficency cm ROL va wrlh pilte peogrelal in 3 520 LOLIT LL Se 08 Gali 1ra AAF lela wa oe ed ed rama l D SLi well plale papilice Figure 14 6 Quantification plot and results 12 Check the linear fit of the data for each image in the quantification plot A good straight line fi
148. d from Precision Dynamics Co Because the fluorescent signal is significantly bright the background autofluorescent sources are not apparent 270 Living Image Software User s Manual Figure F 13 96 well plate fluorescent reference standard TR 613 Red The fluorescent signal is strong enough to exceed background emissions Imaging parameters DsRed filter set Fluorescence level Low Binning 8 FOV 15 f 1 Exp 4sec Reference Standard TR 613 Red is available through Precision Dynamics Co http www pdcorp com healthcare frs html Animal Tissue Autofluorescence Animal tissue autofluorescence is generally much higher than any other background source discussed so far and is likely to be the most limiting factor in in vivo fluorescent imaging Figure F 14 shows ventral images of animal tissue autofluorescence for the GFP DsRed Cy5 5 and ICG filter set in animals fed regular rodent food and alfalfa free rodent food Harlan Teklad TD97184 Animals fed the regular rodent diet and imaged using the GFP and DsRed filter sets show uniform autofluorescence while images taken with the Cy5 5 and ICG filter sets show the autofluorescence is concentrated in the intestinal area The chlorophyll in the regular rodent food causes the autofluorescence in the intestinal area When the animal diet is changed to the alfalfa free rodent food the autofluorescence in the intestinal area is reduced to the levels comparable to the rest of the body In thi
149. d intensity I is reduced to a relatively simple expression I SK A exp He 1 where S is the absolute total photon flux emitted by the luminescent source and d 1s the source depth The term ppg is the effective attenuation coefficient It is determined by the tissue coefficient of absorption u and reduced scattering u that quantify the two main phenomena light undergoes in tissue The function K A is a more complex expression that is derived from the model and includes terms that describe the effect of the tissue air boundary on the light propagation Both py and the function K are dependent on the wavelength Equation shows that if the total integrated intensity ROI measurement is measured at several wavelengths it is proportional to an exponential function of the product of the depth and the optical property uy Therefore the steps to planar spectral image analysis include e Acquire two or more images at different wavelengths e Measure the total integrated intensity on each image e Fit the measured values to the exponential function of Equation 1 The results of the fit are the total flux of the luminescence source S and the source depth d Y Caliper ifeSciences Appendix G Planar Spectral Imaging 277 G 2 Optical Properties Planar spectral image analysis requires prior knowledge of the tissue optical properties at the wavelength used at image acquisition The two main optical parameters ar
150. dade E REDS eee es 171 13 6 Managing Results aoaaa 172 13 7 Point Source Fitting Tools 2 2 8 s 2044844 4844444 9 Sew eee eee Oe SY 173 14 3D Quantification Database 2 2 2 ee 179 14 1 Preparing amp Imaging the Samples 2 2 ee 179 14 2 Creating a Quantification Database 2 180 14 3 Managing Quantification Results 2 183 15 3D Reconstruction of Sources 2 2 eee ee 185 15 1 Reconstructing Luminescent Sources 2 1 o 188 15 2 Reconstructing Fluorescent Sources aoao 198 15 3 3D Reconstruction Results 2 200 15 4 Checking the Reconstruction Quality 2 0 204 15 5 Meastirins SOUICES 244 fi 4244 4 645 5 wR ee H Eee Be eee eh eee eee Be 206 15 6 Displaying Luminescent amp Fluorescent Sources on One Surface 0 209 15 7 Exporting a 3D Scene 2 462 465 4a ee bee OEE REE REORDER ERE EES 209 15 8 3 TOOISOVEIVIEW ava ee Ge ub eav Sb Re Gene REE E EEK E EE eee SS 211 15 0 OOS SUr a Ge ae ee eee OES ES Eee REEYE REED EE CERRO 211 15 10 3D Tools Source noanoa 213 15 11 3D Tools Registration 2 214 15 12 3D Tools Animate lt 4 6 lt 28 5 ee ee eee ees eee aun dee EES Eee ee ee Bes 221 15 13 DLIT FLIT Troubleshooting oaoa 2 227 Appendix A IVIS Acquisition Control Panel 2 2 eee eee es 229 mA COMUOLPaNel 44 454 6 54 Gen4 ee ee Ge eeeeetacee ee eee ee eae ee ts 229 A2 Manually Setting th FOCUS i 5 24 8 4 sadra td doa peara aeaa t
151. data For more details on measurement units see page 247 Use Saved If this option is chosen data are displayed using a user specified color palette For Colors 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 38 Living Image Software User s Manual Table 5 4 Image window Sequence view continued Item Description Layout Choose a display option for the images in a sequence For example here Is Film Strip mode Options Sort by Options for ordering images in the sequence window Default Order in which the images are stored in the folder TimeStamp Ascending order of the image acquisition time UserlD Ascending alphanumeric order of the user ID Display Choose the types of information to display with each image In this example exposure time and binning factor are displayed on each image Click to show or hide the image label information Figure 5 13 Opens all of the images in the sequence Closes all open images Opens the Edit Sequence dialog box that enables you to add or remove images from the sequence Enables you to export the active image as a graphic file for example png dcm 7 Cali Y a Iper Chapter 5 X Ray Imaging 39 feSciences Table 5 4 Image window Sequence view continued Item Description ij Creates a preview picture snapshot of the
152. data for the reconstruction 191 Quantify the number of cells in a luminescent source or dye molecules in a fluorescent source 179 Display luminescent or fluorescent sources on one surface 188 Export source voxels or source surfaces 188 Kinetic Imaging Acquire ventral kinetic data using the Ventral Imaging Chamber 59 Spectral Unmixing A spectral unmixing wizard for image sequence setup 149 Distinguish the spectral signatures of up to four different fluorescent or luminescent reporters The Spectra window displays the spectra plots of the unmixed results Add other spectra 152 to the plot from the Living Image source library or the spectrum associated with an ROI View user selected probes in the composite view 151 Apply signal thresholds 149 ROIs Automatically draw ROIs on all images in the Sequence View window 102 General Activity log tracks session duration for each user and IVIS statistics for example the 19 number and type of acquired images fluorescent lamp usage X ray tube usage 2 Living Image Software User s Manual 1 2 About This Manual This user manual explains how to acquire image data on an IVIS imaging system and analyze images using the Living Image software The manual provides detailed instructions and screenshots that depict the system response sometimes the screenshots in the manual may not exactly match those displayed on your screen For more details on your IVIS imaging system please see the appropria
153. e editor 3 Click the Imaging Wizard button tssraweed The Imaging Wizard is not available until the IVIS Imaging System is initialized and the demand temperature is locked Y Caliper Chapter 5 X Ray Imaging 33 Select the type of subject and probe from the drop down lists Sequence Editor pred pra ad on seat tna 3 emer GE uate Cl meee Double click here to set aia Imaging Wizard up a bioluminescence image sequence Ceo fe coir Kr uE ey faci Sarai ae cr If this screen does not appear when the wizard starts click Restart Wizard restat wea ON the wizard screen to restart the wizard CATA E HE a a a LE a Double click here to set up a fluorescence image sequence dii besti bac ae rena ce ect Mace ee Figure 5 7 Opening the sequence editor and starting the Imaging Wizard 4 In the wizard choose bioluminescence or fluorescence imaging Figure 5 7 5 Inthe next wizard screen choose the type of image sequence that you want to acquire Step through the rest of the wizard 6 To include an X ray image or photograph in the sequence put a check mark next to the X ray or Photograph option when you set the imaging parameters in the wizard Figure 5 8 34 Living Image Software User s Manual Inspr Subject Capcoure Paai fe bubo Settings fe Luminescent o Paia E kay Fiii col Wia C Ogm Fizu Subject Height 1 50 5 on Foour use atid heig
154. e the BlackRed color table represents the source intensity photons sec from low to high using a color scale from black to red If Reverse is chosen the source intensity photons sec from low to high Is represented using the color scale from red to black Log Scale Choose this option to apply a logarithmic scale to the photon density scale Y Caliper mae Chapter 15 3D Reconstruction of Sources 167 15 10 3D Tools Source Use the source tools to e Adjust the appearance of sources in DLIT GADLIT or FLIT reconstructions e Make source measurements page 160 e Export voxel measurements csv Surface Source Ragiitration Animate C Smpmnce view Sow Sgecha Diis Soure Surface i l j Fu E a7 i k m le a HE gt F ra ati seyi T a 4 0 5 tj h E Cpa Liat E Displey viele Threshokd J Ssa S Gradation 5 owed size 031 6 Display vosek as Sinouthing 505 Cubes i Cier Table co fis Reverse Log Stake Merarel HARE Tia Flas 2 20 ae Waimes aA mn Host Organs Lindner Center oF Mase Sood ct ok Pont var Genter if met ii x SOO Fett Height 26 4 mm Figure 15 29 Source tools and example DLIT reconstruction Table 15 10 Source tools Item Description Display Source Choose this option to display the source surfaces reconstructed using DLIT Surface GADLIT or FLIT A surface will be wrapped around the currently displayed
155. e Export button e in the image window to view the current folder path setting Figure B 3 Window Size amp Position Applies the active image window size and position settings to subsequently opened image data M ost Recently Used Dataset History Applies the active image window size and position settings to subsequently opened image data Y Caliper feSciences Appendix B Preferences 237 Table B 1 General preferences continued Item Description Display ROI Label As Photons Select the type of measurement in photons to show in the Measurement ROI label Counts Select the type of measurement in counts to show in the ROI label Some of the general preferences specify how the main application window is organized To undock the Tool Palette click on the palette title bar and drag it a distance greater than its width To dock the Tool Palette in the main window drag the palette to the right or left side of the window and release G Pis E _ aL in gumi r i wk a e E k UU t amp P unom s i ro a Bpa Seah Tei Fete EH IFFT rris b Lrj sires Te wel i Fe Si 3 y E I iat Alpe HE eee ee T Tamla i os Fima gered Lec Bn E E eee T Mumla Ije epin C Fam bare raie E i om Tool palette a i aah Sram tning gatear Activity window Phin ir Sema Cre ei ec n Figure B 3 Main application window 238 Living Image Software User s Manual B 2 User Preferences Preteens Cerea
156. e and overwrites previous results Pataturie Delete Deletes the database from the system Baber WEGUAHT_I eam ae Load Opens quantification results from the system path Save Saves the quantification results to a system database that is available for DLIT or FLIT reconstruction Overwrite Saves the results to the selected database name and overwrites previous results 138 Living Image Software User s Manual This page intentionally blank Y Caliper feSchences 1 5 3D Reconstruction of Sources Reconstructing Luminescent Sources 2 0 6 2 ee ee ee ee 142 Reconstructing Fluorescent Sources 2 08 2 eee eee ees 152 3D Reconstruction Results 1 2 ee 154 Checking the Reconstruction Quality 2 852 a 158 Measuring Sources 1 a ee 160 Displaying Luminescent amp Fluorescent Sources on One Surface 163 Exp riing a 3D SCENE is ee chee et EOS eR ESD ERS HOH KH es 163 JD Tools UVGIVIEW gc hk eee EERE EERE REED ER EES Se Ew BH 165 3D Tools Surface 1 ee tards Dasi aa ri 165 SU Tols SOUS ga kc beet E DSK TSE ER HOO HEE Ow ES SO 167 3D Tools Registration aaoo ee Ree ER RERE REE RSE E ESD ES 168 3D Tools Animate eh aw Rea RES ROHR eee wee OS 175 DLIT FLIT Troubleshooting 1 2 eee ee ee 181 The Living Image software provides algorithms for 3D reconstruction of luminescent or fluorescent sources tomographic analysis For more details on the
157. e and click the or arrow 5 To update a key frame a Select the key frame and adjust the 3D view b Click the 4 button 6 To delete a key frame a Select the key frame that you want to remove b Click the button and select Delete Current y Caliper heh iii Chapter 15 3D Reconstruction of Sources 181 15 13 DLIT FLIT Troubleshooting Issue Solution No sources in solution e In DLIT or FLIT this can occur if the surface is not correct That is if a Surface Is imported into the 3D View from another source other than from the Structured Light Analysis In FLIT if there is very little signal detected at the surface and the Background box is checked in the Params tab then it is possible that only the background fluorescence is simulated Surfaces are spiky e The most common source of spiky surfaces are folds in the animal Skin or animal fur which corrupt the desired smooth lines projected on the animal from the laser galvanometer Choose the Fur Mouse option for Subject Smoothing the surface by using the Smooth feature in the Surface Topography tools can help improve the surface Toal Palette t L gt Manar Spectral maging J Surface Topography Drie Recondtruchion Subject Fur More we Reconrriuct Grierialion Corga ka Drie orecectharey Level Medan Smooth Feios Sawe Renis hame Doran surface sa Dei Liw GADLIT 30 Reconstruction
158. e and pH dependent The luciferase spectra included in the software are only valid for measurements performed at 37 C and pH 7 0 7 5 If you use other temperature or pH conditions for an experiment the associated luciferase spectral curve is required for planar spectral image analysis For more information on the pH and temperature dependence of the luciferase spectrum please contact Caliper Corporation 278 Living Image Software User s Manual G 4 An Example of Planar Spectral Imaging Melanoma cells were injected intravenously into the tail vein of nude mice After 13 days metastases developed in the lungs kidney and hind limb bone An image sequence was acquired on the IVIS Imaging System 200 Series using filters at six wavelengths from 560 to 660 nm in 20 nm intervals NOTE When using the 560 nm and 580 nm band pass filters tissue optics result in a larger attenuation of light due mainly to hemoglobin absorption A longer exposure time is recommended at these wavelengths Figure G 2 shows the metastasis sites The signals from the lungs and right kidney are well defined in both animals However in the lower back area of the left mouse the signals are in close proximity causing an artifact in the planar spectral analysis Se CEZ00RI7 15150449 08 Unite Protone Dupe Gerke Figure G 2 Metastatic sites in nude mice Mice were imaged 13 days after a tail vein injection of 5x10 B16F10 melanoma cells Imaging parameter
159. e details see Quantifying Image Data page 251 If the image is displayed in photons you can compare images with different exposure times f stop setting or binning level When an image is displayed in terms of efficiency the fluorescent image is normalized against a stored reference image of the excitation light intensity Efficiency image data is without units and represents the ratio of emitted light to incident light For more details on efficiency see page 253 Fluorescent Efficiency The detected fluorescent signal depends on the amount of fluorophore present in the sample and the intensity of the incident excitation light At the sample stage the incident excitation light is not uniform over the FOV It peaks at the center of the FOV and drops 266 Living Image Software User s Manual of slowly toward the edges Figure F 7 To eliminate the excitation light as a variable from the measurement the data can be displayed in terms of efficiency Figure F 8 TOR RI T 60 E i E 2 40 BE ames CY 10 FY 15 20 fO 20 0 15 1 5 0 5 10 15 Widltli ca Figure F 7 Illumination profiles for different FOVs on an IVIS Imaging System 100 Series measured from the center of the FOV A TAGS 10114517_010 Choose Radiant Efficiency to enable a more quantitative comparison of fluorescent signals E part heraa Reda iaer pole irta aye Cader Satake Pin a 2 H Piar Se Figure F 8 Fluorescent image dat
160. e 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 Choose this option to automatically acquire a photograph The illumination lights at the top of the imaging chamber are on during a photographic image so that the system can acquire a black and white photograph of the sample s Note You can adjust the appearance of the photographic image using the Bright and Gamma controls See page 99 Choose this option to acquire an X ray image Choose this option to take a structured light image an image of parallel laser lines scanned across the subject when you click Acquire The structured light Image is used to reconstruct the surface topography of the subject which is an Input to the Diffuse Luminescence Imaging Tomography DLIT algorithm that computes the 3D location and brightness of luminescent sources When this option is chosen the f stop and exposure time are automatically set to defaults for the structured light image f 8 and 0 2 sec respectively The Spatial resolution of the computed surface depends on the line spacing of the structured light lines The line spacing and binning are automatically set to the optimal values determined by the FOV stage position and are not use
161. e on photograph 9 Chapter 2 Overview of Imaging amp Image Analysis Y Caliper feSciences Table 2 2 Image types Example Imaging Mode Description Structure A structured light image of parallel laser BA scanned across the subject The surface topography of the subject is determined from the structured light image K Hi Wisp j JA TTINA ETI aa X ray An exposure of the subject using the X ray energy source on the Lumina XR The X ray image is displayed as a grayscale image X ray image Overlay Fluorescent Image on X ray image Kinetic A series of images captured on the IVIS Kinetic Imaging System that enables visualization of luminescent or fluorescent signals in real time Play back kinetic data in real time or view a particular frame s 10 Living Image Software User s Manual 2 3 About Image Sequences A sequence is a collection of images that are grouped together in a single folder A sequence may include images that are acquired during the same session and are intended to be grouped together For example images taken at different time points or an image sequence for DLIT or FLIT 3 D tomographic analysis Images that were acquired during different sessions can also be grouped together to form a sequence for more details see page 98 For example a time series could be constructed from images acquired on different days following an experimental treatment So
162. e page 160 Export Voxels Enables you to export the voxel measurements in their x y and z coordinates and source intensities csv file 15 11 3D Tools Registration Use the registration tools to e Display organs in the surface page 170 e Manually adjust the location or scale of organs in the surface page 170 e Check the organ fit page 172 e Import an organ atlas page 173 You can check the organ fit in the 3D View window page 172 YP Caliper Hatclances Chapter 15 3D Reconstruction of Sources 169 7 aD Tonks Sulaa Source Regetrabon Animata C Sequence view OAD view Soot Poser alin Tracts ag till gt ga ra ir ee uj SAA __ E Chaplin Corgan A Demy Ergan Allas Femsle Loorsal Ongar EA Darena EA bladder EI bore E Piren afp m EA Mi EA Bitri E yelni enir e rr saetornlnec Ly SIE Height 264 mm Perspective Figure 15 30 3D registration tools and surface with fitted organs skin not displayed Table 15 11 Registration tools Item Description Use this tool to manually adjust the scale of location of organs For more details see page 170 Fits the organs to the surface using a linear transformation that keeps the ia Shape of the atlas surface am E Fits the organs to the surface using linear transformation and volume H deformation If you manually change the location orientation
163. e segment endpoint to a user selected position Width Sets the line width Position Line position pixels X Min Displays the minimum and maximum value of the x axis Use the arrows X Max to change the x axis min or max If photons is selected in the image window the x axis units pixels If counts is selected in the image window the x axis units cm To display the range available for the Min or Max place the mouse pointer over the Min or Max edit box Y Min Displays the minimum and maximum value of the y axis Use the amp arrows Y Max to change the y axis min or max To display the range available for the Y Min or Y Max place the mouse pointer over the Min or Max edit box Click to reset the X and Y Min and Max values to the defaults Full Scale Select this option to display the full X and Y axis scales Logarithmic Scale Select this option to apply a log scale to the y axis Enables you to choose the grid line pattern to display in the line profile Es window pem Exports the line profile data to a csv or txt file H Copies the line profile graph to the system clipboard E Opens the Print dialog box 88 Living Image Software User s Manual Making Measurements To measure distance with the measurement cursor 1 Open an image and in the Image Information tools click the Distance Measurement Cursor button A measurement cursor 4s appears on the image Figure 7 16
164. e the e Absorption coefficient u that defines the inverse of the mean path before photons are absorbed by the tissue e Reduced scattering coefficient u that defines the inverse of the mean path before photons are scattered isotropically in the tissue The effective attenuation coefficient uz is a function of the absorption and reduced scattering coefficients Hef SHa U s M 2 Calculation of the function K in Equation requires all three coefficients u4 H s and Mey as input The function K includes a term called the effective reflection coefficient to account for the reflection of light at the air tissue boundary due to a mismatch in the index of refraction The tissue index of refraction is generally assumed to be close to 1 4 The model assumes that the tissues are optically homogeneous and the Living Image software provides several factory set tissue optical property values to choose from G 3 Luciferase Spectrum Analyzing spectrally filtered images requires knowledge of the spectral dependence of luminescent light emission The luciferase luminescence spectrum was measured in vitro at 37 C and pH 7 in various cell lines This spectrum is used to normalize the photon flux values that the software measures at each wavelength Source spectra for several reporters are included in the database including firefly click beetle renilla and bacteria Figure G 1 The firefly luciferase spectrum is temperatur
165. e you move the mouse 7 5 Correcting Image Data Use the Corrections Filtering tools to subtract background or apply corrections to the image data For more details on sources of background see Appendix E page 255 You can also apply smoothing and soft binning to the image data For more information on binning and smoothing see Appendix C page 245 Tool Palette gt Image Adjust Corrections Filtering Read Bias Subtraction Read Bias Subtraction and Flat Field Flat Field Correction Cosmic Correction Adaptive FL Background Subtraction Correction are default mandatory corrections in photons mode In counts mode these corrections can be cleared Binning Smoothing None gt Point Source Fitting gt FLIT 3D Reconstruction Figure 7 10 Tool palette Corrections Filtering tools 82 Living Image Software User s Manual Table 7 4 Tool palette Corrections Filtering tools Tool Description Read Bias Select this check box to subtract dark background from the image data If Subtraction Dark a dark charge image is available for the imaging conditions the dark Charge Subtraction background image including read bias noise will be subtracted Otherwise only read bias noise will be subtracted For more details on background see Appendix E page 255 Note In Radiance Photons mode dark background or read bias subtraction is a mandatory default In counts mode the check box can be cleared Fl
166. ect around the x y or z axis use a click and drag operation Click to hide or show the x y z axis display in the 3D view window Click to hide or show coronal sagittal and transaxial planes through the surface in the 3D view window Click to show or hide a bounding box around the surface Click to show or hide a grid under the surface E MR Select this tool from the drop down list to change the view perspective top bottom left right front back or perspective view For examples of the views see Figure 12 6 Se 4 Select this tool from the drop down list to display the perspective view k Click to show or hide measurement cursors in the coronal sagittal or transaxial views E ii After you perform DLIT or FLIT analysis click a voxel in the 3D reconstruction i then click this 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 Changing the View Perspective Figure 12 6 shows examples of the available views You can view the surface from different perspectives by doing one of the following e Select w to change the view Figure 12 4 e Alternately click the surface in the 3D View window then press the V key to cycle through the different views of the surface e Select to display the perspective view Figure 12 4 ii Y Caliper Chapter 12 Recon
167. ectors The back illuminated CCD has the highest efficiency particularly in the 600 800 nm region of the spectrum the area of greatest interest for in vivo imaging Back lkmmated E front kminatad amp maga Intensfer Quantum Efficiency 300 400 600 800 000 Wavelength nm Figure C 1 Quantum efficiencies Bialkali photocathode A back illuminated CCD and front illuminated CCD m IVIS systems use back illuminated CCDs Lens Aperture IVIS Imaging Systems are equipped with a high light collection f 1 lens The sensitivity of the IVIS Imaging System can be adjusted by changing the f stop setting that controls the lens aperture The detected signal scales approximately as 1 f stop For maximum sensitivity select f 1 the largest aperture setting on the VIS Imaging System Figure C 2 This provides the greatest light collection efficiency but results in the minimum depth of field for the image The depth of field refers to the depth over which the image appears to be in focus and is determined by the f stop and the field of view FOV At f 1 the depth of field ranges from 0 2 cm at FOV 3 9 cm IVIS Imaging System 200 Series only to 2 cm at FOV 25 cm You can use the manual focus option on the Control panel to easily assess the depth of field at any f stop and FOV setting For more details on 246 Living Image Software User s Manual manual focusing see page 36 Generally f 1 is recommended for low light luminescen
168. ectral imaging Mace 9 Thi Andos Properties Hatu Special Hesut Fl Digah ime Total Fha ji PO ae 1S Bee a R rd E Pe Lissas Fa renan A eo i Save Nacuki i After the analysis is completed usss sein 2 lt Daig click a button to display Save Max G die graphical results sll Linear Fit Plat ter EOI I Iniensbiy m Lavaide Plot bie EOI I Log Home Fox E jo Moemabred Fux a x 0 0 20 F a TAJ D O40 030 im wal Figure G 4 Planar spectral analysis results To estimate the cell count divide the absolute photon flux by the flux per cell This is best determined by making independent in vitro measurements of the cell line used in the experiment 280 Living Image Software User s Manual The Plot of Linear Fit Results is weighted by the uncertainty of the measured images and takes into account the uncertainty in the determination of the optical properties The precision of the method is largely determined by the known precision of the optical properties In most cases the relative uncertainty in the depth determination is equal to the relative uncertainty in the optical properties An analysis of the dorsal and ventral views of the mouse left lung in Figure G 5 results in total flux values that are very similar The measured depth values are also close indicating that the cells are distributed about the same distance from the front and back of the animal D t bi w p
169. ectronic dark charge and read bias corrections that are applied to the raw CCD data The Image Adjust tools and zoom feature are helpful for selecting an appropriate area for an ROI By setting the image minimum close to zero and zooming in on a background area in the image you can determine where naturally occurring background luminescence or autofluorescence is present For more details on the Image Adjust tools and the zoom feature see page 99 and Magnifying or Panning in the Image Window page 81 To measure background corrected signal 1 Draw one or more measurement ROIs on the subject For more details see page 64 2 Draw an average background ROI on the subject a Select Average Bkg ROI from the Type drop down list b Click the Square 0 or Circle button and select 1 The ROI is added to the image For more details on adjusting the ROI position or dimensions see page 73 and page 74 NOTE The average background ROI and measurement ROI do not need to be the same shape or size because the software computes the average intensity signal in each ROI 3 Use one of the following methods to associate the average background ROI with one or more measurement ROIs Method 1 Draw a subject ROI that includes the measurement ROI and the average background ROI For details on how to draw a subject ROI see page 64 ii ae E Jepi Cee oF e nai m Deewe 1 s iy 66 Living Image Software User s Manual Method
170. ee ee 27 Acquire an Image Sequence That Includes X Ray Images 32 Measuring Relative Density 0 ee ee ee 39 X ray images can be acquired on the Lumina XR Imaging System An X ray image luminescent or fluorescent image and a photograph can be acquired at the same time You can choose two images to create an overlay Figure 5 1 a ferme AA A Dorh Pah Figure 5 1 Example overlay fluorescence image on X ray image 5 1 Acquire an X Ray Image This section explains how to acquire an X ray image on the Lumina XR Imaging System For information on including a luminescent or fluorescent image in the acquisition see Chapter 4 page 5 1 Start the Living Image software double click the icon on the desktop 2 Initialize the Lumina XR Imaging System and confirm or wait for the CCD temperature to lock For more details see page 17 3 In the control panel put a check mark next to X Ray Figure 5 2 To enable X ray acquisition verify that the X ray enabling key on the front of the Lumina XR is set to ON and the orange X ray enable button has been depressed and is illuminated 28 Living Image Software User s Manual VIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter X ray acquisition settings d Field of views System Status o X Rays will be produced Idle when energized Acquire Subject height iso ll 50 a 0 Sequence Setup
171. elect Measurement ROI from the Type drop down list 3 Click the Contour button and select Kinetic ROI The create tool appears on the image FQ b G a E ommen ee Map Fa Gji rbir fe Figure 8 12 ROI create tool 4 Use the ring amp to move the create tool to the area where you want to draw the ROI then click Create The ROI and label appear on the image NOTE When drawing kinetic ROIs on kinetic data with multiple sources it is recommended that you start with the brightest source then the next brightest and so on in order to create ROls that can be distinguished based on the signal strength 5 If itis necessary to adjust the ROI boundaries or change any of the auto ROI parameters use the slider or aa arrows Threshold Specifies the minimum per cent of peak pixel intensity that a pixel must have to be included in an ROI identified by the software ON Click Done to hide the create tool The kinetic data playback starts and shows the ROI in each image 68 Living Image Software User s Manual NOTE After the ROIs have been created right click an ROI to view a shortcut menu of ROI commands Ctrl click for Macintosh users The shortcut menu provides easy access to many functions for managing ROIs and viewing ROI properties 7 To measure intensity in the ROI click the Measure button in the Tool Palette The Kinetic ROI Measurements table appears Figure 8 13 OO To view R
172. emperature box turns green when the temperature 1s locked at the demand temperature 90 C or 105 C for IVIS Systems cooled by a Cryotiger unit indicating the instrument is ready for operation and image acquisition The demand temperature for the CCD camera is fixed Electronic feedback control maintains the CCD camera temperature to within a few degrees of the demand temperature The default temperature of the stage in the imaging chamber is 37 C but may be set to a temperature from 25 40 C 18 Living Image Software User s Manual FIIS Acquisition Control Panel Imaging Mode Exposure Time Excitation Filter Emission Filter Fstop Binning Temperature Status P X Rays will be produced when energized Acquire Sequence Setup Initialize Temperature box color indicates Field of View Ic Demand Measured Service fic on Camera Temp 90 ggo Stage Temp 37 0 oO Subject height o co cm 2 j ce Focus fuse subject height Temperature Click the temperature box to view the demand and measured temperatures of a System not initialized the CCD camera and stage ha System is initialized but CCD camera temperature is out of range and not ready for imaging ibe System is initialized and CCD camera is at or within acceptable range of the demand temperature and locked The system is ready for imaging Figure 3 4 IVIS acquisition control panel NOTE The
173. en cycled to the imaging chamber or the camera controller a component of some IVIS Imaging Systems The initialization procedure moves every motor driven component in the system for example stage and lens to a home position resets all electronics and controllers and restores all software variables to the default settings Initialization may be useful in error situations For further details on instrument operation see your IVIS Imaging System hardware manual To initialize the IVIS Imaging System 3 Start the Living Image software double click the icon on the desktop 4 Inthe control panel that appears click Initialize You will hear the motors move VIS Acquisition Control Panel Exposure Time Binning F Stop Excitation Filter Emission Filter Imaging Mode Field of View System Status Idle AcquIre Ba Jm Subject height cm Sequence Setup Figure 3 3 IVIS Acquisition control panel NOTE The control panel is only available on the workstation that controls the imaging system The items available in the control panel depend on the particular IVIS Imaging System and the imaging mode selected luminescent or fluorescent Image Setup or Sequence Setup mode 3 3 Checking the System Temperature The temperature box in the IVIS acquisition control panel indicates the temperature status of the charge coupled device CCD camera Figure 3 4 After you initialize the system the t
174. encil 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 Open the Data Preview window and click Start in the Analyze tab Figure 15 6 2 Click an image in the Data Preview window 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 Y Caliper Chapter 15 3D Reconstruction of Sources 147 4 In the window that appears enter a new Threshold value The new Threshold appears in the Analyze tab 5 To reset the Threshold to the default value for the selected images click Restore Threshold Tool Pabette Data Preview window WT 00c0074145507 S00 lyst 1 C eee ko aves ra Gute Preview gt RIM Teale gt gt Hans Spectral Treachery gt Surlace Topar gt Point Source Pibbing DLIT 30 Reconstruction The red outline Anae Properties Renk indicates the Se Se A Thum icin Select Filters Fie Ci o oF aR c aE aE I El ean A LATI AF Hotel ri E gt Spectr al Larinia E Image Lita Set the Threshold here ak free image selected for data adjustment Thresh T a3 as 65
175. enu e Select the data row and click Load e Double click the thumbnail The image s and Tool Palette are displayed Open data is highlighted in green in the browser Figure 7 2 W Caliper Chapter 7 Working With Data 73 ae ju t CB MF mecu w C Apply te al E Liring imaget Drescer HOO CC SOOM E oc 4 Seu a Se ee oo ee oe igabanbacin Prarie Filet Caicos Li rira Hain Contig ORT Ha Pac erate be tat aie ae Spe ih Link Counts C tee Saeed Colors Tp T btn A i LET n Barile pF y o DLT 30 Becenstruction ie GALUT 20 Moriri ie gt Spectr al Unaribeing Figure 7 2 Image sequence opened M ultiple data sets can be open at the same time Table 7 1 Living Image browser Item Description Hide Browse View Closes the browser table Close Preview Closes the image preview box Label Set A drop down list of the available label sets which specify image information column headers that is displayed in the Living Image browser Add to List If you choose this option the data that you select in the Browse for Folder box is added to the Living Image browser If this option is not chosen the data that you select in the Browse for Folder box replaces the contents of the Living Image browser except for loaded data Browse Opens the Browse For Folder box View The name of the Living Image browser configuration the column headers and their order in the browser Configure Opens
176. erly 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 180 Living Image Software User s Manual To save an animation setup 1 Click Save 2 In the dialog box that appears select a directory and enter a file name xkf To record the animation to a movie 1 Click Record 2 In the dialog box that appears choose a directory enter a file name mov mp4 avi and click Save To edit an animation setup 1 Open a reconstruction 2 Open an animation setup To select a predefined setup make a selection from the Preset drop down list To select a saved user defined setup a Click Load b In the dialog box that appears select an animation setup xkf 3D Tools Surface Source Registration Animate Preset Animations Presets Spin Cv on Axis wt Frame Factor 1 B Animation Setup Time Scale o Key Frame 1 Key Frame 2 Key Frame 3 Key Frame 4 bd Key Frame 5 Key Frame 6 Frames Per Second Total Duration secs Figure 15 41 List of key frames in the selected animation 3 To add a key frame a Adjust the position of the reconstruction in the 3D view using an image tool for example or 4 For more details on the image tools see page 145 b Click the button 4 To reorder a key frame in the sequence select the key fram
177. escent 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 M ax The maximum pixel intensity associated with the color scale for an image Pixels greater than the maximum value are displayed in the maximum color Opacity Controls the opacity of the fluorescent or luminescent image Color Table Tools selecting and modifying the color scale associated with an image Color Scale Type Choose BlackLevel to show black at the low end of the color scale Choose WhiteLevel to show white at the low end of the color scale gum Click the drop down arrow to select a color table for the image data For more details on color tables see Pseudocolor Images page 249 Reverse Choose this option to reverse the selected color table Logarithmic Scale Choose this option to apply a log scale to the relationship between numerical data and the color range in the color table A log scale increases the range of meaningful numerical data that can be displayed 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 7 9 Rendering Intensity Data in Color The c
178. escription sf Enables you to select a spectrum to add to the graph From Library Choose this option to select a probe from the Living Image database The library includes spectra of different sources obtained using excitation and emission filters From ROI Choose this option to display a spectrum calculated for a user selected ROI w Deletes the spectrum selected in the spectrum list from the plot Type The type of spectrum UM X A spectrum generated by the spectral unmixing algorithm LIB A user selected library spectrum The library includes spectra obtained of different sources obtained using excitation and emission filters ROI A spectrum calculated for a user selected ROI Y Caliper a p Chapter 11 Spectral Unmixing 107 ifeSchences Table 11 4 Spectra window continued Item Description Name The spectrum identifier used by the unmixing algorithm The name cannot be modified Label The spectrum name in the spectrum plot key Double click the label to edit it n a es ee SRR Se ea The plot line color for a particular spectrum For the UM X type spectrum it is also the color in the composite image Double click a color swatch to open the color palette that enables you to change the plot line color for the spectrum Normalized Normalizes ROI spectra to library spectra Legend Click to show or hide the spectra plot legend Red Shift Adjusts library and ROI spectra to compensate for tissue absorption
179. escription of the preset animations After a preset animation is selected a list of the key frames appears 3D Tools Surface Source Registration Animate Presel Animations Presets Spin CW on Axis Frame Factor 1 4 Animation Setup Time Scale E key Frame 1 Key Frame 2 Key Frame 3 Key Frame 4 Key Frame 5 Key Frame 6 KE ame Frames Per Second Total Duration secs Figure 15 38 Key frames for the selected animation 6 To view the animation click Play NOTE You can view multiple animations sequentially For example if you select Spin CW on X Axis and Spin CW on Y axis from the Presets drop down list the animation shows the 3D reconstruction spinning clockwise on the x axis then spinning clockwise on the y axis 178 Living Image Software User s Manual Creating a Custom Animation To create an animation specify a custom animation setup or edit an existing setup 1 Open 3D reconstruction results 2 Select properties to display in the 3D View window for example organs sources surface or photon density maps 7 3D Tools Surface Source Registration Animate Preset Animations Presets Spin Cy on amp Axis Frame Factor 1 Animation Setup Time Scale o m Play Record Frames Per Second 10 li 4 it Total Duration secsi 5 Load cave Figure 15 39 3D animation tools 3 In the 3D tools click the Animation tab If nec
180. essary clear the key frame box click the 6 button and select Delete All 5 To capture the first key frame click the Ff 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 7 or 4 For more details on the image tools see page 145 7 Click the f button The second key frame is added to the key frame box m T Y Caliper Chapter 15 3D Reconstruction of Sources 179 YE 3D Tools Surface Source Registration Animate Spin CW on xis ha Frame Factor Preset Animations Presets Animation Setup Time Scale a H key Frame 1 key Frame 2 Key Frame 3 Key Frame 4 Key Frame 1 Key Frame 2 Frames Per Second Total Duration secs Key Frame 3 Key Frame 4 Figure 15 40 Example key frames for a custom animation 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 180 Click a key frame to display the associated 3D view and the time stamp position in the time scale 0 100 at which the frame occurs in the animated sequence 9 Confirm the defaults for FPS frames per second and Total Duration length of animation or enter new values FPS X Total Duration No of frames generated to create the animation The number of generated frames should be 2 to the number of key frames Otherwise the frames may not be prop
181. ession you are prompted to enable the autosave function 14 Living Image Software User s Manual Living Image 4 0 Do vou want bo enable auto saving of acquired data For this session 7 This can be changed anytime From the Acquisition menu Yes Figure 4 12 Autosave prompt 15 To enable autosave click Yes in the prompt and choose a folder in the dialog box that appears If autosave is enabled all images acquired during the session are automatically saved to a user selected folder You can choose a different folder at any time select Acquisition Auto Save on the menu bar Image acquisition proceeds During acquisition the control panel Acquire button becomes a Stop button To cancel the acquisition click Stop in the control panel The image window and Tool Palette appear when acquisition is completed Figure 4 8 page 10 4 4 Acquire a Sequence Using the Imaging Wizard To acquire an image sequence first specify the acquisition parameters for each image in the sequence editor Figure 4 14 The Imaging Wizard provides a convenient way to do this for some imaging applications Table 4 3 The wizard guides you through a series of steps prompting you for the information that the software needs to set up the sequence in the sequence editor If you don t use the wizard you can set up a sequence manually for more details see page 21 5 i Birime ae ieee k EE FA Ge Er PFU ae AFFETTE i
182. etails on the Imaging Wizard see page 26 DLIT is the recommended algorithm for 3D reconstruction of luminescent sources If you plan to manually set up the sequence Table 15 2 shows the recommended image sequence Analyzing more images usually produces more accurate results At a minimum the sequence must include data from at least two different emission filters 560 660 nm e Emission filter 1 Photographic luminescent e Emission filter 2 Luminescent image e One structured light image Table 15 2 Recommended image sequence for DLIT or GADLIT analysis Image Type Emission Filter Options 560 580 600 620 640 660 Photograph J Select the Reuse option in the control panel Structured light J Luminescent J J J J J J It is recommended that the binning level be the same for all of the luminescent images For more information on the DLIT algorithm and user modifiable parameters see Appendix H page 283 Y Caliper feSciences Steps to Reconstruct Luminescent Sources Using DLIT 1 Load an image sequence Chapter 15 3D Reconstruction of Sources 143 2 Generate or load a surface using the Surface Topography tools For details on generating the surface see page 113 3 In the Tool Palette choose DLIT 3D Reconstruction The Analyze tab shows the data that the algorithm automatically selects for the reconstruction Figure 15 2 For more details about the Threshold see page 146 Toal Falete _
183. ette Spectral Unmixing E Analyze Options Results Spectral Unmixing Results Item Value Mumber of Iterations 20 Number of Components 2 Number of Wwayelenghts 7 Humber of Samples 2541 Lack of Fit ofP24 2 6175 Lack of Fit SEP 3 02533 Divergence counter 0 Maximum Iterations 200 Denoise PCA Medium Normalization Equal Heightidefault Mon negativity method nnls Weighting mode None Column Weighting mode Mone Row Weighting mode None Save Results Name SPUM_6 wt Figure 11 8 Tool palette Spectral unmixing tools Results tab Table 11 5 Spectral unmixing tools Results tab Item Description Spectral Unmixing Results Number of The number of iterations that the algorithm used Iterations Number of The number of components unmixed Components Number of The number of wavelength pairs used in the analysis Wavelengths Number of Samples The number of pixel samples used in the analysis Lack of Fit PCA The fitting residue compared to the data filtered by principal component analysis Lack of Fit EXP The fitting residue compared to the experimental data Divergence The number of divergences that occurred Counter Maximum The maximum number of iterations allowed Iterations Denoise PCA Indicates how much of the data was filtered by principal component analysis Normalization The normalization method used in the analysis Non negativity The non negativity method used in the analysis Method
184. ew C Use Bkg Offset _ Replace ROIs Restore Defaults gt Planar Spectral Imaging gt Surface Topography gt Point Source Fitting gt BLIT 3D Reconstruction gt GADLIT 3D Reconstruction Figure 8 24 Removing ROIs from the system 80 Living Image Software User s Manual 8 9 Managing the ROI Measurements Table The ROI Measurements table shows information and data for the ROIs created during a session The ROI measurements can be displayed in units of counts or photons or in terms of efficiency For more details see Quantifying Image Data page 247 Viewing the ROI Measurements Table 1 Click the button Alternately select View ROI Measurements on the menu bar Tool Palette La Image Adjust gt Corrections Filtering I gt Image Information V7 ROI Tools T RO kesari Refresh DER BOOT Fiesa renens iraga Murer Rial img Leper Total Count Arg counts Eider Counts Min Counts Max Counts Bod Pook wate rar a a TTia 001 Overly Sheet betii 4 H er l L Pieet Aeri i a ASit i Dela n Taoousaa5 Oot Roli Overlay HM WDMI Lae aH Tae WHE i ALI 140009 EA eon Spratling N H Ae T Ht A eet AHA A 7 E au A Peet AENEHY H UIST O03 RO Overly BRT AAE Meee See eHe gl S M4etd Bales TLISMEORI45507 O04 ROLI Gwele Ecleete OSSeeD4 Gorea SiTe E Sde 40 S 6 bentd SHer TASOA Oo RO Ovelay HHO Le eH LI ie See LEH ig d E Li Cumieira Saber rate Image irbes Pol C
185. f formatting options for the Image Layout window For example the 2x2 layout style provides 4 separate layout areas in the window A different image can be pasted into each layout area Layout Style Layout Ze wl re _ To apply notes to an image enter text in the annotation box and press Enter se Drag the text to the location of interest in the image mE 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 7 11 Editing an Image Sequence You can add or remove individual images from a sequence Only individual images not an image sequence can be added to a sequence 1 Open the image sequence that you want to edit 2 If you plan to add images to the sequence browse for the images that you want to add in the Living Image browser For more details on browsing see page 71 If you plan to add images to the sequence browse for the images that you want to add in the Living Image browser For more details on the browser see page 71 3 In the image window click the Edit button Browser Images BIZ0080 241445930 DA20081 107162612 4200901 20143585 Single images in the Living Image Browser TLTZ0050624145507 006 E 7 that can be added to the sequence Images in the JW20090727135249 active sequence JW200
186. f the propagation of light through tissue 276 Living Image Software User s Manual Optical Properties of Mouse Tissue Firefly Luciferase Spectra Ex vivo muscle tissue integrating sphere measurement PCShA Luc cells ae ma l EE 9 a Us Tissue Transmission 0 8 1 Window Oe ln wire bd 0 6 j4 E 0 3 E oA Li w 0 no i50 cer Fatal ro 400 500 G00 Foo B00 VI a ee ice Fe Le Wavelength nm Wavelength nm Figure G 1 Optical Properties of M ouse Tissue and Firefly Luciferase Spectra The luminescent signal from firefly luciferase right is emitted from wavelengths of 500 700 nm which spans a region of the spectrum where there are major contrasts in the optical properties of mouse tissue left The firefly spectrum was measured at 37 C using PC3M cells Diffusion Model of Light Propagation Through Tissue Light propagating through tissue undergoes scattering and absorption The diffusion model assumes that scattering is the predominant phenomenon and the reduced scattering coefficient u gt gt absorption coefficient u This is valid mostly for wavelengths in the red and near infrared part of the spectrum The model also assumes that the light is produced by a single point source and that the tissues are optically homogeneous Under these conditions if we model the animal surface as flat and infinite in extent and integrate the light that is collected over the animal surface the total integrate
187. ferences General Acquisition Tissue Properties Start Up Defaults Show Activity Window on Warnings Errors C Dock Tool Palette Left Fight Save Settings C Save Float corrected image Window Size Color Selections Width 5 Restore Defaults Folder Locations Window Size amp Position Height 65 Most Recently Used Dataset History Apply Individual Color Scale For Sequences Display ROI Label 45 Measurement Show Transillumination Locations Photons Total Flux w Show Od d Opti Counts Total Count wt Figure 13 1 General Preferences 13 3 Point Source Fitting Point source fitting is performed separately on each image in a sequence 1 Open the image sequence that you want to analyze ganik A g E unan sa C Appt to all 7 TLT2005041 61627633 S60 Tool Palette a t Adjust C Sequence view L cowie Spectra e m BC vw sects EP arrear J Point Saami Filly Anar PE ag asias Peng TEOREME Kia ef et Tee Pieri Source Arey Select Singe bmige Source iger Ex Dm aaa ala Select an image for analysis e pribral Uir Figure 13 2 Select an image for point source fitting 2 In the Analyze tab of the Point Source Fitting tools select an image in the sequence 3 In the Surface Topography tools generate or load a surface For more details on generating the surface see page 113 Jie eT i Y Caliper Chapter 13 Point Source Fitting 123 NOTE Conf
188. focus A Smaller FOV results in a narrower depth of field Select the FOV by choosing a setting from the drop down list For more details on the calibrated FOV positions A E see Table A 3 page 233 Click to move the stage to a position for cleaning the imaging chamber below the stage Only available on the IVIS Imaging System 200 Series and IVIS Spectrum Click to move the stage from the cleaning position back to the home position Note This check box is only available on an IVIS System that includes the XFO 24 lens option When the XFO 24 lens is installed choose the XFOV 24 option For more details on how to install the XFO 24 lens see the XFOV 24 Lens Instructions Or ALERT If you remove the XFO 24 lens from the system be sure to remove the check mark from the XFOV 24 check box 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 1 IMPORTANT ALERT The IVIS System has a protection system to prevent instrument damage however always pay close attention to subject height particularly on the IVIS Imaging System 200 Series For example it is possible for a large subject 10 cm ventral d
189. from the Field of View drop down list For more details on the field of view see page 227 oy T WY Caliper Chapter 4 Luminescent or Fluorescent Imaging 13 9 Set the Focus e Select use subject height from the Focus drop down list and use the arrows or the keyboard arrows to specify a subject height cm or e Select Manual focus from the Focus drop down list For more details on manual focusing see page 229 10 If necessary click mageSetup in the control panel to operate in single image mode NOTE In single image mode the Sequence Setup button appears in the control panel Click this button to set up sequence acquisition 11 To acquire an overlay image coregistered images for display after acquisition put a check mark next to Overlay NOTE To view the subject s inside the chamber before image acquisition take a photograph Uncheck the Fluorescent option choose the Photograph and Auto options and click Acquire 12 When you are ready to capture the image click Acquire The information entered here appears in the image label Figure 4 8 page 10 lg Has a aen Ga ae ps a pa Figure 4 11 Edit Image Labels box 13 In the Edit Image Labels box enter information about the image and click OK NOTE You can enter image label information at any time during or after acquisition If you do not want to enter image information click Cancel 14 If this is the first image of the s
190. g With Data aoaaa a 71 TA Opena e Ddi sssr apace en eee ee eeeneetasese orean rauser 71 7 2 About the Image Window amp Tool Palette aoaaa 0000 ee ee 75 7 3 Viewing Image Information o oo 77 7 4 Adjusting Image Appearance aoaaa 79 7 5 Correcting Image Data aoaaa 81 Living Image Software User s Manual 7 6 Viewing Intensity Data amp Making Measurements 2 2 eee ee ee ee a 83 7 7 Creating a Transillumination Overview 2 1 ee a 90 7 8 Overlaying Multiple Images aoaaa a 91 7 9 Rendering Intensity Data in Color a oa a a 93 7 10 Exporting or Printing Images oaoa ee 95 7 11 Editing anImage Sequence s sss sse cesando s etes dr as Try aktera 97 7 12 Creating an Image Sequence from Individual Images oaoa a 98 8 Working With ROI Tools 0 00 2 eee a 101 8 1 About ROIS oaaao 101 8 2 Quick Guide Drawing Measurement ROIs on an Image or Sequence 102 Bo LOLONG eeeeaet ee dae eeeueea Gea ene eew eeu tee donee bau he ee 104 8 4 Measurement ROIs aoaaa aaa ee 106 Bo DUDiECDROIG 425 444 2 a4 Gees BESS hE EH eee SSE Eee BA 110 8 6 Measuring Background Corrected Signal oaoa 110 Ba EORR 4424 coe u Gee eee ee een Fee eee be ee eee Se GS 112 8 8 Managing ROM 4 46646 846 88266608 dd anidar dind dakaka red REESE 115 8 9 Managing the ROI Measurements Table n oaa aaa 0002 eee eee eee 126 9 Mage MAIN s e wake ee ee A ae ea Oe ee ee ee ee ee
191. ge Information tools click the Line Profile button 2 A line appears on the image and the Line Profile window appears Fle Ede View Tok acquisition Windows Pep or OQ tL Be A OP nts cots I Apel to al Ted Fache x TLIO aT _ OO Terie Alpi r gt Derrections Fillbering i 77 inpe eicarrmabion b Ail BB us e w T D arga wahia Hih dibo lne 0 Y SS a ige bata es Corn ie eee a eme a mae _ gt Print Source Fitting DUTT 30 Reconstruction GADLIT 30 Reconsbraction Line Profile Window Une Grientation ZEEE Width 1 gt Postion 138 L gy th amp x Mr 0 Shaw 739 ym a Y Mae 117 m F Ful Seale C Legere Scale TLT200S0ha4 145507 _O0e5 Crrenlay iO Counts J Figure 7 15 Viewing a line profile of pixel intensities 2 To view the line profile at another location in the image put the mouse pointer over the line When the pointer becomes a drag the line over the image The blue line determines the pixel intensities that are plotted in the line profile graph The line profile is updated as you move the line move over the image Caliper Y ee p Chapter 7 Working With Data 87 ifeSchences Table 7 7 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 lin
192. ge background ROI selected from the drop down list at the top of the dialog box The Bkg ROI tab shows a drop down list shows all average background ROIs in the click number selected above that can be linked to a user specified measurement ROI or subject ROI selected from the drop down list at the top of the dialog box Info tab Information about the ROI selected in the image ID User entered information about a subject ROI YP Caliper Prp Chapter 8 Working With ROI Tools 73 Moving an ROI Table 8 3 ROI Properties dialog box Item Description Label Label name of the selected subject ROI Lock Position Choose this option to lock the position of the ROI selected in the image XC X coordinate of the ROI selected in the image Yc Y coordinate of the ROI selected in the image Lock Size Choose this option to lock the dimensions of the ROI selected in the image Width Width pixels or cm of the ROI selected in the image for more details on setting the units see RO Dimensions page 82 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 anew value or click the up down arrows Line Color Specifies the color of the ROI line To select a line color click the Browse button ig Done Click to close the ROI Properties box and apply any new settings including e Linkage between a measurement ROI and subject
193. ground filters Point Source Fitting Spectral unmixing DLIT FLIT Analysis Optional requires premium filters Lumina optional yes yes no optional no no Lumina XR optional yes yes no optional no no 100 Series optional yes yes no optional no no 200 Series yes yes yes yes yes yes yes Spectrum yes yes yes yes yes yes yes Kinetic optional yes yes no optional no no Y Caliper feSciences 2 4 Image Display amp Analysis Chapter 2 Overview of Imaging amp Image Analysis 11 You ll find most of the tools for adjusting image display and analyzing images in the Tool Palette The Tool Palette automatically appears when acquisition is finished or when you open image data Its contents depend on the type of image data that is active Figure 2 4 shows the tools that are available for an image or a kinetic sequence Figure 2 5 shows the Tool Palette for an example image sequence Click a section of the Tool Palette to show or hide the tools Overlay image Image Adjust Tools page 79 Tune the photograph brightness gamma Similar to contrast or Opacity Set the image display color scale minimum and maximum Select a color table for image display Toal Falete CE Color Seane Min Maxi GRETA Pua Corrections Filtering Tools page 81 e Subtract dark background from the image data
194. gt Surface Topography gt Point Source Fitting E DLIT 3D Reconstruction gt GADLIT 3D Reconstruction Figure 8 22 Name and save the ROIs to the system 2 Click Save The ROI s from the image are saved to the system and can be selected from the Name drop down list To load ROIs on an image 1 Open an image 2 In the ROI tools make a selection from the Name drop down list and click Load NOTE If you load ROI s onto an image then draw additional ROIs the Save button changes to Overwrite If you want to save this collection of ROIs using the existing name click Overwrite You can delete ROIs from an image or permanently remove ROIs from the system To delete ROIs from an image 1 In the ROI tools click the x button Y Caliper Chapter 8 Working With ROI Tools 79 Tool Palette Figure 8 23 Removing ROIs from an image 2 In the drop down list that appears select a delete command The specified ROIs are deleted from the image NOTE This does not delete ROIs saved to the system global save To permanently remove ROls from the system 1 Select the ROI s that you want to delete from the drop down list of saved ROIs 2 Click Delete Figure 8 24 Tool Palette gt Image Adjust p gt Corrections Filtering r gt Image Information e ROI Tools oo ely x C Apply to Sequence ne Measurement ROI ka Save ROIs ame ROT 1 KS4 a ee orere Previ
195. h of normalized intensity versus the effective attenuation coefficient usp the optical property of the tissue selected in the Tissue Properties drop down list along with the linear fit to these data determined by the spectral analysis code Plot Intensity Displays a graph of normalized intensity versus wavelength Intensity is normalized by the selected source spectrum and filter transmission properties Export Opens a dialog box that enables you to save the results to a text file txt Save Results Name A drop down list of saved results Includes the default name for new unsaved analysis results Splm_ lt name gt Delete Deletes the selected results Load Opens the selected results Save Saves the analysis results results name appears in the Name drop down list 10 3 Viewing Graphical Results 1 In the Results tab select an ROI 2 Click Plot Intensity or Plot Linear Fit The linear fit graph plots the logarithm of the intensity normalized to the selected source spectrum and the filter transmission properties against the optical property of the tissue Uep The slope of the line is the source depth If any of the measured points in red deviate significantly from the straight line fit then the analysis results may be suspect The horizontal error bars represent the uncertainty in the optical properties usually estimated at 10 The vertical error bars represent noise in the image a i Y Caliper Chapter 1
196. hape on the image 56 Automatic The software automatically locates and draws an ROI s on the image 61 To do this the software locates the peak pixel intensities in the image and searches the neighborhood around a peak pixel A pixel is included in the ROI if the pixel intensity is greater than the threshold a user specified percentage of the peak pixel intensity Free draw Draw line segments that define the ROI 63 Manually Drawing a Measurement ROI 1 Open an image or image sequence and in the ROI tools select Measurement ROI from the Type drop down list 2 Select the ROI shape a Click the Circle O Square O or Grid button E 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 ROI s and intensity label s appear on the image If you are working with a sequence open an image to show the ROI intensity gt Comections Filtering e naage Informakion q ROL Tools o 0 t 3 Y irira a Sine i Thre Wa Ekg arf Aipa ROl Rehe Chef gais Saree Load a Planer Spectral Imaging n a4 Piim enire HEL irg DLIT 30 Reconstruction gt GADLIT 30 Reconstruction _ Special Lining Figure 8 6 Placing two circular ROIs on the image 3 Adjust the ROI position YP Caliper iaGelaricns Chapter 8 Working With ROI To
197. he different types of autofluorescence The following examples illustrate sources of autofluorescence including microplates other materials and animal tissue Microplate Autofluorescence When imaging cultured cells marked with a fluorophore be aware that there is autofluorescence from the microplate as well as native autofluorescence of the cell Figure F 9 shows autofluorescence originating from four different plastic microplates The images were taken using a GFP filter set excitation 445 490nm emission 515 575nm White polystyrene a Clear polystyrene Black polystyrene Figure F 9 Examples of microplate autofluorescence emission The black polystyrene plate emits the smallest signal while the white polystyrene plate emits the largest signal Imaging parameters GFP filter set Fluorescence level Low Binning 8 FOV 15 f 1 Exp 4sec Two types of autofluorescent effects may occur Overall glow of the material Usually indicates the presence of autofluorescence 268 Living Image Software User s Manual Hot spots Indicates a specular reflection of the illumination source Figure F 10 The specular reflection is an optical illumination autofluorescence signal reflecting from the microplate surface and is not dependent on the microplate material Specular Reflection Specular Specular E Reflection Reflection Specular Reflection Figure F 10 Specular reflection The four symmetric ho
198. he graph repeat step 6 to step 7 COND M 8 8 Managing ROIs In the ROI Properties box you can view information about an ROI change the position of the ROI on the image and edit the ROI label or line characteristics Viewing ROI Properties 1 To view ROI properties do one of the following e Double click an ROI in the image e Right click the ROI and select Properties from shortcut menu that appears e Select the ROI then select View Properties on the menu bar The ROI Properties box appears for more details see Figure 8 17 2 To view properties for another ROI click the ROI in the image Alternately select an ROI from the ROI drop down list in the ROI Properties dialog box Figure 8 15 70 Living Image Software User s Manual Fle EE Wess Took Acquisition Window Hep ae 2 oe BB RP ure cones v C Appr toal A TT05044145507 00i IE A ROL Properties n x SERN lonia Overy noe ROL RA ROI selected in ROLLabe FOL 2 the image To Ee eee view properties Se ee for another ROI LIS OO select another wen EKG sal ROI from the drop down list or click an ROI in the image RO 20KG 1485 207605 7 Lock Posin ae Yel pix 107 67500 m Yet px 165 31250 Angie deg 0 0000 Lock fine With pie 7 00000 Hina pate 17 00 Copy ATADI Dph ROM Comeet ho nna Sat Ekg AOS bo BSG Sal Blog Cd bi r Line Sire E A Sek Subject ROL bo Subject Lre Color M C Bek Subje
199. here is not visible Figure E 1 High Reflectance Hemisphere and a plastic marker pen Left Photographic image Right Photograph with luminescent overlay The hemisphere is illuminated by phosphorescence emitted from the pen If any part of the hemisphere exhibits what appears to be luminescent emission it is actually the light reflected from a source illuminating the hemisphere Observe the side of the hemisphere that is illuminated to help determine the source location In Figure E 1 the pen appears very bright due to phosphorescent emission that is also illuminating the portion of the hemisphere next to the pen If the pen had been outside the 258 Living Image Software User s Manual field of view it would not have been imaged and the source of the phosphorescence would be less obvious However the illumination of the hemisphere would still be very apparent and indicative of a light pollution problem OL ALERT Handle the High Reflectance Hemisphere by its black base plate while wearing cotton gloves provided by Caliper Skin oils can phosphoresce and will contaminate the hemisphere Latex gloves and the powder on them may also phosphoresce If the hemisphere becomes contaminated contact Caliper technical support for a replacement There are no known agents that can clean the hemisphere To check the hemisphere for contamination take several images of the hemisphere rotating it slightly between images A glowing fingerprint f
200. hich the subject is in focus A smaller FOV results in a narrower depth of field but gives a higher resolution image Select the FOV by choosing a setting A B or C from the drop down list For more details on the calibrated FOV positions see Table A 3 page 229 Table 5 3 Imaging Wizard Item Description Focus Drop down list of focusing methods available Use subject height Choose this option to set the focal plane at the specified subject height Manual Choose this option to open the Focus Image window so that you can manually adjust the stage position For more details on manual focusing see page 36 Options Time Series Study Choose this option to specify the number of segments to acquire and atime delay between segments This option is useful for acquiring data for kinetic analysis Note The sequence specified in the sequence editor is called a segment Restart Wizard Returns the wizard to the starting screen F TES gai Earla s P rel lasni werd a E C ii yai Settings man ujue AE E OE Tere aoe i A L i Beie Ed ea ORCE la eet ape Reuse photograph amp reuse X ray options Figure 5 9 Control panel and sequence editor 7 Complete the rest of the Imaging Wizard When you complete the wizard the sequence information appears in the sequence editor Figure 5 10 aves ee amas Meche Exposure Tit E bre ny cr f iiir Hayag Settee EE i i nar rites
201. how Advanced Options is selected in the Preferences Combines the images of a FLIT sequence into a single image intensities are Summed that can be analyzed using tools in the Tool Palette Opens the Image Math window for the active data Opens a dialog box that enables you to acquire a dark charge measurement Opens a dialog box that enables you to select an instrument luminescent background This background measurement is subtracted from luminescent images 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 Opens a dialog box that enables you to view the dark charge measurements for the system Opens a dialog box that enables you to remove the dark charge measurements from the system Opens a dialog box that enables you to acquire background images or schedule or disable automatic background acquisition Starts a measurement of the instrument fluorescent background Opens a dialog box that enables you to select an instrument fluorescent background measurement for the active image data If the Sub Fluor Bkg option is chosen in the control panel the background measurement Is subtracted from the image data Opens a dialog box that enables you to select a fluorescent background measurement
202. ht Chiki O Tie Series ity Cancel Bleck Hei Figure 5 8 Imaging Wizard imaging parameters Table 5 3 Imaging Wizard Item Description Imaging Subject Choose the type of subject from this drop down list Exposure Parameters The Auto Settings exposure option is the default To manually set the exposure parameters select the M anual Settings option For more details on the exposure parameters see page 225 Ee Luminescent To include a photograph or X ray image in the ta Photograph acquisition put a check mark next to the Photograph or X ray option a Ae grap y Op In the control panel the Photograph and X ray Reuse option is selected by default Figure 5 9 This means the same X ray image and photograph will be used if camera conditions do not change for example binning or F Stop If you do not want to reuse the X ray image or photograph you can manually edit the image sequence in the sequence editor for more details see page 24 Alternately remove the check mark next to Reuse in the control panel before you begin the Imaging Wizard Field of View Sets the size of the stage area to be imaged by adjusting the position of the stage and lens The FOV is the width of the square area cm to be imaged A smaller FOV gives a higher sensitivity measurement so it is best to set the FOV no larger than necessary to accommodate the Subject or area of interest The FOV also affects the depth of field range in w
203. i i i A ra this box to display the associated 3D view and time stamp position in the time scale 0 C 100 at which the frame occurs in the animation Click _ _ to view the animation composed of the key frames Figure 15 36 Individual 3D views key frames in the preset animation Spin CW on Y Axis Use the animation tools to e View apreset animation derived from a factory loaded animation setup page 176 e Create a custom animation generated from your custom animation setup page 178 e Save an animation setup page 180 e Record an animation to a movie file page 180 e Edit an animation setup page 180 Table 15 12 3D animation tools Item Description Time Scale The time stamp of a key frame in the animation on a time scale of 0 100 For example if the animation is 10 sec long and includes five key frames Key frame 1 Time stamp 0 first frame of the animation Key frame 2 Time stamp 25 frame occurs 2 5 seconds after the start of animation Key frame 3 Time stamp 50 frame occurs 5 0 seconds after the start of animation Key frame 4 Time stamp 75 frame occurs 7 5 seconds after the start of animation Key frame 5 Time stamp 100 last frame of the animation Presets A drop down list of predefined animation setups Key frame A 3D view The software interpolates the key frames to create intermediate frames in real time then generates an animated sequence from all of the frames
204. ic ROI plot 114 draw 114 113 L lens aperture 245 line profile 86 Living Image browser 73 Living Image software starting 15 luciferase spectrum 277 luminescence reconstruct 3D sources 188 194 luminescent imaging single image 21 24 M magnify image 81 manual focusing 233 234 234 save data 41 manual conventions 2 manual sequence setup 37 41 maximum vs time graph tracking infusion 291 maximum vs time graph 60 measurement ROI automatically draw 107 109 Index 299 measurement ROIs 102 106 109 free draw 109 measurements 88 measuring relative density 55 57 menu commands 293 miscellaneous material autofluorescence 268 270 multiple reporters per photograph 91 93 N NNLS weighted fit 201 normalization 262 0 open image data 74 optical density 264 optical properties for planar spectral imaging 277 organ atlas import 219 organ display 216 219 organ registration tools 214 220 overlay 250 overlaying images 91 93 p pan image 81 PCA biplot 157 PCA explained variance 157 photon density 204 photon density map measured 205 simulated 205 photon density maps 204 photon radiance 253 photons 252 pixel 249 pixel data 84 planar spectral analysis optimizing precision 281 planar spectral image analysis 139 141 planar spectral imaging 275 281 diffusion model 276 intensity graph 142 linear fit graph 142 luciferase spectrum 277 optical properties 277 sequence requirements 139
205. ick Cancel 50 Living Image Software User s Manual Kinetic Acquisition window oO amp Bw Pee Hee 1 Image window HI E fil als HERRER i Tool palette Figure 6 5 The Edit Image Labels box Image window and Tool Palette appear when you save an image or kinetic stream NOTE You can edit and analyze kinetic data in the Image window 6 4 Playing Kinetic Data In the image window you can e Play kinetic data e Select and view a particular image e Select a range of images and extract as a separate kinetic data set Caliper Y p Chapter 6 Kinetic Imaging 51 Current image Current image number top Slider position To select a particular image enter anew number or move the top Slider Start frame image in aE Use the bottom sliders to End frame image in the selected the selected data range select a range of data for data range right slider position left slider position viewing or export Figure 6 6 Image window kinetic data Fe Cot are Eee Wiem Pep ae iu E A E u O ra wm AHR eee IEA mae Hi ch erini E Jah b araa lehed cat of 34 hams f Figure 6 7 Image window selecting kinetic data for export Put the mouse arrow over a bottom slider to view a tooltip that shows the number of selected images frames 52 Living Image Software User s Manual Table 6 4 Image window kinetic data Item Description
206. if the Size minimum size is set at 50 then ROIs created on the image must be greater than 50 pixels in size Preview If this option is chosen the software draws the ROI each time a parameter Is changed ROI parameters can be saved without drawing the ROI Use Bkg Choose this option to measure background corrected signal This is typically used Offset to remove natural animal background luminescence and should not be confused with the dark charge and read bias background corrections that are applied by default to the raw CCD data to remove electronic noise before any measurements For more details see page 64 Replace If this option is chosen all auto ROIs are replaced when new ROI s are created ROIs Restore Restores the factory set defaults for the auto ROI parameters Defaults Save Load Click to display or hide the tools that enable you to save load or delete auto ROIs in the active data Note The save function saves parameters the not actual ROIs This means that when you load saved auto ROI parameters the software draws a new ROI using the saved values Threshold Lower Limit Minimum Size 60 Living Image Software User s Manual 8 4 Measurement ROIs To obtain the intensity signal in a user specified area of an image draw a measurement ROI on the image There are three ways to draw measurement ROIs Drawing Method Description BES Page M anual Places one or more ROIs circular square or grid s
207. igh No Z 1 50 15x23 2 11 Acquire a fluorescent image at 6 W Auto Medium 2 640 700 High No lc 1 50 15x23 2 10 3 the remaining transillumination 7 W Auto Medium 2 640 700 High No iE 1 50 15x23 2 10 acato 6 tad Auto Medium 2 640 700 High No Z 1 50 15x23 2 9 v is Ee Number of Segments li ia Delay oo i min Apply to All X Remove i Update Insert Add Figure 15 12 Example sequence setup for FLIT Steps to Reconstruct Fluorescent Sources Load an image sequence 2 Generate or load a surface For details on generating the surface see page 113 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 15 2 For more details about the Threshold see page 146 Tool Palette Gomecthone Filteri Lo ROI Tools Gurl ane Topgal Apatia Preeti Rea Sages Sere i a oe The Shae Torre Ln Select Imay Sources Fu E Threshold Hi HE zl Hyra m g 5 1 Elos m3 5 1 Fluo 745 5 0 EJ os HE A Hlg mS 5 1 Plas or m3 j 3 1 Fluo 45 5 1 EJ p HE 6 2 Mii M g 5 1 eJ ii oS 5 1 EA at Ta inay hga Riedbarce Figure 15 13 FLIT 3D Reconstruction tools Analyze tab W Caliper a Pp Chapter 15 3D Reconstruction of Sources 153 feSciences 4 Select the type of image displayed in the Sequence View window Radiance or NTF Efficiency
208. ii hE j LLC E stan feed nage E 4 j gt A Figure A 2 Opening the M anual Focus window 2 To mark the center of the camera in the window put a check mark next to Display CCD Center Select the size of the step increment that the stage moves Coarse Normal or Fine 4 Click Up or Down to move the stage and change the focus If necessary select another F stop setting from the drop down list and adjust the light level using the arrows 6 Click Update to apply the settings 234 Living Image Software User s Manual The resulting focal plane cm above the stage is automatically entered in the Subject height box 7 Click OK when the image is focused Y Caliper Appendix B Preferences General Preferences aoaaa a a a a ee ee ee aa ee ee 236 User Preferences aoaaa aa a a a aaa we 238 ACQUISITO s tk KERR EKA REARS ETHER SEGAL EKG EE HKG DS A99 Tete 20 KORRES RSD penan sha pesna nenne ae ee e Tissue Properties 1 a a a ee ee ee ee ee es 248 You can manage user Ds and specify defaults for some parameters that are associated with the user ID selected at the start of a new session To view the user modifiable preferences after you log on select Edit Preferences on the menu bar Preferences General Acquisition Optical Properties Start Up Defaults Show Activity Window on Warnings Errors C Dock Tool Palette Left Fight Save Settings Save float corrected image Window Size
209. ilable the number of fluorophore molecules or cells per source can be determined in addition to the total fluorescence yield The input data to the FLIT algorithm for 3D reconstruction of fluorescent light sources includes e A surface that defines the surface of the subject 140 Living Image Software User s Manual e A sequence of images acquired at different transillumination source positions using the same excitation and emission filter at each position Use the Imaging Wizard to acquire the images The input data to the DLIT or GADLIT algorithm for a 3D reconstruction of luminescent light sources includes e A surface that defines the surface of the subject e A sequence of two or more images of the light emission from the surface of the subject acquired at different filter bandwidths Table 15 1 Use the Imaging Wizard to acquire the images Table 15 1 IVIS System filters for luminescence amp fluorescence tomography IVIS Imaging System Filters Bandwidth nm 200 Series 6 emission filters 550 670 nm 20 Spectrum 10 excitation filters 415 760 nm 20 18 emission filters 490 850 nm Figure 15 1 shows an example 3D reconstruction workflow j Y Caliper ii Chapter 15 3D Reconstruction of Sources 141 Load an image sequence see page 142 Generate or load a surface for image sequence requirements Ted Pakelbe E E E gt Planar Spectral Imaging Surface Lopography Wass Seo Q sequence Wes S
210. illumination provides an excitation light source located above the stage For more information about fluorescent imaging see page 257 I Start the Living Image software double click the FZ icon on the desktop 2 Initialize the IVIS Imaging System and confirm or wait for the CCD temperature to lock For more details see page 17 3 In the control panel put a check mark next to Fluorescent and select Auto exposure click the arrow When you select Auto exposure the software automatically determines the binning and F Stop settings Alternately you can manually set the exposure binning and F Stop For more details on these control panel settings see page 225 IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter F Field of Views System Status Idle Acquire Gee An Subject height 1 50 Siem Sequence Setup Figure 4 5 Acquisition control panel i j Y Caliper Chapter 4 Luminescent or Fluorescent Imaging 9 Select an excitation and emission filter from the drop down lists For more information about the standard filter sets see Table F 1 page 260 5 Puta check mark next to Photograph and select Auto exposure click the arrow 6 Make a selection from the Field of View drop down list For more details on the field of view see page 227 J Set the Focus e Select use subject height from the Focus drop down list and u
211. image or thumbnails that the Living Image browser displays when data are selected For more details on the browser see page 71 2 akj foe Leser Cee Pee ee Cee ee Le ee i tl Preview picture of the selected data 5 3 Measuring Relative Density As density increases tissue absorbs more X ray energy and appears darker in a raw Lumina XR X ray image Figure 5 14 To enable measurements of this X ray absorption the raw X ray image is mathematically transformed resulting in an image where denser material appears lighter and intensity measurements are proportional to the tissue density Figure 5 14 A opona a0 _ooF fel A dogoni taaa 007 Figure 5 14 X ray images raw left and in absorption units right The Living Image software displays transformed X ray images by default To display raw X ray images remove the check mark next to X Ray Absorption in the Corrections Filtering tools Figure 5 15 40 Living Image Software User s Manual Tool Palette gt Image Adjust Corrections Filtering Read Bias Subtraction Flat Field Correction Cosmic Correction If this option is not selected no mathematical transformation is L applied to the data and raw X ray AE RN A E a E Figure 5 15 Corrections Filtering tools When the X ray data has been corrected for absorption you can evaluate relative bone density by comparing the signal intensities of measurement ROIs The ROI intensity increases with in
212. ing Image browser is open makes it the active window and displays it on top of all other open windows Displays the online help Displays the online help index Displays the license information Displays a list of tool plug ins and tool palette plug ins Click this button then click an item in the user interface to display Information about the item Table J 2 Keyboard shortcuts Keys Shortcut Description Ctri B Opens the Living Image browser Ctri C Copies the active image to the system clipboard Cti D Arranges open windows in a cascade Ctri O Displays a dialog box that enables you to open data Ctri P Open the Print dialog box Ctri S Saves the active file or window Ctri T Tiles the open windows Ctri W Closes the active window Shift F1 Changes the mouse pointer to the What s This tool he7 Click this button then click an item in the user interface to display information about the item Macintosh users use the Cmd key apple key instead of the Ctrl key 296 Living Image Software User s Manual This page intentionally blank Y Caliper feSciences Index Numerics 3D quantification database 179 183 create 180 182 manage results 183 samples 179 3D reconstruction fluorescent sources 198 200 luminescent sources 188 194 reconstruct particular regions 194 3D reconstruction results DLIT or FLIT 200 201 GADLIT 202 203 3D surface 159 165 3D tools 211 226 Animate tab 221 226
213. int source in a user specified region of interest Analyzes a sequence of luminescent images acquired using different emission filters Figure 2 5 Tools palette for an example luminescent image sequence Image sequence luminescent or fluorescent Analyses Requiring an Image Sequence Surface Topography page 159 Analyzes structured light images to reconstruct the animal surface A surface Is required for point source fitting and 3D reconstruction of luminescent or fluorescent sources Inside a subject Point Source Fitting Advanced Option Estimates the optical properties of tissue the location and power of a point source or the fluorescent yield of fluorophores DLIT 3D Reconstruction page 189 A 3 dimensional reconstruction of the Subject that estimates the depth and intensity of a luminescent light emitting source FLIT 3D Reconstruction page 198 A 3 dimensional reconstruction of the Subject that estimates the location and intensity of a fluorescent light emitting source ROI Tools page 101 e Measure counts or photons ina user specified region of interest ROI and compute measurement Statistics for example average min max standard deviation e Measure efficiency in the ROI and compute measurement statistics for fluorescent images only Spectral Unmixing page 147 Extracts the signal of one or more fluorophores from the tissue autofluorescence Distingui
214. intentionally blank Y Caliper ifeSciences 3 Getting Started Starting the Living Image Software 0 ee ee ee ee ee 15 Initializing the IVIS Imaging System a ee ee ee ee 17 17 Checking the System Temperature 0 2 eee eee ee ee About the IVIS Acquisition Control Panel amp Auto Exposure Feature 18 Tracking System amp User Activity 1 2 a a a This chapter explains how to start the Living Image software and initialize the IVIS Imaging System After it is initialized the imaging system is ready to acquire images 3 1 Starting the Living Image Software The Living Image software on the PC workstation that controls the IVIS Imaging System includes both the acquisition and analysis features The Living Image software on other workstations includes only the analysis features For information on installing the software see the Installation Guide included on the Living Image CD ROM By default the software is installed at PC C Programs Caliper Life Sciences Living Image Macintosh Applications Caliper Life Sciences Living Image All components of the IVIS Imaging System should be left on at all times due to the long cooling time required to reach operating demand temperature It is also important to leave the system on to enable automatic overnight electronic background measurements Periodically rebooting the computer is permissible and does not affect the camera operation To
215. ion is about 0 7 OD 0 15 and blocking outside of the bandpass region is typically in the OD 7 to OD 9 range The band gap is defined as the gap between the 50 transmission points of the excitation and emission filters and is usually 25 50 nm There is a slope in the transition region from bandpass to blocking Figure F 5 A steep slope is required to avoid overlap between the two filters Typically the slope is steeper at shorter wavelengths 400 500 nm allowing the use of narrow band gaps of 25 nm The slope is less steep at infrared wavelengths 800 nm so a wider gap of up to 50 nm is necessary to avoid cross talk Fluorescent Filters and Imaging Wavelengths Eight excitation and four emission filters come standard with a fluorescence equipped IVIS Imaging System Table F 1 Custom filter sets are also available Fluorescent imaging on the IVIS Imaging System uses a wavelength range from 400 950 nm enabling a wide range of fluorescent dyes and proteins for fluorescent applications For in vivo applications it is important to note that wavelengths greater than 600 nm are preferred At wavelengths less than 600 nm animal tissue absorbs significant amounts of light This limits the depth to which light can penetrate For example fluorophores located deeper than a few millimeters are not excited The autofluorescent signal of tissue also increases at wavelengths less than 600 nm Table F 1 Standard filter sets and fluorescent dyes and pr
216. ired during the session are automatically saved to this folder You can choose a different folder at any time select Acquisition gt Auto Save on the menu bar This section explains how to manually save data if you do not want to use the autosave feature l Turn off the autosave feature select Acquisition on the menu bar and remove the check mark next to Auto Save After you acquire an image or image sequence click the Save button fal Alternatively select File Save on the menu bar In the dialog box that appears select a directory and click OK 26 Living Image Software User s Manual NOTE The software automatically includes the user ID and a date and time stamp with the data 4 7 Exporting Image Data You can save the active image view in different file formats for example bmp dcm 1 Open an image or image sequence 2 Click the Export Graphics button mg Unie Cows g Omnis o nite aie TY Te A are de Arm i Lei ey bee fee oe Comet Dor bin Hi Pan m hi Figure 4 25 Exporting an image to a graphic file 3 In the dialog box that appears select a directory choose a file type and enter a file name 4 Click Save NOTE To export a sequence to DICOM dcm format select Export DICOM on the menu bar This creates a directory that contains the dcm files and a Sequencelnfo txt Y Caliper feSciences 5 X Ray Imaging Acquire an X Ray Image 2
217. irm that the surface is good quality If necessary regenerate the surface using a higher level of smoothing 4 Click the Params tab The default starting values for the source location power and tissue optical properties are displayed The software automatically selects the correct model type for the image data Toal Palelthe nahra o Params Properties Results Moga Type Deohurrenecence s Angie Lime deg 70 Spatis Fiter o1 Paracnebers slakir value minim inn Fire Figure 13 3 Point Source Fitting tools Params tab For more details on the tools see page 127 5 To fix a parameter starting value click the unlocked icon so that it becomes a closed lock amp 6 If you want to construct the source only in a region of interest make a selection from the Mask drop down list Confirm the angle limit and spatial filter defaults or enter new values To specify different starting values for the optical properties a In the Properties tab make a selection from the Tissue Properties drop down list b Confirm the internal medium index of refraction or enter a new value 9 In the Params tab click LM Fitting The source appears on the surface and the Results tab displays the point source fitting results For more details on the Results tab see page 130 124 Living Image Software User s Manual File Edt mea b kgia Wire Harki SUUGA P moes veed
218. ity included in the data The tissue properties for modeling the photon propagation Source Spectrum The emission spectrum of the type of luminescent source Photon Density Maps Click to view the photon density maps For more details see page 158 Export Results Opens a dialog box that enables you to save the results csv Save Results Name The default name for the active DLIT or FLIT results Select results from this drop down list Delete Click to delete the selected DLIT or FLIT results Load Click to load the selected DLIT or FLIT results Save Click to save the active DLIT or FLIT results Overwrite If you reanalyze saved results click to save the new results and overwrite the previous results 156 Living Image Software User s Manual GADLIT Results Tool Palette gt ROI Tools Z GADUTT aD Reconstruction Analyze Properties Results Reconstructed Sources Rendering Sources All Results Parameters Fitted value The total number of sources 2 source 1 strengthiphotons 5 67e 10 source 1 location x direction mm 3 45 Y direction mm 22 69 z direction mm 13 57 source 1 volume 117 00 mm3 Jsource 2 strength photon s 4 50e 10 source 2 location Photon Density Maps W Export Results Save Results Name GADLIT_45507 w gt 3D Tools gt Spectral Unmixing Figure 15 17 GADLIT results Choose all sources or a particular sour
219. iving Image software Fi amp initialize the IVIS Imaging System pees al Ome arate ne Tar TE wees awe eee ted ie En aier Beg ee r hi fee Tum Foai is abed hg oe er HLE aaa Gena h ae ey ee DAE j ee ee ea ee eed Le Inthe control panel e Click Sequence Setup e Click the Imaging Wizard button imagna wizara Inthe Imaging Wizard double click Bioluminescence 4 or Fluorescence and step through the wizard Bg Inthe control panel click Acquire Sequence Imaging Wizard Enter image label information optional Theimage window appears and displays the images as they are acquired Figure 2 2 Steps to acquire an image sequence YP Caliper feSciences Chapter 2 Overview of Imaging amp Image Analysis 7 2 2 Imaging Modes Table 2 1 shows the imaging modes that are available on IVIS Imaging Systems Table 2 2 shows examples of the different types of images You can acquire e Single images for example a luminescent image and a photograph After acquisition the Living Image software automatically coregisters images to generate an overlay image e An image sequence a collection of images that are grouped together in a single folder Figure 2 3 Table 2 1 IVIS Imaging Systems amp imaging modes Imaging Mode IVIS Imaging System Lumina Il Lumina XR 100 Series 200 Series Spectrum Kinetic pren ww Luminescent V V V Vv Vv Vv _ Fluorescent V
220. ize 21 0 MB Pixel spacing rm Slice dimension pixels Width Height Axial range rom Min Max Solid mesh Hollow mesh Figure 15 27 3D Scene Exporter dialog box 4 Inthe Browse For Folder dialog box that appears choose a folder for the DICOM files and click OK During the export operation the 3D View window displays the each slice in the export For example if Transaxial Slice is selected for export then the transaxial windowpane cycles through a display of each exported slice Table 15 8 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 Choose this option to export source voxels without any smoothing or Original resolution binning The original resolution of the source voxels is the resolution obtained after DLIT or FLIT reconstruction approximately 1mm resolution Total Slices Parameters that determine the number and resolution of the slices to Slice spacing export Pixel spacing Slice dimensions Solid mesh The intensity of the pixel
221. l Subtraction window measured using the background filter to the autofluorescent signal measured using the excitation filter in a region on the animal where no fluorophore is present Compute k from ROI This option is useful for subtracting fluorescence background Draw the same ROI in both images on an area considered background In the Compute k from ROI drop down list select the same ROI in each image with Photo from Choose this option to display the new image in overlay mode using the selected photographic image This option is only available if one of the selected images is an overlay Display Result for Opens the image generated by image math in an image window M easuring 90 Living Image Software User s Manual 9 2 Subtracting Tissue Autofluorescence To remove tissue autofluorescence from image data the IVIS Imaging System implements a subtraction method using blue shifted background filters that emit light at a shorter wavelength Table 9 2 Table 9 2 Example emission excitation and background filters for acquiring data that will be corrected for tissue autofluorescence Emission Excitation Filter Background Filter LEE Colcol LAL ELLET E Filter Primary Image Fluorophore Passband nm GFP 515 575 445 490 410 440 DsRed 575 650 500 550 460 490 Cy5 5 695 770 615 665 580 610 ICG 810 875 710 760 665 695 The objective of using a background filter is to excite
222. l Liner Huu herr Dpt Frop ri Lkr Settings Enashineg r D ESA y Mem Usar DD Delete Le r ID Prelate Da aus Libi net Living Iriage Universal Cat Lebel Choices Ceefeulk Linde Courts ka Figure B 4 User preferences Table B 2 User preferences Item Description User s Settings Existing User ID The user ID displayed in the log on dialog box at startup New User ID Opens the Add New User box A new user is added to the Existing User ID drop down list Delete User ID Deletes the user selected from the Existing User ID drop down list Preferences Edit User label Choices Opens a dialog box that enables you to edit the Defaults Living Image Universal label set Default Units Choose counts or radiance photons for image display YY Caliper feSciences B 3 Acquisition Preferences ener al Lier Angistri There Mio Expire Camera Settings Luminescent Fluorescent Auto Expogure Preferences Tad felererce Targat Cour Firg Pralerarmce Sato Preberere Esmaa Tine CELL ka Range Yaka Esp Tiere sa Biia ir MEET Mir Pika fl Pl Appendix B Preferences 239 Dpt Frop ri FiS Epi flucrescert Ome Lusrairesec ental ao S P Trara fhorami S000 F Torche Defa Figure B 5 Acquisition preferences Auto Exposure Table B 3 Auto exposure settings Item Description Luminescent Fluorescent Auto Exposure Preferences First Preference Second Preference Third P
223. l background autofluorescence leakage pattern The image represents the minimum background level that a fluorophore signal of interest must exceed in order to be detected Figure F 11 Light from black Lexan This image shows the typical ring like structure of light from a sheet of black Lexan a low autofluorescent material that may be placed on the imaging stage to prevent illumination reflections Imaging parameters GFP filter set Fluorescence level High Binning 16 FOV 18 6 f 2 Exp 5sec Other laboratory accessories may exhibit non negligible autofluorescence The chart in Figure F 12 compares the autofluorescence of miscellaneous laboratory materials to that of black Lexan For example the autofluorescence of the agar plate with ampicillin is more than 180 times that of black Lexan Such a significant difference in autofluorescence levels further supports the recommended use of black polystyrene well plates NOTE It is recommended that you take control measurements to characterize all materials used in the IVIS Imaging System Autofluorescence Relative to Black Lexan eat Jl be PPO OK y ee oo Ea F 12 Comparison of autofluorescence of various laboratory materials to that of black Lexan Despite the presence of various background sources the signal from most fluorophores exceeds background emissions Figure F 13 shows the fluorescent signal from a 96 well microplate fluorescent reference standard TR 613 Red obtaine
224. le you click the first and last file in the selection ey N y Caliper Chapter 7 Working With Data 99 To select non adjacent images in the browser e PC users Press and hold the Ctrl key while you click the images of interest in the browser e Macintosh users Press and hold the Cmd key apple key while you click the images of interest in the browser 3 Click Load as Group The image thumbnails are displayed together in an image window For details on how to save or export the image data see Chapter 3 page 36 Organizing Images When multiple image windows are open you can organize them in a cascade or tile arrangement Choose Window gt Cascade or Window Tile on the menu bar fn be cee i fe a be ieee Oe flue amp 3 p unis p i an al Th et ee ed Ps le ee eee eli E A Fy P p ay Sd ee Gia ch TENDS a BN j j i Figure 7 28 Image windows cascade top or tiled bottom 100 Living Image Software User s Manual This page intentionally blank Y cole 8 Working With ROI Tools ADOUt ROIS aa srar rsss br aSr RSF kES aniar ER ES RE EG 55 Quick Guide Drawing Measurement ROIs on an Image or Sequence 56 Se gr EEEE EE E EE EERE EEE 58 Measurement ROIS aoaaa a a a 60 Suplee ROIS ace ec dbbe deeded eee SRD RES EER EO HOHE OS 64 Measuring Background Corrected Signal 2 0 58 ee eee ees 64 POUCH ss ee echt eeecuePee ee hh eevee eRR ERE BHC E
225. librated units for cell numbers or molecule quantities in picomole units 4 Well Plate Quantification Windor Tool Palette gt ROI Tools gt Surface Topography gt Point Source Fitting FLIT 3D Reconstruction ber Sequence LLAMA LOS SOU Chk ELS Lae ee Finnie Tye Well Pleke Toa G Dya malnnudes C Crit Per errant e akarod wels GD 4 Analyze Properties Results Appl bo Sequence Tissue Properties Muscle w Weel Plat Histon Pots Rhesus e Quantification Plot Tissue Properties k Catabare WPEANT_ Fluor Calib WPOUANT_1 t Fecihatien Ere sann Fatit CT Croat Section in Lin Q E Hi 100 G a irt 1 465 520 oot e407 1 115 03 2 ABS gan ae ee 4 5 ie 7 465 el gt EP be dcall 4 465 pi Doe Oi pal Save the results with the image sequence Hare 5 PESH _ SFF T ne i SLAB _ eite toad Save the results to a database that is Casket Lint CAPE Vari LEAT available for DLIT or FLIT analyses Wavelength rn gt 3D Tools gt Spectral Unmixing TapE Figure 14 7 Save the quantification results Table 14 2 Managing quantification results Item Description Sequence Delete Removes the active quantification results from the image Horns hom gP sa sequence etete tosa i Load Opens quantification results from the sequence path Save Saves the quantification results with the selected image sequence Overwrite Saves the results with the selected image sequenc
226. lters Ideally bandpass filters transmit all of the wavelengths within the bandpass region and block absorb or reflect all wavelengths outside the bandpass region This spectral band is like a window characterized by its central wavelength and its width at 50 peak transmission or full width half maximum Figure F 5 shows filter transmission curves of a more realistic nature Because the filters are not ideal some leakage undesirable light not blocked by the filter but detected by the camera may occur outside the bandpass region The materials used in filter construction may also cause the filters to autofluoresce Band ap f Ei taticii 100 Emission m O Fa Excitat on Emission Filter Filter 1 0_ rans mis spon p m oO oO paar 0 001 Wavelength nm Figure F 4 Typical excitation and emission spectra for a fluorescent compound The graph shows two idealized bandpass filters that are appropriate for this fluorescent compound Senaration 20 nm Waveberngihi Figure F 5 Typical attenuation curves for excitation and emission filters 264 Living Image Software User s Manual In Figure F 5 the vertical axis is optical density defined as OD log T where T is the transmission An OD 0 indicates 100 transmission and OD 7 indicates a reduction of the transmission to 10 For the high quality interference filters in the IVIS Imaging Systems transmission in the bandpass reg
227. m Due to the increase in binning the latter image has a four fold increase in sensitivity compared to the former A charge coupled device CCD is a photosensitive detector constructed in a two dimensional array of pixels After an image is acquired each pixel contains an electrical charge that is proportional to the amount of light that the pixel was exposed to The software measures the electrical charge of each CCD pixel and assigns a numerical value counts For more details on counts and other measurement units see Appendix D page 249 The resulting image data comprise a two dimensional array of numbers each pixel contains the counts associated with the amount of light detected IVIS Imaging Systems are equipped with a CCD that ranges from 1024x 1024 to 2048x 2048 pixels in size and thus have a high degree of spatial resolution At binning 1 each pixel is read and the image size number of pixels is equal to the physical number of CCD pixels Figure C 3 feSciences y7 Caliper p Appendix C Detection Sensitivity 247 Binning Binning Binning CCD pixel Signal 4 times larger Signal 16 times larger Spatial size doubled Spatial size quadrupled Figure C 3 A small segment of the CCD At binning 2 4 pixels are summed together at binning 4 16 pixels are summed At binning 2 four pixels that comprise a 2x 2 group on the CCD are summed prior to read out and the total number of counts for the group is recorded
228. m in the table Copy Selected Copies the selected row s in the table to the system clipboard All Copies all rows in the table to the system clipboard Refresh Updates the ROI Measurements table for example after you draw new ROIs move an ROI and close or open image data Configure Displays the Configure Measurements box that enables you to specify and organize the data categories column headers for the table Export Displays the Save Measurements box so that the data can be saved to a txt or csv file Note Grid ROI measurements exported to a csv file can be opened in a Spreadsheet application like Microsoft Excel Close Closes the ROI Measurements table YP Cali feSciences Chapter 8 Working With ROI Tools 83 Configuring the ROI Measurements Table You can customize the data and information column headers in the ROI Measurements table Several predefined categories are available in the Measurement Types Click Attributes and ROI Dimensions drop down lists 1 To reorder the columns drag a column header left or right in the table 2 To change the measurement units make a selection from the Measurement Types drop down list H RII Mearurenects Ai Hala Tete Courts Avg Couns dey Counts Min Counts Macc Count PO Piik rod Pe a image Hureber Ai ES eo TRO aT mul PMO aT Bol 1 ThE cae EAN OO RA Z Se e S KAE ear _O0 Pole EALAR ELI IPPs L viriy verly Ce
229. maging of subjects with very dim sources Sets the size of the camera lens aperture The aperture size controls the amount of light detected and the depth of field A larger f stop number corresponds to a smaller aperture size and results in lower sensitivity because less light is collected for the image However a smaller aperture usually results in better image sharpness and depth of field In kinetic mode the photographic and luminescent or fluorescent image are acquired at the same F Stop For more details on f stop see Appendix C page 241 46 Living Image Software User s Manual Table 6 1 Kinetic acquisition settings continued Item Description EM Gain M ultiplies the signal in real time This option is useful for boosting low signals above the background noise For kinetic imaging the EM gain may be set to 50 100 or 250 For conventional 16 bit still image acquisition EM gain may be set to Off 50 100 or 250 Excitation Filter A drop down list of fluorescence excitation filters For fluorescent imaging choose the appropriate filter for your application GFP DsRed Cy5 5 or ICG For luminescent imaging Block is selected by default If you select Open no filter is present Lamp Level Sets the illumination intensity level of the excitation lamp used in fluorescent imaging Off Low High and Inspect Low This setting is approximately 18 of the High setting Inspect Turns on the illumination lamp so that y
230. me binning f stop and field of view As a result images of the same subject acquired during the same session have the same signal amplitude regardless of the camera settings because the radiance on the animal surface does not change The advantage of working with image data in photons mode is that camera settings can be changed during an experiment without having to adjust the images or the measured ROI data Images or ROI data can be quantitatively compared across different IVIS Imaging Systems Caliper Corporation calibrates the camera settings of each IVIS Imaging System at 600 nm The response of the CCD is relatively flat 10 over the range from 500 700 nm which includes the spectral variation found in bacterial or firefly luciferase Therefore calibration is accurate over this range Efficiency The fluorescent signal detected from a sample depends on the amount of fluorophore present in the sample and the intensity of the incident excitation light The excitation light incident on the sample stage is not uniform over the field of view FOV At FOV 10 there is a slightly dished illumination profile due to the close proximity of the stage to the illumination reflectors while the profiles for the other stage locations are peaked near their center The illumination intensity profile varies by up to 30 across the entire FOV Figure D 4 100 80 p T 60 k pe 40 FOV 10 FOV 15 20 ome FOV 20 ome FOV 25 0 15 10 5 0
231. me types of analyses are performed on an image sequence see Table 2 3 The sequence requirements number and type of images depend on the type of analysis Table 2 4 shows the types of analyses that are possible on the different IVIS Imaging Systems Table 2 3 Analyses that require an image sequence Analysis Planar spectral image Display multiple fluorescent or luminescent reporters Subtract tissue autofluorescence using blue shifted background filters Point Source Fitting Spectral unmixing DLIT FLIT Table 2 4 IVIS Imaging System capabilities Description Page Computes the total flux and average depth of a luminescent source 10 below the surface Uses the Image Overlay function to display multiple luminescentor 91 fluorescent images on one photographic image Uses the image math feature to subtract a background image from the primary image Estimates the optical properties of tissue the location and power of a point source or the fluorescent yield of fluorophores Removes tissue autofluorescence from a fluorescence image Reconstructs the brightness and 3D location of luminescent sources Reconstructs the brightness and 3D location of fluorescent sources IVIS Imaging System 133 167 147 189 198 Acquire a Sequence for Planar spectral image analysis Displaying multiple fluorescent or luminescent reporters Subtracting tissue autofluorescence using blue shifted back
232. mended that you subtract the tissue autoluminescence from the image data For more details on subtracting tissue autoluminescence see Appendix E page 258 Itis also recommended that you inspect all images in the sequence to confirm that the luminescent signal is greater than the tissue autoluminescence If the luminescent signal does not exceed the tissue autoluminescence at a particular wavelength do not include that wavelength in the analysis 282 Living Image Software User s Manual This page intentionally blank WY Caliper Appendix H DLIT amp FLIT Reconstruction of Sources Determining Surface Topography 2 2 0 8 eee ee ee ee 283 Converting Light Emission to a Photon Density Map 284 Defining the Linear Relationship Between a Source and Photon Density 285 Determining the Best Approximate Solution to the Linear System 286 Source amp Tissue Properties 1 a a a a a a 286 Diffuse Tomography DLIT is a technique that analyzes images of the surface light emission from a living subject to generate a three dimensional 3D reconstruction of luminescent light source distribution inside the subject Fluorescent Tomography FLIT analyzes images of surface light emission to generate a 3D reconstruction of fluorescent light source distribution inside the subject To reconstruct luminescent sources the Living Image software requires a photograph a structured light image and luminescent im
233. more details Se see page 89 Click this button to display hide a scale on the x and y axis of the image window HE 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 84 Living Image Software User s Manual Table 7 5 Tool palette Image Information tools continued continued Tool Description Image Binning The binning applied to the image Note If soft binning is applied to the image data and the binning level is changed from 8 to 16 the new binning is indicated as 8x2 Width Height The FOV dimensions If the selected units 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 P5 The x y pixel coordinates at the upper left corner of the crop tool hi OR The x y pixel coordinates at the A end of the distance p The x y pixel coordinates at the lower right corner of the crop tool OR The x y pixel coordinates at the B end of the distance The width and height of the image crop tool OR Ax Ay from the A to B end of the distance measurement cursor For more details see page 88 and 88 Viewing X Y Coordinates amp Intensity Data 1 Open an image
234. mple that may or may not be part of a sequence can be selected for grouping into a new sequence Mm Lwin mee Uroatei Chek umber Ex Filter EMPker Ohuminaton Mode User IC User Gow l Eeoa 0S AD Peleti NEM 2 No 4 APEB Testing Scan Emission at EGE OOPS GEIS D03 S o elei PE NOA APAID Terg Sean Emisaon at Erbi Groner oan ray 003 auth emu SEM fe APAADI Teeny Scan imaan at ftir S AE HCOTO NIST SE YFM No 4 AFGE Testing Scan Emission at Betis iG odorantiosis7 20l a5 ea feleti itt DPN Noa Arid Testing Scan Emission at Exits dab toaooron0 157 002 eS K KEME Maa AFH Teir Scan Erissa at Ext hb recooroszi0n 157 003 te Be Roellecthen Hi SFM Nod APSO Treating Sean free di Erith M tsbrozporoazioe1 97 cos 605 700 Reflects MFM No 4 AF GRO Testing Scan Cmesmon at Crit steer 157 chia l sere l i istic TAPLO Testing Tiai Lire eag Lethe HEAP ION ET i ane T Telienti He YEMA No4 APEA Tritingy Scan Enivam a Pte Speers ee 71 R SEM 2 No 4 APSSO Testing Scan Emission ot Egi recocraxz9e1 167 oon os yuo Releawa Pet STM No ANLA Tostra Sean Umeson at Ext eE Images loaded in the browser as part of a sequence highlighted pink in this example These images can also be selected for grouping into another sequence Figure 7 27 Living Image browser 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 whi
235. n Choose this option to display the transillumination setup window that Setup enables you to select the locations for image acquisition using bottom IVIS Spectrum only illumination that originates beneath the stage y Caliper Per ne Appendix A IVIS Acquisition Control Panel 233 Table A 3 Typical field of view FOV settings FOV IVIS Imaging System Setting Lumina Spectrum Kinetic FOV cm A 5 5 10 4 4 B 7 5 7 5 15 6 5 6 5 7 C 10 10 20 13 13 10 D 12 5 12 54 25 22 5 19 5 lt 22 5 19 5 12 E 246 N A N A 22 5 26 22 5 26 24 a Position D is not available for X ray imaging on the Lumina XR b Available with removable lens option XFOV 24 Not available on the IVIS Lumina XR c Some imaging systems may have the FOV in parentheses FOV 19 5 and 26 were replaced by FOV 22 5 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 B Menual Focus Wilodow 4 WS Acquisition Control Panal fo IE Dika ej Pees Cipier Tire saigi F Ei 1 maed Per CA ao oe Cepia CCD Center le Il Uedste u Ce i E 4 Pid of ew C System Stabus ihe Serena LLA i i Suiberk Feed oi cn Faar se subject h ow lege abu Lihai irii ai
236. n density at the start of the analysis Ending ChiSqure Error between the measured and simulated photon density at the end of the analysis Parameters M uaEm cm Absorption coefficient at the emission wavelength M usEx cm Reduced scattering coefficient at the excitation wavelength M ueff cm Effective attenuation coefficient Mueff 3Mua Mua Mus Diff cm Diffusion coefficient Diff Mua Mus 3 Y Caliper i i j p Chapter 13 Point Source Fitting 131 ifeSchences Table 13 4 Results tab continued Item Description X location of the source X coordinate of the source location Y location of the source Y coordinate of the source location Z location of the source Z coordinate of the source location Misfit improvement The error between the measurement and the calculated fit percentage 0 0 132 Living Image Software User s Manual This page intentionally blank YP Cali per cts 1 4 3D Quantification Database Preparing amp Imaging the Samples 2 2 ee ee ee ee ee 133 Creating a Quantification Database 00208 ee eee 134 Managing Quantification Results 0 00 eee ee ee 137 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
237. n to the default position and size click the Reset button then g7 button 10 To turn off the transform tool click the Transform tool button i To check the organ fit 1 Check the fit in the coronal sagittal and transaxial windowpanes 2 Click the Change view toolbar button amp The Top view is displayed Sequence view SD View photons sec Source Intensity Subject Height 26 4mm Perspective Figure 15 33 Skin pink fitted to surface gray 3 Press the V key or the button to display alternative views of the surface r T W Caliper Chapter 15 3D Reconstruction of Sources 173 UD A Bottom Front Back Left E Right Figure 15 34 Alternate views of the surface Importing an Organ Atlas An organ atlas iv dxf or stl one organ per file consisting of segmented organ surfaces derived from an MRI or CT scan can be imported into the Living Image software for registration with the animal surfaces derived from IVIS data Organ files must be segmented from MRI or CT 3D volumetric data in third party medical imaging analysis software NOTE The imported atlas must include a surface skin file which delineates the animal surface The file name must include the word skin for example rat skin iv 1 Load a DLIT or FLIT image sequence that is associated with the mouse comprising the organ files in iv dxf or stl format 2 Select File Import Organ Atlas on
238. nalysis Tissue Source The tissue properties and source spectrum selected in the Properties tab Select Filters In the Filter box select the acquisition wavelengths for the images in the selected sequence To select non adjacent wavelengths press and hold the Ctrl key while you click the wavelengths Macintosh users press and hold the Cmd key while you click the wavelengths ROI List A drop down list of the ROIs in the active image 96 Living Image Software User s Manual Table 10 1 Planar spectral imaging tools continued Item Description Analyze Click to perform the spectral analysis Properties tab Tissue Properties Drop down list of the absorption and scattering properties of various tissues Internal medium index Tissue index of refraction that is automatically specified when you of refraction select a tissue property Source Spectrum Drop down list of luminescent sources Plot Tissue Properties Click to display graphs cm vs nm of the absorption coefficient u effective attenuation coefficient uss and reduced scattering coefficient u s Source Spectrum Click to display the spectrum of the selected luminescent source intensity versus wavelength normalized to one Results tab Spectral Results ROI Name of the analyzed ROI Depth mm Estimated depth of the point source Total Flux phot s Estimated total photon flux from the point source Plot Linear Fit Displays a grap
239. nd 1 FR Signal Backpround FSO Min detectable cells 3 8 010 Adin etette cells A00 Figure F 15 Fluorescent left and luminescent right images of stably transfected dual tagged PC3M luc DsRed cells The images show the signal from a subcutaneous injection of 3x106 cells in an 11 week old male Nu nu mouse When you make ROI measurements on fluorescent images it is important to subtract the autofluorescence background For more details see Subtracting Tissue Autofluorescence page 136 F 6 Subtracting Instrument Fluorescent Background The fluorescence instrumentation on an IVIS Imaging System is carefully designed to minimize autofluorescence and background caused by instrumentation However a residual background may be detected by the highly sensitive CCD camera Autofluorescence of the system optics or the experimental setup or residual light leakage through the filters can contribute to autofluorescence background The Living Image software can measure and subtract the background from a fluorescence image Fluorescent background subtraction is similar to the dark charge bias subtraction that is implemented in luminescent mode However fluorescent background changes day to day depending on the experimental setup Therefore fluorescent background is not measured during the night like dark charge background is After you acquire a fluorescent image inspect the signal to determine 1f a fluorescent background should be subtra
240. nd Theory of Operation System Components The VIS Imaging System 200 Series and VIS Lumina offer built in fluorescence imaging capability as standard equipment The IVIS Imaging System 100 and 50 Series use the XFO 6 or XFO 12 Fluorescence Option to perform fluorescence imaging The fluorescence equipment enables you to conveniently change between luminescent and fluorescent imaging applications Figure F 1 For more details see the IVIS Imaging System 200 Series System Manual the IVIS Lumina System Manual or the XFO 6 or XFO 12 Fluorescence Option Manual Erich ead 7 filter wheel iil features Fe itatrenti Fil best wheel Eacitation Emasion Filter Wheel Filter Wheel Light source Asem reat visible IVIS Lumina IVIS Imaging System 200 Series IVIS Imaging System 50 or 100 Figure F 1 Fluorescent imaging hardware A 150 watt quartz tungsten halogen QTH lamp with a dichroic reflector provides light for fluorescence excitation The relative spectral radiance output of the lamp reflector combination provides high emission throughout the 400 950 nm wavelength range Figure F 2 The dichroic reflector reduces infrared coupling gt 700 nm to prevent overheating of the fiber optic bundles but allows sufficient infrared light throughput to enable imaging at these wavelengths The Living Image software controls the illumination 262 Living Image Software User s Manual intensity level off low or high The illumination intensity a
241. nd select Export Results on the shortcut menu 2 In the dialog box that appears choose a folder for the results enter a file name txt and click Save 126 Living Image Software User s Manual 13 6 Managing Results Tool Palette gt ROI Tools gt Planar Spectral Imaging gt Surface Topography Point Source Fitting Analyze Params Properties Results Error Estimation Starting Chive 1 246 09 Ending chiz 2 44e 07 Point Source Fitting Results Parameters Fitted valu 5 500 5 660 13 570 0 030 x location of the source rim 13 405 Y location of the source rm 6 897 zZ location of the source mim 8 522 Photon Density Maps Export Results Save Results Name LMFIT_1 s Delete Load BLIT 4D Reconstruction gt GADLIT 3D Reconstruction gt 3D Tools gt Spectral Unmixing Figure 13 7 Point Source Fitting tools Results tab To save results 1 Select the results of interest LMFIT_ lt name gt from the Name drop down list 2 Click Save The point source fitting results are saved with the image To view results 1 Select the results of interest from the Name drop down list 2 Click Load To delete results 1 Select the results that you want to delete from the Name drop down list 2 Click Delete To copy selected results 1 Right click the results row of interest and select Copy Selected from the shortcut menu that appears The selec
242. nd the CCD camera reaches operating demand temperature locked 6 1 Kinetic Acquisition 1 Start the Living Image software double click the Fy icon on the desktop 2 Initialize the IVIS System and confirm or wait for the CCD temperature to lock For more details see page 17 3 If you are acquiring ventral images requires the subject be placed in the Ventral Imaging Chamber choose the VIC option in the control panel 4 When you are ready to begin imaging click Kinetics in the control panel The Kinetic Acquisition window appears A TV Auguri imi Ciri ral Han LM aain Friik Pier Firik Filer Fali of View D SPOS FE vic ize f Subject height 100 Pee ice sau ec eel Figure 6 1 Control panel top and Kinetic Acquisition window bottom 5 Select the type of data to acquire and set the acquisition parameters For more details on the acquisition parameters see Table 6 1 page 45 44 Living Image Software User s Manual Kinetic Acquisition Control Pavel Select the type data to acquire luminescent or fluorescent amp choose the Overlay option to acquire photographs After acquisition choose the type of data to display EAA Tessas 36 innir 1 i PM der To adjust a setting using the thumb 5 wheel put the mouse arrow on the arenen Mar wheel then click and hold the Enia ia mouse button while you move the Open mouse arrow left or right PL Liri Liai Baeri L t
243. ned using the Load as Group function in the LIving Image browser Default Order in which the images are stored in the folder TimeStamp Ascending order of the image acquisition time UserlD Ascending alphanumeric order of the user ID Display Choose the types of information to display with each image In this example exposure time and binning factor are displayed on each image Click to show or hide the image label information Figure 4 19 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 Y Caliper feSciences Chapter 4 Luminescent or Fluorescent Imaging 21 Table 4 4 Sequence View window continued Item Description Creates a preview picture snapshot of the image or thumbnails that the Living Image browser displays when the data Is selected For more details on the browser see page 71 Ehi irim eb eet HE a ii Fai jA kaha FT EN eel e AP iF p n ka sE pie he Come ate Sel dita yee D Be E ee ee i fel 1 joel St theme gle Fe iF p haa a eee L bai aa T SA HHN kiip Er Cor es EN E i rwie e a linen Tre vanes Laii j fo Aji lal tomas omit ors chee Lane C Page Si ee ee ee ee ee ee ei tot Preview picture of the selected data 4 5 Manually Set Up an Image Se
244. nescent or fluorescent image Pixels less than the color scale minimum do not appear in the image Pixels single image i l greater than the color scale maximum are displayed in the maximum color Options Layout Choose a display option for the images in a sequence Default Dynamic image or Film Strip For example here is Film Strip mode sequence A Sort by Options for ordering images in the sequence window Default Order in which the images are stored in the folder TimeStamp Ascending order of the image acquisition time UserlD Ascending alphanumeric order of the user ID Display Choose the types of information to display with each image In this example exposure time and binning factor are displayed on each image Opens all of the images in a sequence Closes all open images of a Sequence Y Caliper feSciences Table 7 2 Image window continued Chapter 7 Working With Data 77 Item Description z Opens the Edit Sequence dialog box that enables you to add or remove images f from a sequence The Tool Palette appears when you open an image or sequence lod Palette Lo Image Adjust gt ROI Took gt Planer Spectral Imaging os Gaare Tepngeaphy Post Source Hitting gt GUT 3D Foenenevbrue bin ARLIN JU Fenton o Spiral L Figure 7 5 Tool palette Tool Paktte s if Treas Adi Click to expand a tool ea ho bhebr rat Caper it bee gee Pin Fea Coker E
245. nge a color click the button that opens the color palette TT AO06 74145507 _ 006 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 242 Living Image Software User s Manual Preferences Curae A Tat Coker ees Surface Cor ee __ Tent Cole fT Poet tore Da auks Palora Del aults Color Paella T E o rawe Defis oe Caed i Figure B 8 3D view preferences Table B 6 3D view preferences Item Description Color Theme Background Color Predefined color schemes available for the 3D View window Click the button to restore the defaults for the selected color theme 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 Color Palette Settings that modify the display of the surface and text in the 3D View window Source voxels Choose a color table for voxel display
246. ning the summing is performed digitally on the stored image data not on the electronic charge before readout as in hardware binning Although soft binning does not improve the signal to noise ratio for read noise it may enhance the signal visibility because it reduces the statistical scatter of nearby pixel contents Usually hardware binning is preferred but if it is not possible to take another image applying soft binning to the data may provide a worthwhile solution C 3 Smoothing Smoothing is a filtering method that reduces noise in the image data To apply smoothing the software replaces the intensity of each pixel with the average intensity of a nearby pixel neighborhood that includes the pixel Figure C 4 shows a 3x3 pixel neighborhood Smoothing does not change the pixel size and helps e Eliminate outlier pixel values that are extremely high or low e Reduce noise fluctuations in the image to help reveal small signals Center pixel value the mean value of the nine pixels in the 3x3 neighborhood Figure C 4 3x3 pixel neighborhood Y Caliper ifeSciences Appendix D Image Data Display amp Measurement WieGe Uae 2 52 eS ewe RS HERS SOE CHE CE RS EE Ow 249 Quantifying Image Data i hb ame ew eH RHEE KH RH OH eH ED HS 251 PIAL FIENO ss eS EH ERA DBE SEK EERE E EERE EEE HES 254 Cosmic Ray Corrections 6 i vw ee2iGes ev bPeGevvd ee ee aD wwe 254 D 1 Image Data Scientific Image Data Scientific image data is
247. nning f stop or field of view setting All IVIS Imaging Systems include a CCD digitizer that is a 16 bit device which means that the signal count range is from zero to 65 535 Sometimes the displayed signal count may appear outside of this range due to corrections applied to the image data for example background corrections 252 Living Image Software User s Manual Photons Select Counts Photons or Efficiency for the image data 111 0050824145907 009 Lint Cire e re ET delay i ewe ag wj FY R Measurements Food Manan Ratnesh pai Pid Rel mat Lay Total Colt ais ok tou Matis Ma Goria TLS Soo Oe POL Teig AMO SO E eh Lire i e Ei Le i HEH g Praia 7H JAAHA aH aH TFHA Cupi eti Bima Prj Emane Altri dee Ft T ee Ea l tiei Counts eo ore Ea Contiguen Expat Diri In counts mode the ROI measurements table includes Total Counts Sum of all counts for all pixels inside the ROI Average Counts Total Counts Number of pixels or superpixels Quantity ROI Pixels Number of binned pixels inside the ROI Area CCD pixels Number of unbinned CCD pixels inside the ROI Figure D 2 ROI measurements counts mode When image data is displayed in photons the photon emission from the subject or radiance is displayed in photons sec cm sr Counts are a relative measure of the photons incident on the CCD camera and photons are absolute physical units that measure the photon emission fr
248. nsity and wavelength of the excitation source and the type of tissue Autofluorescence can occur throughout the animal but is strongest at the surface where the excitation light is strongest Dee OCD Array gt Imaging Lens Liege Autoiluorescence Photon Scattering hean free path I me hiaturmimescent eH Fluorescent Source Figure F 6 Illustration of the in vivo fluorescence process At 600 900 nm light transmission through tissue is highest and the generation of autofluorescence is lower Therefore it is important to select fluorophores that are active in the 600 900 nm range Fluorophores such as GFP that are active in the 450 600 nm range will still work but the depth of detection may be limited to within several millimeters of the surface Specifying Signal Levels and f stop Settings Fluorescent signals are usually brighter than luminescent signals so imaging times are shorter typically from one to 30 seconds The bright signal enables a lower binning level that produces better spatial resolution Further the f stop can often be set to higher values f 2 or f 4 is recommended for fluorescence imaging A higher f stop improves the depth of field yielding a sharper image For more details on the f stop see Lens Aperture page 245 F 4 Image Data Display Fluorescent image data can be displayed in units of counts or photons absolute calibrated or in terms of efficiency calibrated normalized For mor
249. nts organic compounds plastic tape and plastic containers Contaminants such as animal urine can be phosphorescent To help maintain a clean imaging chamber place animal subjects on black paper for example Artagain black paper Strathmore cat no 445 109 and change the paper frequently Cleaning the imaging chamber frequently 1s also helpful OL ALERT Use only cleaning agents approved by Caliper Many cleaning compounds phosphoresce Contact Caliper technical support for a list of tested and approved cleaning compounds If it is necessary to introduce suspect materials into the imaging chamber screen the materials by imaging them Acquire an image of the material alone using the same settings for example FOV and exposure time that will be used to image the sample to determine if the material is visible in the luminescent image Microplates white black or clear plastic can be screened this way Screen all three types with a test image White plates appear extremely bright by IVIS Imaging System standards and interfere with measurements Black or clear plastic microplates do not phosphoresce making them better choices The High Reflectance Hemisphere provides a more definitive way to determine the presence of an undesirable light source Figure E 1 It is a small white hemisphere that is coated with a non phosphorescent material A long exposure image of the hemisphere should produce a luminescent image in which the hemisp
250. nued Item Description Overwrite If you reanalyze saved results click to save the new results and overwrite the previous results Managing Reconstruction Results Toal Falete ROI Tools gt Surface Topography raria Proparlias Rhesus CLIT Anaha ferais iUad Key aha Final visel dine mn 1 25 Humber of weds Reduced Chaz Jakig wine bel Harf best hotel surf sarge Phresh ares Yoel site shaling vaue S Linien Saef ace Samping Damping Reduction Photon henaky Maps simi Hatut hamai OLIT Results name GADLIT 30 Reconstruction gt 30 Tonks gt Spectral Urimixing Figure 15 18 3D analysis results To save 3D results 1 In the Results tab of the DLIT FLIT 3D reconstruction tools confirm the default name or enter a new name 2 Click Save The results are saved to the sequence click number folder and are available in the Name drop down list To open 3D results 1 In the Results tab make a selection from the Name drop down list 2 Click Load The 3D results appear in the 3D View window To copy user specified results to the system clipboard 1 In the Results tab select the text of interest 2 Right click the results table and select Copy Selected from the shortcut menu that appears To copy all results 1 In the Results tab right click the results table and select Copy All from the shortcut menu that appears All of the results table is copied to the sys
251. o optimize image display and Suppress background noise To display the histogram using the full intensity range of the image click Full in the Histogram window 2 To edit the minimum or maximum bin intensity enter a new value in the Min Bin or Max Bin box or click the arrows r 3 To edit the number of bins enter a new value in the Bins box or click the arrows NOTE In the Overlay display mode the histogram plots the luminescent data To obtain a histogram of the photograph select Photograph from the Display drop down list Table 7 6 Histogram window Full Displays the histogram using the full intensity range of the image Min Bin The lowest intensity bin M ax Bin The highest intensity bin Bins The total number of bins pE Opens a dialog box that enables you to export the histogram csv Copies the histogram to the system clipboard 86 Living Image Software User s Manual Line Profile Table 7 6 Histogram window continued Item Description Opens the print dialog box The line profile plots intensity y axis at each pixel x axis along a user specified line in the image The line profile is automatically updated when you change the line position NOTE In the Overlay display mode the line profile plots the luminescent data To obtain a histogram of the photograph select Photograph from the Display drop down list To display the line profile 1 Open an image and in the Ima
252. objective of the background filters is to excite the tissue autofluorescence without exciting the fluorophore The background filter image is subtracted from the primary excitation filter image using the Image Math tool and the appropriate scale factor thus reducing the autofluorescence signal in the primary image data For more details see Chapter 9 page 136 The assumption here is that the tissue excitation spectrum is much broader than the excitation spectrum of the fluorophore of interest and that the spatial distribution of autofluorescence does not vary much with small shifts in the excitation wavelength Figure F 17 shows an example of this technique using a fluorescent marker In this example 1x 10 HeLa luc PKH26 cells were subcutaneously implanted into the left flank of a 6 8 week old female Nu nu mouse Figure F 18 shows the spectrum for HeLa luc PKH26 cells and the autofluorescent excitation spectrum of mouse tissue It also shows the passbands for the background filter DsRed Bkg the primary excitation filter DsRed and the emission filter DsRed Figure F 17 shows the IVIS images using the primary excitation filter the background excitation filer as well as the autofluorescent corrected image The corrected image was obtained using a background scale factor of 1 4 determined by taking the ratio of the autofluorescent signals on the scruff of the animal The numbers shown in the figures are the peak radiance of the animal
253. ock Position option NOTE The ROI position cannot be changed until the Lock Position option is cleared Editing ROI Dimensions There are two ways to resize a circle or square ROI e Drag a handle on the ROI e Edit the settings in the ROI Properties box NOTE You cannot change the size of an ROI that was created using the auto ROI or free draw tool To resize an ROI using a handle 1 Select the ROI and put the mouse pointer over a handle m on the ROI 2 When the pointer becomes a arrow drag the handle Y Caliper To resize an ROI using the ROI Properties box 1 Double click the ROI in the image Chapter 8 Working With ROI Tools 75 The ROI Properties box appears and displays the positions and dimensions of the selected ROI H R I Properties PH EGI AOH Laii EEG Saas By ot Sugno Into H Ue os BG For Fubune Pacts in aTa 145507 _006 Grtine sequence C Lock Position Xol pie 120 47354 Yelwed o0 Ande deg 0 0000 Sl C Lock Sise with po eraa S Figure 8 19 ROI Properties dialog box Dimensions of the ROI selected in the image 2 Enter a new width or height value in the ROI Properties box 3 To lock the current ROI size choose the Lock Size option NOTE The ROI size cannot be changed until the Lock Size option is cleared 76 Living Image Software User s Manual
254. olor to each numerical value and plotting the array so that each pixel is filled with the color that corresponds to its numerical value A color table defines the relationship between the numerical data and the displayed color For example a grayscale color table assigns black to the smallest number in the array white to the largest number and shades of gray to the values in between Figure D 1 The resulting image is equivalent to a black and white photograph An illuminated photographic image acquired on an IVIS Imaging System is an example of a grayscale pseudoimage The reverse rainbow color table is also commonly used and assigns violet to the smallest number on the array red to the largest number and all of the spectral colors of the rainbow to the values in between Figure D 1 250 Living Image Software User s Manual Overlays Color table associated with the data A photograph is a gray Overlay mode pseudocolor image of the scale pSeudoimage luminescent data is overlaid on a photograph Figure D 1 Example pseudoimages A pseudocolor scheme is typically used to display the numerical contents of scientific image data like the luminescent or fluorescent images acquired on an IVIS Imaging System The pseudocolor scheme makes it easy to see areas of bright light emission The amount of light emission can be quantified using measurement ROIs For more details page 102 Measurement data is independent of the colors displa
255. olorize 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 The colorize tool is only available if Show Advanced Options is selected in the general preferences see page 236 94 Living Image Software User s Manual To view colorized intensity data 1 Open an image sequence aD ote E Sapri Py ad lieramnag Figure 7 23 Microplate images Images were acquired using different combinations of excitation and emission filters The samples are quantum dot nanocrystals 700 or 800 nm 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 7 24 A HX 200704201 71444 SEO D Sequence view Sparta Culoice Yew USAT Hani MaR Coa Bane Figure 7 24 Colorize view Caliper Prp Chapter 7 Working With Data 95 Table 7 11 Colorize tools Item Description Colorize Color Range The color map indicates the color range of the selected camera setup from Short to long wavelength The two sliders determine the lower and upper limits of the color range that is used to render color The parts of the color map outside the selected range are not used in the color rendering process By default the entire color range is selec
256. ols 61 a Place the mouse pointer over the ROI When the pointer becomes a f click the ROI b Drag the ROI 4 Adjust the ROI dimensions a Place the mouse pointer over the ROI When the pointer becomes a click the ROI b Place the mouse pointer over an ROI handle so that it becomes a Drag the handle to resize the ROI NOTE You can also change the ROI position or size using the adjustment controls in the ROI Properties box see Moving an ROI page 73 and Editing ROI Dimensions page 74 5 Click the Measure button Y The ROI measurements and table appear For more details on the table see Managing the ROI Measurements Table page 80 For information on how to save ROIs see page 116 Automatically Drawing Measurement ROls The Living Image software can automatically identify all of the ROIs in an image or image sequence that meet the auto ROI parameter thresholds or draw one ROI at a user specified location 1 Open an image or image sequence and in the ROI tools select Measurement ROI from the Type drop down list 2 Click an ROI shape button Circle Square m or Contour and select Auto All from the drop down list The ROIs appear on the image or sequence thumbnails ae amp D g MP unia C Apply to al Fy TILT Ae 14 5507 So M Dapa ie Spectra Unda Cots tse Sarai akri Options Fito w 3 s aaa Y pe Meroe fae Pita beng POT St
257. om the subject The radiance unit of photons sec cm sr is the number of photons per second that leave a square centimeter of tissue and radiate into a solid angle of one steradian sr Figure D 3 Surface Radiance Tissue _ _ _ _ __ 2 Solid Angle units of steradians Figure D 3 Isotropic radiation Isotropic radiation from a cell is called photon flux photons sec When cells occur in tissue photon emission from the tissue surface Is called surface radiance photons sec cm2 sr Caliper Appendix D Image Data Display amp Measurement 253 ifeSciences A steradian can be thought of as a three dimensional cone of light emitted from the surface that has a unit solid angle Much like a radian is a unit of arc length for a circle a steradian is a unit of solid angle for a sphere An entire sphere has 4r steradians Lens systems typically collect light from only a small fraction of the total 4m steradians When image data is displayed in photons mode the units change to photons sec cm sr These are units of photon radiance on the surface of the animal A very important distinction between these absolute physical units and the relative units of counts is that the radiance units refer to photon emission from the subject animal itself as opposed to counts that refers to photons incident on the detector Measurements in units of radiance automatically take into account camera settings for example integration ti
258. on 2 2 261 Ee Piller P e was bc ea eee kee ee eee Gee eee eee ee eeeeeeesess 263 F 3 Working with Fluorescent Samples aoaaa 264 F4 Image Data Display 2 2224 44 24 4954 R464 aadd dtsia SEEGER ESS EG 265 F 5 Fluorescent Background 2 1 ee 267 F 6 Subtracting Instrument Fluorescent Background 2000002 eee 271 F 7 Adaptive Background Subtraction a oa aaa 212 F 8 Subtracting Tissue Autofluorescence Using Background Filters 273 Appendix G Planar Spectral Imaging a 2 eee eens 275 G 1 Planar Spectral Imaging Theory aooaa ee 275 GZ Optical Properties 25222 4 4 445 N ee BESS eS EES ee eRe Se ae eee Sees 271 G L citerase SpecHuUM e a see ranega eeun E SOR e kak EN e S ag G 4 An Example of Planar Spectral Imaging 2 a 278 G 5 Optimizing the Precision of Planar Spectral Analysis 0 0 2 000000 281 Appendix H DLIT amp FLIT Reconstruction of Sources 283 H 1 Determining Surface Topography aoao a a a 283 H 2 Converting Light Emission to a Photon Density Map 004 284 H 3 Defining the Linear Relationship Between a Source and Photon Density 285 H 4 Determining the Best Approximate Solution to the Linear System 286 H 5 Source amp Tissue Properties oaoa a a 286 Appendix I IVIS Syringe Injection System 0 2202208 289 I 1 Controlling the Infusion Pump 2 0
259. onduct an imaging test using the High Reflectance Hemisphere Figure E 1 A more subtle source of external illumination is the possible presence of light emitting materials inside the imaging chamber In addition to obvious sources such as the light emitting diodes LEDs of electronic equipment some materials contain phosphorescent compounds Do not place equipment that contains LEDs in the imaging chamber Phosphorescence is a physical process similar to fluorescence but the light emission persists for a longer period Phosphorescent materials absorb light from an external source for example room lights and then re emit it Some phosphorescent materials may re m je Ww Caliper Appendix E Luminescent Background Sources amp Corrections 257 emit light for many hours If this type of material is introduced into the imaging chamber it produces background light even after the chamber door is closed If the light emitted from the phosphorescent material illuminates the sample from outside of the field of view during imaging it may be extremely difficult to distinguish from the light emitted by the sample IVIS Imaging Systems are designed to eliminate background interference from these types of materials Each system is put through a rigorous quality control process to ensure that background levels are acceptably low However if you introduce such materials inadvertently problems may arise Problematic materials include plastics pai
260. or example will rotate with the hemisphere while a glowing spot due to external illumination most likely will not E 3 Background Light From the Sample Another source of background is the natural light emitted from a sample that is not due to emission from the source of interest in the sample This type of background may be due to a material associated with the experimental setup For example the cell culture medium may phosphoresce Materials should be screened so you can identify and eliminate problematic materials If a background source is phosphorescent and the phosphorescent lifetime is relatively short you can try keeping the sample in the dark for a long period before imaging to reduce background light emission Occasionally there is no way to eliminate the natural light emission of the sample The natural light emission associated with living animals autoluminescence is a major area of interest in in vivo luminescent imaging Most animals exhibit a low level of autoluminescence Usually this is only a problem when looking for very low signals at the highest levels of sensitivity Caliper has conducted tests to try to minimize the source of the background light emission in mice Test Description Observation Test 1 Subject animals were housedin Background emission levels were not reduced A the dark 12 hours prior to imaging phosphorescent component in mouse fur or skin is not the source of light emission Test 2
261. or scale of an organ s you t can click Reset to restore the default size and position of the selected organs 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 170 Drawing styles for the organs see Display Organs Shading styles for the organs see Display Organs Opacity Adjusts the opacity of the organ display Organ Atlas Choose a type of organ atlas 170 Living Image Software User s Manual Table 15 11 Registration tools continued Item Description Click to select all organs in the database and display them on the surface Click to clear the selected organs and remove all organ diagrams from the Surface Displaying Organs With the Reconstruction 1 Load reconstruction results and confirm that the surface is in the perspective view 2 3 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 i Rigid registration Performs linear transformation but keeps the shape of the atlas surface A Full registration Performs linear transformation and volume deformation For an optimum fit when there is a large difference between the orientation or size of the atlas org
262. orescent filters 264 fluorescent imaging efficiency 262 f stop 265 normalization 262 single image epi illumination 24 26 focus manually 233 234 234 G GADLIT sequence requirements 188 GADLIT results 202 203 graphic image data 249 H help 20 High Reflectance Hemisphere 257 histogram 85 I image adjusting appearance 79 cascade 99 correct or filter 81 82 correcting filtering tools 81 82 export 95 97 histogram 85 information 77 line profile 86 magnify or pan 81 measurements 88 pixel data 84 print 95 97 tag 90 tile 99 image data browse 71 74 colorize 93 95 export 42 open 74 save manually 41 image layout window 96 97 image math 133 135 image overlay tool 91 93 image sequence application specific 10 create from individual images 98 99 edit 97 98 include X ray images 48 55 manual setup 37 41 image window 75 Y Caliper feSciences imaging modes 7 9 imaging system capabilities 10 Imaging Wizard 30 34 import organ atlas 219 surface 164 165 information about an image 77 infusion pump 289 infusion syringe injection system control panel 290 infusion pump 289 initialization See system initialization instrument fluorescent background background instrument fluorescent background 271 IVIS Imaging System fluorescence imaging components 261 263 K kinetic acquisition settings 61 acquisition window 60 kinetic data acquire 59 save 65 66 view and edit 63 64 kinet
263. orsal height to contact the top of the imaging chamber if you set the subject height 0 and choose a small FOV 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 36 232 Living Image Software User s Manual Table A 1 IVIS acquisition control panel continued Item Description Temperature The temperature box color indicates the temperature and status of the system L System not initialized iy System initialized but the CCD temperature is out of range Ii System is initialized and the CCD temperature is at or within acceptable range of the demand temperature and locked The system is ready for imaging Click the temperature box to display the actual and demand temperature of the CCD and stage For more details see page 17 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 Click to display the Sequence Editor so that you can access the Imaging Wizard Setup specify and manage sequence acquisition parameters or open sequence acquisition parameters xsq For more details on setting up an image sequence see page 23 Image Setup Click to close the
264. ory and click Save ifeSchences Y C li Y alliper Chapter 9 Image Math 89 A folder of data is saved to the selected location AnalyzedClickInfo txt ClickInfo txt luminescent and photographic TIF images To export the image to a graphic file 1 Click the Export button mg Figure 9 2 2 In the dialog box that appears select a directory enter a file name and select the file type from the Save as type drop down list 3 Click Save Table 9 1 Image Math window Item Description Color Ranges for A and B Full Choose this option to set the Max and Min values to the maximum and minimum data values in the image Auto When this option is chosen the software sets the Min and M ax values to optimize image display and suppress background noise The Min and Max settings can be manually adjusted to further optimize the image display for your needs Note The color scale does not affect the image math result Color Ranges for Result Full See above Image Auto See above Min 0 Choose this option to set the minimum data value to zero Results Drop down list of mathematical functions that can be used to generate the new image including A B k A B k A B k A B k if Counts B gt k Useful for fluorescence tomography k Image Math window A user specified scaling factor applied in the results function k Fluorescent Background The software computes k the ratio of the autofluorescent signa
265. oteins used with IVIS Imaging Systems Name Excitation Passband Emission Passband nm Dyes amp Passband nm GFP 445 490 GFP EGFP FITC DsRed 500 550 DsRed2 1 PKH26 CellTracker Orange Cy5 5 615 665 Cy5 5 Alexa Fluor 660 Alexa Fluor 680 ICG 710 760 Indocyanine green ICG GFP Background 410 440 GFP EGFP FITC DsRed Background 460 490 DsRed2 1 PKH26 CellTracker Orange Cy5 5 Background 580 610 Cy5 5 Alexa Fluor 660 Alexa Fluor 680 ICG Background 665 695 Indocyanine green ICG F 3 Working with Fluorescent Samples There are a number of issues to consider when working with fluorescent samples including the position of the subject on the stage leakage and autofluorescence background signals and appropriate signal levels and f stop settings Tissue Optics Effects In in vivo fluorescence imaging the excitation light must be delivered to the fluorophore inside the animal for the fluorescent process to begin Once the excitation light is absorbed by the fluorophore the fluorescence is emitted However due to the optical y Caliper ad p Appendix F Fluorescent Imaging 265 feSciences characteristics of tissue the excitation light is scattered and absorbed before it reaches the fluorophore as well as after it leaves the fluorophore and 1s detected at the animal surface Figure F 6 The excitation light also causes the tissue to autofluoresce The amount of autofluorescence depends on the inte
266. ou can manually inspect the excitation lamp Note M ake sure that the filters of interest are selected in the filter drop down lists before you select Inspect The Inspect operation automatically positions the selected filters in the system before turning on the lamp Subsequent changes to the filter popup menus will have no effect until another Inspect operation is performed Photograph Light Controls the brightness of the lights at the top of the imaging chamber Level that are used to acquire photographic images Accumulate Select this option to view the cumulative intensity signal in real time When this option is chosen the software computes and visualizes the cumulative signal in each frame Color Scale Auto If this option is chosen the software chooses the color scale minimum and maximum Note Do not choose this option if the Accumulate option is selected Minimum A user specified threshold for the color scale minimum that is applied to the data if the Auto option is not selected Intensity signals less than the minimum are not displayed Maximum A user specified threshold for the color scale maximum that IS applied to the data if the Auto option is not selected File Size Displays the file size of the kinetic stream dcm being acquired The file size display is only available in the Kinetic Acquisition panel Save Click to select an option for saving the data Save Current Image Saves the currently selected frame
267. outside the subject image Y Chapter 12 Reconstructing a 3D Surface 115 Figure 12 3 Data mask purple 8 Ifitis necessary adjust the threshold value so that the mask fits the subject image as closely as possible To change the threshold do one of the following e Press the left or right arrow keys on the keyboard e Move the Threshold slider left or right e Click the 1 Click Finish arrows or enter a new value in the box The surface and 3D tools appear in the Tool Palette For more details on the Tool Palette see page 207 TEENE Oe WL 20050674177348 S10 Sequence view LZ Siew Seeetra Figure 12 4 3D view and 3D tools in the toolbar and Tool Palette 116 Living Image Software User s Manual Table 12 1 3D view toolbar Tool Description Image Tools A drop down list of tools for viewing and working with the surface Select ie to e Click and display measurement dimensions in the coronal sagittal or transaxial view in the 3D view window e Drag a measurement cursor in the coronal sagittal or transaxial view and display measurement dimensions For details on measurement cursors see page 88 Fop fa Select ay 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 M to rotate the subj
268. p Wak Hp m i gL H t Ag E U ee Saalace lepagaphp r Mi CP fe fet ae oth a ee a ae AO amp te WP line Phire ie il Gl Lites Peto Dipi mmi eine u es La et Titel Fin jahi he Foo ee L E Pi rama IF Phir iraerniy Supe Fale Fi Eps mi ee ey e Beian feeeperte OUT Figure G 5 Planar spectral analysis results Top Dorsal view of the left lung bottom ventral view of the left lung y7 Caliper Per nc Appendix G Planar Spectral Imaging 281 G 5 Optimizing the Precision of Planar Spectral Analysis The accuracy of the planar spectral analysis is highly dependent on the quality of the e Measured data for the firefly luciferase spectrum and the tissue optical properties e Fit of the experimentally measured total flux at each wavelength to Uep effective attentuation coefficient In general more experimental values produce a better fit of the data It is particularly important to be able to extract signals at all wavelengths to optimize the quality of the fit If the software detects no signal above the animal background level at 560 nm and 580 nm the wavelengths that absorb the most light the dynamic range of the optical properties is reduced and with it the precision of the fit If a luminescent signal is dim or buried deep in the tissue it may barely exceed the tissue autoluminescence at the shorter more absorbing wavelengths 560 and 580 nm In this case it is recom
269. pata krp a Be sii Select tissue and source Reconstruct and view source measurements properties Figure 15 1 Basic workflow for 3D reconstruction of sources 142 Living Image Software User s Manual 15 1 Reconstructing Luminescent Sources General Considerations Animal Requirements The best surface topography reconstruction is obtained from nude mice It is possible to perform 3D imaging on white or light colored furred mice if the fur is reasonably smooth over the mouse surface Therefore it is recommended that you comb the fur before imaging to eliminate any fluffy areas that may trigger artifacts during the surface topography reconstruction In this case it is recommended that you shave the animals or apply a depilatory 3D reconstructions are currently not possible on black or dark colored furred mice Luminescent Exposure vs Luciferin Kinetic Profile It is important to consider the luciferin kinetic profile when you plan the image sequence acquisition The DLIT algorithm currently assumes a flat luciferin kinetic profile Therefore to optimize the signal for DLIT 3D reconstruction carefully plan the start and finish of image acquisition and ration the exposure time at each emission filter so that the sequence is acquired during the flattest region of the luciferin kinetic profile Image Sequence Requirements Use the Imaging Wizard to setup the image sequence required for DLIT or GADLIT analysis For more d
270. pen the kinetic acquisition control panel When the kinetic control panel is open the Acquisition menu is unavailable 2 Set the volume and flow rate 290 Living Image Software User s Manual Infusion Pump Control Infusion Pump Control Panel volume Flow Fate Syringe Type Diameter L Auto Start After Start Mow 10 uLjmin BD icc M 633 mr C Auto Stop After Acquisition Stop Mow Figure 1 2 Volume amp flow rate settings 3 Make a selection from the Syringe Type drop down list the associated syringe diameter is automatically entered To enter a custom syringe a Select Custom from the drop down list b Click OK in the dialog box that appears F Information Claret not bren for pyri type Citim LJ Fee enter avad dameter for the syringe selected Figure 1 3 Confirmation message for a custom syringe c Enter the syringe diameter in the infusion pump control panel NOTE Custom syringe information that is entered in the infusion pump control panel is not saved to the system 4 To automatically start the infusion pump after data acquisition begins choose Auto Start After and enter the amount of seconds For example enter 10 to start infusion 10 seconds after acquisition begins To manually start infusion click Start Now 5 To automatically stop infusion choose Auto Stop After Acquisition To manually stop infusion at any time click Stop Now If the auto stop option is not chosen and
271. pir i al A TLT70060406164950_SE0 Tool Palette Speetral Urri d ere Ea ba Lirie Leese iz Spcekr HEEJ E Show Label F ndaka ka Ehe all i aaa rrr ol borsin Fa huvber of Corgon 2 Huber of Waert 7 Birri ot Sunred Pb Lack off Pi ef Pe celia Lock of FE YER a he oc Dreergere A Coed ee a Placa Thee ating Pa Praha quel ie ae T a Hor pesgi nether Wiehe fee a io Column Meaghiting made Hone Fipe Weahin mde Hene i E G Figure 11 4 Spectral unmixing results 7 To analyze an unmixed image double click the image YP Cali er ifeSciences Chapter 11 Spectral Unmixing 105 The image appears in a separate image window and the Tool Palette is available This enables you to make ROI measurements and image adjustments that are saved with the image 8 To adjust the composite image double click the composite image The composite image is displayed in a separate window 4 11120060406164950_ SEQ Sequence View Spectra Unmixed Images Composite Units counts Image List ja TissueAF UMA 2 Photograph Image Adjust Min 3058 amp ty Max 611654 Color Brightness C Logarithmic Scale Label Tissue4F Figure 11 5 Composite image window Table 11 3 Composite image window Units The type of data displayed in the composite image Image list A list of the images that comprise the composite background component s probe s and a photograph M in M ax Sets the minimum
272. play the color scale minimum and maximum Display Image Min Choose this option to display the minimum and maximum signal M ax 6 3 Saving Kinetic Data The IVIS Kinetic instrument enables you to acquire a real time data stream which can generate very large files The file size limit for DICOM data is 2GB Kinetic data acquisition automatically stops when this file size limit is reached Table 6 3 shows how binning conditions affect the total number of frames that can be collected in overlay or luminescent fluorescent only mode Table 6 3 Frames collected per 1 GB DICOM file Binning Level Frame Size Overlay Mode Luminescent or DICOM File Size Fluorescent Only Total Frames Collected Binl 2 MB 250 500 Bin2 512 KB 975 1950 1 GB Bin 4 128 KB 3900 7800 Bin 8 32 KB 15600 31250 Bin 16 8 KB 62500 125000 To save data 1 In the Kinetic Acquisition window click the Save button and select a save option Save Current Image Saves the currently displayed frame Save Accumulated Saves the accumulated signal for the selected frames tiff Image Note It is not necessary to select the Accumulate option to save an accumulated image Save Kinetic Data Saves the data photographic frames all luminescent or fluorescent frames and read bias in DICOM format dcm 2 In the Edit Image Labels box that appears enter information for the image label and click OK If you do not want to enter label information cl
273. playing data click the Stop button 4 To view the cumulative signal during playback choose the Accumulate option If the accumulated image maximum exceeds the current color scale range use the image adjust tools to adjust the color scale Exporting Kinetic Data You can select a range of images for export to DICOM format includes photographs intensity signal and read bias or to a movie 1 In the image window click the button Figure 6 7 2 If you want to select a particular range of data for export use the frame range selection to select the data Use the left slider to select the start image and the right slider to select the end image in the data range of interest The top slider automatically moves to denote the location of the current image with respect to the selected data range 3 To export the selected data to a movie a Click Extract and choose Save as a Movie b In the dialog box that appears select a folder enter a name for the movie and choose the file format for example mpg4 y Caliper a PET nes Chapter 6 Kinetic Imaging 53 Exporting an Image from a Kinetic Data Set 1 To select an image move the frame slider or enter a frame number in the spin box Export Graphics button EE i k mirr m r lm fi oe Lore dou pee remi fa Sere Spin box Figure 6 8 Image window selecting an image for export 2 Click Extract and choose Extract Current Image A new image window
274. ple luminescent x ray and photograph additional drop down lists appear so you can conveniently choose any two images to overlay Display FF Luminescent on BY Photograph 8 Living Image Software User s Manual Table 4 1 Image window continued Item Description Info Click to display or hide the image label The image label includes information you enter in the Edit Image Labels dialog box Figure 4 2 and other information automatically recorded by the software at Opens a dialog box that enables you to export the active view as a graphic file ij Creates a preview picture snapshot of the image or thumbnails that the Living Image browser displays when the data is selected For more details on the browser see page 71 giia te aii o iE e UEI Mabi a Fe je ee ates tr oa eemi Ol p 28 tie Se G 1i Fai jii Tha FAT LEN eel e AP iF pan ba i eS He marti a 5H cep 14 See TO LED edie diene oe 1 pl ba a ee es TL DA DE eters er Cori Tes E OS ea erim tne sen Pesan Lebel het a fo mddan ond i dome sa coe Longe Cee Si eee ee eT ee eT eel le Preview picture of the selected data Color Scale Provides a reference for the pixel intensities in a luminescent or fluorescent image Pixels less than the color scale minimum do not appear in the image Pixels greater than the color scale maximum are displayed in the maximum color 4 2 Acquire a Fluorescent Image With Epi IIlumination Epi
275. pography Point Source Fitting Analyze Params Properties Results Model Type Bioluminecence w Angle Limit deg Spatial Filter Parameters starting values ara F Starting parameter values for the analysis vom o E amo pw E Power phaton s E Restore Defaults Mask Mo mask Statistics Weighting LM Fitting gt BLIT 3D Reconstruction gt GADLIT 3D Reconstruction gt Spectral Unmixing Figure 13 9 Point Source Fitting tools Params tab Table 13 2 Params tab Item Description M odel Type Transmission This model assumes that the light source comes from under the animal and is transmitted through the entire subject Bioluminescence Model for luminescent data Transillumination Fluorescence Model for fluorescent data acquired with bottom illumination Epi illumination Fluorescence Model for fluorescent data acquired with top illumination Angle Limit deg The angle limit refers to the angle between the object surface normal and the optical axis The optical axis can be considered to be a line perpendicular to the stage The surface normal is a line perpendicular to a plane tangent to the surface point For example in a dorsal view of a mouse the highest point on its back would have a normal line perpendicular to the stage In this case the angle is zero The side of a mouse abdomen would have a normal line parallel to the stage so the angle here would be close to 90 The software
276. pr dc ed n a Peed j Pdi AN 3 Brresestrenieess De ED ee cee fh al i hay Figure 8 2 Open an image from a sequence to see the ROI intensity measurements Y Caliper Chapter 8 Working With ROI Tools 57 4 Ifitis necessary to adjust the ROI boundaries change any of the auto ROI parameters use the slider or 3 arrows Figure 8 3 Toal Falete C Apply to Secure Tari Mesunement RGI spe 2 ESA auto BOL Parameters Threshold Specifies the minimum per cent of peak pixel hal NN boxe intensity that a pixel must have to be included in an ROI identified by the software Point Source Fitting O gt DLIT 20 Reconshrurtion gt GADLIT 30 Reconstruction Spectral Unmiring Figure 8 3 Auto ROI parameters NOTE After the ROIs have been created right click an ROI to view a shortcut menu of ROI commands Ctrl click for Macintosh users The shortcut menu provides easy access to many functions for managing ROIs and viewing ROI properties 5 Click the Measure button in the ROI tools to show the ROI Measurements table RO Measuccninanls E reten imapa raume Ss SSS rae e Counts m TASMA ERAT _ ce TLT20050624145507 WH TLT ANOS 145507 cH TLT 2005062414550 NS JLT ZUe 2414250 Oe TLTZ0050624145507 _006 Pe ak i 1 3
277. quence This section explains how to set up an image sequence if you do not use the Imaging Wizard You can save the sequence parameters in the sequence editor to a Living Image Sequence Setup file xsq For details on image acquisition see Acquire the Image Sequence page 17 To create an image sequence it may be convenient to edit a sequence setup generated by the Imaging Wizard or an existing sequence setup xsq Save the revised sequence setup to a new name _ i Click Sequence Setup in the control panel The sequence editor appears If necessary click the Remove button X Rm and select All to clear the sequence 22 Living Image Software User s Manual eae 3 Choose a subject and probe T Number fsecmente T tele OG ew aytal W temo 4 update C fewest bom nm au Figure 4 21 Choose a subject and probe 4 In the control panel specify the settings for the first luminescence or fluorescence image in the sequence and the photograph For details on the imaging parameters in the control panel see page 225 NOTE If you choose the photograph Reuse option in the control panel Figure 4 22 the IVIS System acquires only one photograph for the entire sequence If this option is not chosen the system acquires a photograph for each image in the sequence Click the Add button _Ga _ The acquisition parameters appear in the seq
278. r modifiable If this option is chosen the system automatically displays the overlay after acquisition is completed for example luminescent image on photograph Turns on the lights located at the top of the imaging chamber Y Caliper ifeSchences Appendix A IVIS Acquisition Control Panel 231 Table A 1 IVIS acquisition control panel continued Item Description Fluor Lamp Level Field of View Service Load XFOV 24 Subject height cm Focus Sets the illumination intensity level of the excitation lamp used in fluorescent imaging Off Low High and Inspect The Low setting is approximately 18 of the High setting Inspect turns on the illumination lamp so that you can manually inspect the excitation lamp Note Make sure that the filters of interest are selected in the filter drop down lists before you select Inspect The Inspect operation automatically positions the selected filters in the system before turning on the lamp Subsequent changes to the filter popup menus will have no effect until another Inspect operation is performed Sets the size of the stage area to be imaged by adjusting the position of the Stage and lens The FOV is the width of the square area cm to be imaged A smaller FOV gives a higher sensitivity measurement so it is best to set the FOV no larger than necessary to accommodate the subject or area of interest The FOV also affects the depth of field range in which the subject is in
279. r the tissue and source you selected make a selection from the Plot drop down 10 To compute the number of cells per source select a luminescent calibration database For details on generating a luminescent calibration database see page 133 11 In the Analyze tab click Start The Data Preview window appears and displays the image data that will be included in the reconstruction Usually no data adjustment is required However it is possible to exclude or include user selected pixel data from the analysis For more details see page 146 Tocd Pakebhe PUG Tanke Flamer Spectral Irei Suntec loper apie l Pte aE ai H T0020 SEO C dapamevsem E a veew Aira Gata Prien E Cai Hr Reto ree iari Parquet eRT see Te arb ee T F Image Late F Medion Fiker Restore Threshold bate Achushment 7 Soleck All cael Reconstruct Figure 15 10 Data Preview window right 12 Click Reconstruct Caliper Chapter 15 3D Reconstruction of Sources 151 K The reconstruction requires about 1 5 minutes depending on the parameter settings and the processor speed When the analysis is finished The 3D View window displays the animal surface and the reconstructed sources In the Tool Palette the Results tab displays the results data and the algorithm parameter values The 3D Tools appear after a reconstruction is generated or loaded For more details on the 3D Tools see page 165 175 For detail
280. ra ipi an bo eE Enie ier mf kPa Oe i Benk api bi Le oreo k Pree BCE ba nm eet Ure bree L Ener Bel P Pemarin Figure 9 1 Opening the Image M ath window 3 In the Image Math window that appears select an image of interest from box A and box B The Image Math window shows a thumbnail of image A image B and the new image 88 Living Image Software User s Manual rs Ineawe Malli Wane Srqucnor TLT ies IMI 14512_ SF A TLT ZS 10114512 O01 TLT AS S TLT ZS 1001 1451 2 _ Cos TET eS L 1451 LOLH TL 200605101 14512_005 TETANOS HST LODE R TUT NS 14512 00 TL ZS 0 145 2_ 0 TAT eS LN S de O T ANA 10114512 04 TAT eS LN HS T ODE TE ARAID IA We Color Scale Limits for A and E C2 Pull E auto Buss Coder irae Linda Co Pall auto _ Mined Forssk a B k k 1 00 0 Connie W fren ROL Ellery tite TEES d Lee ee M Click to export the Image to a graphic file Figure 9 2 Image Math window and new image NOTE For more details on items in the Image Math window see Table 9 1 page 89 4 Select a mathematical function from the Result drop down list 5 To include a scaling factor k in the function enter a value for k 6 To view the new image click Display Result for Measuring To save the new image 1 Click the Save button f Alternatively select File Save on the menu bar 2 In the dialog box that appears select a direct
281. rea Lewis 5 Auto CI Ful e irrita Cika Lista Rnb A E hema T begathri Serle gt RN THs gt Bane Spectral Inega Surlace Topography s Paint Source Fittir F i LE Hi Mii Ferrel ream Riran A ALT Y Hace a E ee gt Spectral Uiiinig The options available in the Tool Palette depend on the type of active image data For an overview of the tools see Figure 2 4 on page lland Figure 2 5 on page 12 7 3 Viewing Image Information At acquisition the software captures image information that includes all of the text information that is associated with an image for example camera parameters and any image label information entered at acquisition Figure 7 6 Click Info to display the image label and acquisition information D fe e m H w UE t ia WP oaos i m GEHI a Ss ett ore Figure 7 6 Image window displaying image information 78 Living Image Software User s Manual Another way to view information about images is available in the View menu 1 Open an image or sequence 2 Select View Image Information on the menu bar The Image Information window appears 3 Choose an image by making a selection from the Sequences drop down list and the Images drop down list Figure 7 7 Drop down list of open sequences Drop down list of images in the selected sequence Choose Individual Images from the Or alist of single images if Individual Images is list to Show the open single images in
282. reference Target M ax Count Minimum Range Values Exp Time Sec Binning F Stop Restore Defaults During auto exposure the software acquires a luminescent or fluorescent image so that the brightest pixel is approximately equal to the user specified target max count If the target max count cannot be closely approximated by adjusting the first preference for example exposure time the software uses the first and second or first second and third preferences to attempt to reach the target max count during image acquisition A user specified intensity The minimum and maximum values define the range of values for exposure time F Stop or binning that the software can use to attempt to reach the target max count during image acquisition Click to apply default settings 240 Living Image Software User s Manual Preferences Ce rar al ener Angker Thina Opita Properties tuto Espoare Canea Settings Dahut bee Dogars Datuk Frage innig Piipa ubo Piar applic Lumea Lurrenescent 1 00 bi w T ote Hedum medum Fher 1 0 Restore Det aul Figure B 6 Acquisition preferences Camera Settings 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
283. rface by this method is for viewing purposes only not for coregistration with 3D reconstructions in Llving Image software To import a surface or other organs for coregistration purposes import an organ atlas For more details see page 215 ifeSchences Y Caliper aA p Chapter 12 Reconstructing a 3D Surface 119 Table 12 2 Surface file types Export Option Description Export Import Surface mesh A native file format of the Living Image software that is yes yes xmh used to exchange 3D surface information between Living Image software and other third party analysis tools It is based on a basic indexed face set format which stores all of the vertex information first then stores the triangle information in terms of indexes into the vertex list AutoCAD DXF Drawing exchange format that is compatible with most yes yes dxf DXF file viewers VRML 1 0 wrl VRML 1 0 wrl Virtual reality modeling language format yes no that is compatible with most VRML viewers Open Inventor iv The ASCII version of the IV file format which is supported yes yes by all IV viewers STL stl or ASCII Stereo lithography binary format compatible with most yes yes format STL viewers binary 120 Living Image Software User s Manual This page intentionally blank y Caliper 1 3 Point Source Fitting Image Sequence Requirements 2 ee ee ee ee ee ee a 121 Displaying the Point Source Fitting Tools
284. s However it is not necessary to subtract the electronic background when making a simple visual inspection of an image The types of electronic background include e Read bias An electronic offset that exists on every pixel This means that the zero photon level in the readout is not actually zero but is typically a few hundred counts per pixel The read bias offset is reproducible within errors defined by the read noise another quantity that must be determined for quantitative image analysis e Dark current Electronic background generated by the thermal production of charge in the CCD To minimize dark current the CCD is cooled during use Read Bias amp Drift Prior to a luminescent image exposure the Living Image software initiates a series of zero time exposures image readout to determine a read bias measurement If a dark charge background is available for the luminescent image the average bias offset for the read bias image stored with the dark charge measurement is compared to the average bias offset determined with the read bias measurement made prior to the image The difference or drift correction is stored with the luminescent image data and is later used to correct minor drift typically less than two counts pixel that may occur in the bias offset since measuring the dark charge background If a dark charge background is not available at the time of the luminescent image exposure the software checks to see if the
285. s high sensitivity binning f stop 1 FOV C 13 cm exposure time 120 seconds at 560 and 580 nm exposure time 60 seconds at all other wavelengths This resulted in signals of 2000 counts on each image To perform the planar spectral analysis draw a measurement ROI that captures the entire signal of each site of interest without including a neighboring metastasis Figure G 3 After the ROI is defined start the planar spectral analysis for more details see page 139 The software e Measures the total flux inside the ROI on each filtered image e Normalizes the data to the luciferase spectrum Plot of Intensity vs Lambda Figure G 4 e Fits the normalized data to the analytical expression in Equation 1 page 276 where S absolute total photon flux emitted by the luminescence source and d source depth Plot of Linear Fit Results Figure G 4 Appendix G Planar Spectral Imaging 279 Figure G 3 Metastatic site ROI includes the signal of the right kidney and separates it from other metastatic sites The Signal coming from the lower back area is spread out due to the presence of two bright spots The dimmer signal in the lower bottom right of the image likely originates from the femoral bone of the animal E Fie Eat view Took Window Hep nC ar T Ag OP iUn Phoon w C App tol E r et w Pa we oT EA Aaa pM Image Indommation gt ROI Tools man i in Zhe U Plane Sp
286. s situation the best way to minimize autofluorescence is to change the animal diet to alfalfa free rodent food when working with the Cy5 5 and ICG filter sets Control animals should always be used to assess background autofluorescence Regular Rodent Food SFP Dsked Wo ICG Alfalfa free Rodent Food GFP DsRed Cys 5 eee ii Figure F 14 Images of animal tissue autofluorescence in control mice Nu nu females Animals were fed regular rodent food top or alfalfa free rodent food bottom Images were taken using the GFP DsRed Cy5 5 or ICG filter set The data is plotted in efficiency on the same log scale Figure F 15 shows a comparison of fluorescence and luminescence emission in vivo In this example 3x 10 PC3M luc DsRed prostate tumor cells were injected subcutaneously y Caliper ad p Appendix F Fluorescent Imaging 271 feSciences into the lower back region of the animal The cell line is stably transfected with the firefly luciferase gene and the DsRed2 1 protein enabling luminescent and fluorescent expression The fluorescence signal level is 110 times brighter than the luminescence signal However the autofluorescent tissue emission is five orders of magnitude higher In this example fluorescent imaging requires at least 3 8x 105 cells to obtain a signal above tissue autofluorescence while luminescent imaging requires only 400 cells Fluorescent Bioluminescent 95210 1oc10 FAe 10 saan al Backgrou
287. s X ray images see Chapter 5 page 43 Sequence Setup Using the Imaging Wizard 1 Click Sequence Setup in the control panel Figure 4 14 The sequence editor appears i If necessary click the Remove button X Rm and select All to clear the sequence editor 3 Click the Imaging Wizard button saa weed The Imaging Wizard is not available until the IVIS Imaging System is initialized and the demand temperature is locked 16 Living Image Software User s Manual i VES Ae jamin Conde Panel E E a A Ta row weamectage _ F F i irga ion Lerrirai Prej E a Sequence Editor ie a nar eens 1 are ile re Biel a Tat Sim T oe ee ane tetera barep ai Lre imil fc i C Maniar at Segments i Tle a we i Gaa a hace Double click here to set Imaging Wizard up a bioluminescence G image sequence Petia ee apti b eee A If this screen does not appear demiaren oren ah a siia When the wizard starts click Restart Wizard estat wird ON the wizard screen to restart the wizard Double click here to set up a fluorescence Image sequence Go tate Lote oe are oe barred oia a Figure 4 14 Opening the sequence editor and Imaging Wizard 4 In the wizard choose bioluminescence or fluorescence imaging 5 Inthe next wizard screen choose the type of image sequence that you want to acquire Step through the rest of the wizard When you complete the wizard
288. s initialized and the CCD camera reaches operating demand temperature locked This chapter explains how to acquire a luminescent or fluorescent image or image sequence This section explains how to acquire an image in the following modes e Luminescent see below e Fluorescent with epi illumination page 8 e Fluorescent with transillumination page 11 4 1 Acquire a Luminescent Image 1 Start the Living Image software double click they icon on the desktop 2 Initialize the IVIS Imaging System and confirm or wait for the CCD temperature to lock For more details see page 17 3 In the control panel put a check mark next to Luminescent and select Auto exposure click the arrow When you select Auto exposure the software automatically determines the binning and F Stop settings Alternately you can manually set the exposure binning and F Stop For more details on these control panel settings see page 225 A IVIS Acquisition Control Panel Imaging Mode Exposure Time Binning F Stop Excitation Filter Emission Filter Field of View System Status Idle Acquire Subject height 150 Shem amp Sequence Setup Figure 4 1 Acquisition control panel 4 Puta check mark next to Photograph and select Auto exposure click the arrow 5 Make a selection from the Field of View drop down list For more details on the field of view see page 227 6 Set the Focus 6 Living Image Software User s Manual i
289. s inside the surface is set to a small value so that the exported surface appears as a solid structure Axial range The axial range min max can be use to select a particular section of the mouse surface or 3D scene for export The default values select the entire surface along the axis selected in the Slice Orientation drop down list Hollow mesh The intensity of pixels inside the surface is set to zero so that the exported surface appears as a hollow empty structure Y Caliper 4eSclences Chapter 15 3D Reconstruction of Sources 165 15 8 3D Tools Overview The Tool Palette includes the 3D Tools after you reconstruct or load a surface or 3D sources The tools are organized by tabs 3D Tools Functions Page Surface Tools Adjust the appearance of the reconstructed animal surface See below and photon density maps Source Tools Adjust the appearance of reconstructed sources make 167 source measurements export voxel measurements Registration Tools Display organs on the reconstructed surface adjust the 168 location or scale of organs on the surface import an organ atlas Animate Tools Display preset animations of the 3D View scene Enables you 175 to create custom animations and record an animation to a movie file 15 9 3D Tools Surface Use these tools to adjust the appearance of the reconstructed animal surface and photon density maps a ue Vier 5 B ee Ser 3D Tools gt Aegre 4 pi i Surface
290. s on managing results for example save load or delete see page 157 3D View toolbar for more details see page 145 Fie Edi We Toos Aonik Aird Hep HAU kt Aa K ures courts me C Anety bo al Pi TT 200506274145507_ S00 Figure 15 11 GADLIT reconstruction results 152 Living Image Software User s Manual 15 2 Reconstructing Fluorescent Sources Image Sequence Requirements Use the Imaging Wizard to setup the image sequence required for FLIT analysis For more details on the Imaging Wizard see page 26 If you plan to manually set up the sequence Figure 15 12 shows the an example image sequence Acquire the images using transillumination on the IVIS Spectrum Imaging System using the same excitation and emission filters from at least four source locations that form a rectangle Imaging Wizard SE C Display Photographic Settings Subject Mone w Probes Mode Exposure Binning Fstop Excitation Emission LampLlevel Structure FOY Height Transillumination De Acquire a fluorescent image Fe e auto Medium 2 640 700 High Yes a 1 50 15x23 2 13 Structured light image and 2 i Auto Medium al 640 700 High No lc 1 50 15x23 2 12 photograph at the first 3 B Auto Medium 2 640 700 High No c 1 50 15x23 2 12 transillumination location 4 jl Auto Medium zl 640 700 High No lc 1 50 15x23 2 11 5 B Auto Medium 2 640 700 H
291. scription Measurement Types Make a selection from the drop down list to specify the type of ROI measurements to include in the table None Excludes ROI measurements from the table Counts Includes Total Counts Avg Counts Stdev Counts Min Counts and Max luminescence Counts in the table Total Counts the sum of all counts for all pixels inside the ROI Avg Counts Total Counts Number of pixels or super pixels Stdev Counts standard deviation of the pixel counts inside the ROI Min Counts lowest number of counts in a pixel inside the ROI Max counts highest number of counts in a pixel inside the ROI For more details on count units see page 247 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 Radiance Photons Total Flux the radiance photons sec in each pixel summed or fluorescence integrated over the ROI area cm x 4r Average Radiance the sum of the radiance from each pixel inside the ROI number of pixels or super pixels pbhotons sec cm2 sr Stdev Radiance standard deviation of the pixel radiance inside the ROI Min Radiance lowest radiance for a pixel inside the ROI M ax Radiance highest radiance for a pixel inside the ROI For more details on photon units see page 248 Radiant Efficiency Epi fluorescence Fluorescence emission radiance per incident fluorescence excitation irradiance p sec cm
292. se the te arrows or the keyboard arrows to specify a subject height cm or e Select Manual focus from the Focus drop down list For more details on manual focusing see page 229 OO If necessary click mageSetup in the control panel to operate in single image mode NOTE In single image mode the Sequence Setup button appears in the control panel Click this button to set up sequence acquisition 9 To acquire an overlay image coregistered images for display after acquisition put a check mark next to Overlay NOTE To view the subject s inside the chamber before image acquisition take a photograph Uncheck the Fluorescent option choose the Photograph and Auto options and click Acquire 10 When you are ready to capture the image click Acquire The information entered here appears in the image label Figure 4 8 page 10 ka ba ii T Figure 4 6 Edit Image Labels box 11 In the Edit Image Labels box that appears enter information about the image and click OK 10 Living Image Software User s Manual NOTE You can enter image label information at any time during or after acquisition If you do not want to enter image information click Cancel 12 If this is the first image of the session you are prompted to enable the autosave function Living Image 4 0 Do vou want bo enable auto saving of acquired data For this session a a This can be ch
293. se this option to acquire a luminescent image Fluorescent Choose this option to acquire a fluorescent image Exposure time The length of time that the shutter is open during acquisition of a photographic or luminescent image The luminescent or fluorescent signal level is directly proportional to the exposure time The goal is to adjust the exposure time to produce a Signal that is well above the noise gt 600 counts recommended but less than the CCD camera saturation of 60 000 counts Luminescent exposure time is measured in seconds or minutes The minimum calibrated exposure time is 0 5 seconds The exposure time for fluorescent images is limited to 60 seconds to prevent saturation of the CCD There is no limit on the maximum exposure time for luminescent images however there is little benefit to exposure times greater than five minutes The signal is linear with respect to exposure time over the range from 0 5 to 10 minutes Integration times less than 0 5 seconds are not recommended due to the finite time required to open and close the lens shutter 230 Living Image Software User s Manual Table A 1 IVIS acquisition control panel continued Item Description Binning F stop Excitation Filter Emission Filter Photograph X ray Structure Overlay Lights Controls the pixel size on the CCD camera Increasing the binning increases the pixel size and the sensitivity but reduces spatial resolution Binning a
294. selected in the Sequences drop down list the Images drop down list A image Infomation Sequences feebatios ape k Ep UATR LOr ete ka O S al Sections Boie g Dever Listed pii Saat mae ra pay pa phahographac image Lites ae Literal Choose the Show All essen spin rv Sections option to Het Info display all categories of image information Key fake Quen Dae Prebry hrir f 0S Quer aire Cate hd ARREA E PEH Aapee PL coaches brig une oreta Eining Factor Dami ree Cr i A D ae T Figure 7 7 Viewing image information 4 To view information of interest select a category in the upper box to show the associated information in the lower box For example select luminescent image in the upper box to show the luminescent image acquisition parameters Editing the Image Label You can edit the image label information after acquisition 1 Open an image 2 Select Edit Image Labels on the menu bar 3 In the Edit Image Labels box that appears Figure 7 8 edit the information You can also select a new label set to apply to the image or sequence YP Caliper ieSelanices Chapter 7 Working With Data 79 de bap Lalki Biri Tall ate fee ae 1 Sees bhara Lee oes Be pM ml fms ffm PO bea i Figure 7 8 Edit Label dialog box 4 When you are finished click OK The image information is updated Nn Save the image to save the updated im
295. selection from the Tissue Properties drop down list Choose the tissue type most representative of the area of interest Muscle is a good choice for a generic tissue type The software automatically sets the internal medium index of refraction based on the selection in the Tissue Properties list Analyze tab Properties tab Results tab Tool Palette Tool Palette Tool Palette gt Image Adjust gt Corrections Filtering gt Image Information Analyze Properties Results gt ROI Tools Sequence Mr saaeeeesd J900 Ske Planar Spectral Imaging Tissue Source Select Filters Analyze Properties Results Analyze Properties Results ROL Depth imm Total Flux iphotisy 154 0 189 1 14e10 1 5969 Imageset lt lt No active ROI selected gt gt Aad stoi 700 200 ss Plot Linear Fit Plot Intensity Save Results ROI List none Analyze eee gt Surface Topography gt Point Source Fitting gt BLIT 30 Reconstruction gt GADLIT 3D Reconstruction Figure 10 4 Planar spectral imaging tools 6 Make a selection from the Source Spectrum drop down list Firefly in this example 7 Click Analyze in the Analyze tab The Results tab displays the computed average depth mm and total flux photon sec of the luminescent point source in the specified ROI s For more details on the results see page 96 Table 10 1 Planar spectral imaging tools Analyze tab Sequence Name of sequence used for the a
296. shes the Spectral signatures of different fluorescent or luminescent reporters when more than one reporter is used in the same animal model y Caliper a p Chapter 2 Overview of Imaging amp Image Analysis 13 ifeSchences Additional tools are available in the menu bar click Tools on the menu bar Kote Description Page Well Plate Analyzes images of known serial dilutions of luminescent cells or 179 Quantification fluorescent dye molecules and generates a quantification database The software uses the quantification database to determine the number of cells in a DLIT source or the number of cells or dye molecules in a FLIT source Image Overlay Displays multiple luminescent or fluorescent images on one 91 photograph Colorize Renders luminescence or fluorescence data in color enabling you to 93 see both intensity and spectral information in a single view The tool provides a useful way to visualize multiple probes or scale probe Signals that are not in the visible range Transillumination Generates an overview image for each filter pair that includes the data 90 Overview for from all of the transillumination locations The overview image can be lt name gt _SEQ analyzed using the tools in the Tool Palette Image Math A method for mathematically combining two images add multiply or 133 Subtract Use image math to remove autofluorescence from a fluorescent image 14 Living Image Software User s Manual This page
297. stogram PCA Biplot The PCA biplot is a visualization tool for principal component analysis It shows a simultaneous display of n observations pixels and p variables wavelengths on a two dimensional diagram g TLT 0040406164750 SEO C sequence ew Spectra Unmined Images Mi Congenarnt l oo VA Compana 2 Figure 11 11 PCA biplot 112 Living Image Software User s Manual This page intentionally blank YP Caliper feSchences 1 2 Reconstructing a 3D Surface Generating a Surface oaoa a a a 114 Manening SUITES s ereke EE KEE ew RE we 118 Export or Import a Surface 2 ee a a 118 A surface is a 3D reconstruction of the animal surface topography derived from structured light images The Living Image software requires a surface to perform some types of analyses Figure 12 1 You can e Save a surface and use it for any of the analyses shown below e Export a surface for viewing in other 3D viewer applications e Import a surface For more details on how the software generates a surface see page 283 Generate a surface for Point Source Fitting DLIT Analysis GADLIT Analysis FLIT Analysis Estimates the optical 3D reconstruction 3D reconstruction 3D reconstruction properties of tissue the of luminescent of luminescent of fluorescent location and power of a sources page 143 sources page 149 sources displayed point source or the as voxels fluorescent yield of page 180 fluorophores page 121
298. structing a 3D Surface 117 P TLT20050624122348_5E0Q Sequence View 3D View ae Subject Height 23 2 nf Perspective View name Figure 12 5 Surface perspective view Bottom Front Back Left Right Figure 12 6 Alternate views of the surface 118 Living Image Software User s Manual 12 2 Managing Surfaces After the surface is saved it can be shared by the Point Source Fitting DLIT or FLIT tools Tool Palette gt Image Adjust gt ROI Tools gt Planar Spectral Imaging Surface Topography Surface Reconstruction Object Nude Mouse w I Orientation Bi Surface Smoothing Level Restore Seiya Re Surface name Name SURFOCE 1 gt Point Source Fitting gt BLIT 3D Reconstruction gt GABLIT 30 Reconstruction gt Spectral Unmixing Figure 12 7 Tool palette Surface topography tools Item Description Name Name of the selected surface Delete Removes the selected surface from the system Load Opens the selected surface Save Saves a surface to the selected name Overwrite Saves the surface and overwrites the previous surface results 12 3 Export or Import a Surface A surface can be shared with other users or viewed in other 3D viewer applications 1 Load a surface 2 Select File Export or Import gt 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 12 2 Importing a su
299. stum from the Plot drop down list to Ft display the selected spectrum tum calb This is required for DLIT Select a tissue or organ from tk drop down list Select a luminescent source spectrum Wavelength rra gt GADLIT 3D Reconstruction Figure H 4 DLIT 3D reconstruction tools Properties tab Y Caliper a Appendix H DLIT amp FLIT Reconstruction of Sources 287 Table H 1 Source spectra Bacteria Bacterial luciferase CB Green Click beetle green luciferase CB Red Click beetle red luciferase Firefly Firefly luciferase hRenilla Sea pansy Renilla reniformis luciferase XPM 2 LED LED in the XPM 2 mouse phantom NOTE The firefly luciferase spectrum is dependent on temperature and pH The data provided are valid only for measurements performed at 37 C and at pH 7 0 7 5 Selecting other temperature and pH conditions for a specific experiment requires the use of the associated spectral curve for the spectral analysis For more information about pH and temperature dependence of the luciferase spectrum please contact Caliper Life Sciences technical support You can view tissue optical property values Mefe Ws Ma in the Tissue Properties drop down list The tissue properties are plotted as a function of wavelength Select the tissue or organ most representative of the source location Muscle is a good choice for general reconstructions in vivo NOTE Default tissue optical properties
300. surements from the source center of mass to the surface Viewing Coordinates 1 In the Coronal Sagittal or Transaxial windowpane click a location in the reconstruction slice The coordinates mm of the position are displayed The coordinates are updated when you press and hold the mouse button while you drag the cursor e Coronal plane Displays the x y coordinates e Sagittal plane Displays the y z coordinates e Transaxial plane Displays the x z coordinates Figure 15 23 Viewing y z coordinates in the sagittal plane Displaying Slices Through a Reconstruction 1 Click a location on a source Alternately click the ce toolbar button draw a box around a source then click Center of mass in the 3D Source tools 2 Click the g toolbar button The Coronal Sagittal and Transaxial windowpanes show a slice through the surface taken by the associated plane 162 Living Image Software User s Manual 3D view Sequence View t elas me MEE ML H i Source Intensity Perspective Figure 15 24 Planes cutting a reconstruction 3 To move a plane put the mouse cursor over a line in the coronal sagittal or transaxial windowpane When the cursor becomes a ji or arrow drag the line The view is updated in the windowpanes as you move the line i j i Y Caliper Chapter 15 3D Reconstruction of Sources 163 15 6 Displaying Luminescent amp Fluorescent Sources on One
301. t images and f 2 or f 4 is recommended for brighter luminescent or fluorescent images Racy POCUS y Figure C 2 Lens f stop positions Left lens wide open at f 1 right lens closed down at f 8 Image Exposure Time The image exposure time also affects sensitivity The number of photons collected is directly proportional to the image exposure time For example an image acquired over a two minute exposure contains twice as many detected photons as an image acquired over a one minute exposure Longer exposure times are usually beneficial when imaging very dim samples However this may not always be true because some types of background dark charge in particular increase with exposure time For more details on backgrounds see Appendix E page 255 An IVIS Imaging System has extremely low background that enables exposures of up to 30 minutes However animal anesthesia issues and luciferin kinetics limit practical exposure times for in vivo imaging to 5 10 minutes Field of View FOV C 2 Binning The FOV indirectly affects sensitivity Changing the FOV without changing the binning or the f stop does not significantly affect sensitivity However CCD pixels are effectively smaller at a smaller FOV higher magnification so that higher levels of binning can be applied without loss of spatial resolution For example an image acquired at binning 4 and FOV 20 cm has the same spatial resolution as an image acquired at binning 8 and FOV 10 c
302. t gives confidence to the results values Large deviations from a straight line could indicate possible issues with the dilution series or errors when entering sample dilutions Table 14 1 Quantification results Item Description Excitation nm The excitation and emission filter wavelengths for the image Excitation and Emission filters will be specified for fluorescent images and the Open filter for Emission will be specified for bioluminescent images Emission nm 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 y Caliper Per ie Chapter 14 3D Quantification Database 137 14 3 Managing Quantification Results The quantification results can be saved with the image sequence and as a calibration database that is made available in the DLIT or FLIT 3D reconstruction tools in the Properties tab When you define the properties for performing a 3D reconstruction and a calibration database is specified the 3D reconstruction results will be displayed in ca
303. t spots on this black polystyrene well plate illustrate the specular reflection of the illumination source Imaging parameters GFP filter set Fluorescence level Low Binning 8 FOV 15 f 1 Exp 4sec Black polystyrene microplates are recommended for in vitro fluorescent measurements Figure F 9 and Figure F 10 show that the black polystyrene microplate emits the smallest inherent fluorescent signal while the white polystyrene microplate emits the largest signal The clear polystyrene microplate has an autofluorescent signal that is slightly higher than that of the black microplate but it is still low enough that this type of microplate may be used Control cells are always recommended in any experiment to assess the autofluorescence of the native cell Miscellaneous Material Autofluorescence It is recommended that you place a black Lexan sheet Caliper part no 60104 on the imaging stage to prevent illumination reflections and to help keep the stage clean The black paper recommended for luminescent imaging Swathmore Artagain Black 9 x12 Caliper part no 445 109 has a measurable autofluorescent signal particularly with the Cy5 5 filter set Figure F 11 shows a fluorescent image of a sheet of black Lexan on the sample stage as seen through a GFP filter set The image includes optical autofluorescence light leakage and low level autofluorescence from inside the IVIS System imaging chamber The ring like structure is a typica
304. t the low setting is approximately 18 that of the high setting Quartz Halogen Lamps with Dichroic Reflectors IR CubOT was EJV Lamp 3400 K 1 i KE Lamp 3200 Relative Spectral Radiance bt fs m i 400 bO BOO 00 1200 1400 6160061800 Wavelength nanometers Figure F 2 Relative spectral radiance output for the quartz halogen lamp with dichroic reflector The lamp output is delivered to the excitation filter wheel assembly located at the back of the VIS Imaging System Figure F 3 Light from the input fiber optic bundle passes through a collimating lens followed by a 25 mm diameter excitation filter The IVIS Imaging System provides a 12 position excitation filter wheel allowing you to select from up to 11 fluorescent filters five filters on older systems A light block is provided in one filter slot for use during luminescent imaging to prevent external light from entering the imaging chamber The Living Image software manages the motor control of the excitation filter wheel Fused Silica Collimating Fiber Optic Bundle Lenses Input Fiber Optic Bundle Excitation Motor Filter Figure F 3 Excitation filter wheel cross section Following the excitation filter a second lens focuses light into a 0 25 inch fused silica fiber optic bundle inside the imaging chamber Fused silica fibers core and clad unlike ordinary glass fibers prevent the generation of autofluorescence The fused silica fiber bundle
305. tains information about the transport of photons through the tissue and the effects of the tissue air boundary By using a planar boundary approximation the Green s function can be calculated analytically as a solution to the diffusion equation Having an analytic expression for G allows Equation 1 to be computed very rapidly H 4 Determining the Best Approximate Solution to the Linear System Once the Green s functions Gj are known the goal is to solve Equation 1 for the source strength S in each voxel The DLIT algorithm attempts to minimize x Equation 2 while requiring that the source strength in each voxel is positive Equation 3 2 1 2 X Fg Pj Wys 2 i S 20 3 A Non Negative Least Squares algorithm is used to find the approximate solution which minimizes In order to reduce the number of variables in the problem the code only uses surface elements with signal above a certain threshold minimum radiance and only keeps the voxels that contribute significantly to these surface elements H 5 Source amp Tissue Properties DLIT analysis of spectrally filtered images requires knowledge of the spectral dependence of luminescent light emission Table H 1 shows the factory set source spectra provided by the software The source spectra is not an input to the 3D reconstruction of fluorescent sources Tool Palette kroos dS Analyze Properties Results Tissue Properties Choose Source Spectrum 7 7Pe
306. te system manual Conventions Used In the Manual Convention Example Menu commands are bolded TO open image data select File Open Dataset on the main bar Toolbar button names are bolded TO open image data click the Open Dataset button l Numbered steps explain how to carry out a procedure 1 To start the Living Image software click the Fz icon on the desktop ie Document names are italicized Note information Caution information Important information Living Image Software User s Guide A note presents pertinent details on a topic or Note Notes may also appear in this format CAUTION A caution note warns you that your actions may have nonreversible consequences or may cause loss of data OL ae ALERT Important information advises you of actions that are essential to the correct performance of the instrument or software feSciences a Y Caliper Chapter 1 Welcome 3 Living Image Help There are several ways to obtain help on the software features e To view a tooltip about a button function put the mouse cursor over the button e To view a brief description about an item in the user interface click the Kf toolbar button then click the item e Press F1 or select Help User Guide on the menu bar to display the Living Image Software User s Manual pdf 1 3 Contacting Caliper Technical Support If you need technical support please contact Caliper at
307. ted Filter Range The wavelength range of the luminescent images in the sequence The two Sliders determine the lower and upper end of the filter range Only the parts of the image that are within the selected wavelength range are colorized By default the entire filter range is selected Color Camera VIS Regular camera setup that mainly renders color in the visible range It is similar to the color response of a commercial digital camera NIR fluorophores appear dark red to invisible using the VIS camera setup NIR A special camera setup that extends the color response into the near infrared range Near infrared fluorophores appear red to purple using the NIR camera setup Log Scale If this option is chosen the dynamic range of the brightness in the image is compressed using a log scale This improves the visibility of dark areas in the image Real Color If this option is chosen the colors are rendered using the wavelengths that directly correspond to the camera setup For example GFP appears green using real color rendering If this option is not chosen the original wavelength range of the image is modified to include the entire visible wavelength range of the camera setup This helps improve the color contrast 7 10 Exporting or Printing Images The Image Layout window Figure 7 25 provides a convenient way to e Annotate and export an image for example bmp e Print an image e Copy an image to the system clipboard 1 To
308. ted results are copied to the system clipboard To copy all results 1 In the Results tab right click the results table and select Copy All from the shortcut menu that appears All of the results table is copied to the system clipboard YY Caliper ifeSchences 13 7 Point Source Fitting Tools Chapter 13 Point Source Fitting 127 If the image sequence does not include a structure light image the point source firing tools do not appear in the Tool Palette Tool Palette Analyze Params Sequence fi Teieseerfefecess Soo Tissue Aurai Select Single Image Source Image Excl Block Block Block Block Block Properties Source Gree Erw L 60 ao 600 620 640 gt BLIT 3D Reconstruction gt GABLIT 3D Reconstruction gt 3D Tools gt Spectral Unmixing MinRadiance 6 14e 05 1 546 06 4 36e 06 6 61e 06 a 22e U6 Results The Analyze tab shows the active image sequence Select an image for analysis Figure 13 8 Point Source Fitting tools Analyze tab Table 13 1 Analyze tab Item Description Image EXWL EmWL M inRadiance X Y Image number in the active sequence Excitation wavelength Emission wavelength Minimum surface radiance used for model fitting x and y coordinates of the bottom illumination source 128 Living Image Software User s Manual Tool Palette gt Image Adjust gt ROI Tools gt Planar Spectral Imaging gt Surface To
309. tem clipboard 158 Living Image Software User s Manual To export results 1 In the results tab right click the results table and select Export Results from the shortcut menu that appears 2 In the dialog box that appears choose a folder for the results enter a file name and click Save The exported results are saved in csv file format 15 4 Checking the Reconstruction Quality To check the quality of a 3D reconstruction it is useful to compare the measured and simulated photon density plots The photon density is closely related to the measured radiance Photon density is the steady state measure of the number of photons in a cubic millimeter Light sources inside the tissue contribute to photon density in other portions of the tissue The reconstruction algorithms first convert the luminescent image of surface radiance to photon density just below the animal surface because this is what can be observed Then the algorithm solves for point source locations inside the tissue which would produce the observed photon density near the surface To view photon density maps 1 After the reconstruction is finished or results are loaded click Photon Density Maps in the Results tab The photon density maps for all wavelengths are displayed Figure 15 19 2 To rotate the surface and view it from a different angle move the thumb wheel to the left or right Use the thumb wheel to rotate the surfaces a Ot w A OP une cons w C
310. ternately select the data and click Open File types 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 ice Got Be E Choose a fibe to oper Look ic C Caper Lie Scrences a 0 cp FE ay Caer ele Inst Hes Liig ump ree Data Figure 7 3 Opening data from the toolbar or menu bar Y Caliper feSciences Chapter 7 Working With Data 75 7 2 About the Image Window amp Tool Palette An image image sequence or kinetic data set is displayed in an image window Multiple image windows can be open at the same time ie iit tes ae UU t A g Petco aie to ml TROTA O L 0 Tes mm em aa E a m a e ec i ___ Double click an image in the sequence to view it in a Separate image window Figure 7 4 Image windows sequence view and single image The options available in the image window depend on the type of active image data Table 7 2 Image window Item Description Units Select the measurement units for the image display from this drop down list The available units depend on the type of image data For more details on measurement units see page 251 Info Click to display or hide the image label The image label includes information you enter in the Edit Image Labels dialog box See page 22 and other information automatically recorded by the software ps Opens a
311. the label and then click to release the label at the new location Figure 8 21 LT A0050674145507_006 liia Emr se Earp harir r F line P ROI Propertins KIR ROL FEEN L wE Edit the ROI BROT stirol ints label here Megi Kurii TETADMSOS SF at ka Rul ae ye e Linck Persihions POI 2 5 324e 05 met pei 119 9500 Yei pix 138 06447 Fergie deg 0 0000 Cider Sain Mi 1177 Boy m JAR Height pls LL teeter Lines ine E a ure coor M C Figure 8 21 Move or edit the ROI label To edit the ROI label 1 Double click the ROI of interest Alternately right click the ROI Ctrl click for Macintosh users and select Properties on the shortcut menu 2 In the ROI Properties box that appears edit the name in the ROI Label box and click Done Figure 8 21 Saving ROIs The software automatically saves ROIs with an image The ROI measurements are saved to the AnalyzedClickInfo txt file associated with the image ROIs are saved per user and can be applied to other sequences To save ROls to the system 1 In the Name drop down list confirm the default name or enter a new name for the ROI s 78 Living Image Software User s Manual Deleting ROIs Tool Palette gt Image Adjust a gt Corrections Filtering m Image Information f ROT Tools oo w Apply to Sequence Preview Use Bkg Offset Replace ROIs Restore Defaults gt Planar Spectral Imaging
312. tion in the image window a m r 2 Photo Brightness Click and move the slider left or right to adjust the brightness of an Adjustment image displayed in overlay or photograph mode Alternatively enter a brightness value Gamma Click and move the slider left or right to adjust the gamma of 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 M ax The maximum pixel intensity associated with the color scale for an image Pixels greater than the maximum value are displayed in the maximum color 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 M anual 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 seq
313. tivity window i hiriaren finn ite Shed moro m teartarg OF Bonar cater l ebdrani te ida aa i ME prieeae jeg OFF Piste pEr Figure 3 6 Activity window 20 Living Image Software User s Manual Software Help 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 _2009 csv located at C Program Files Caliper Life Sciences Living Image Usage There are several ways to obtain help on the software features e To view a tooltip about a button function put the mouse cursor over the button e To view a brief description about an item in the user interface click the k toolbar button then click the item Press F1 or select Help User Guide on the menu bar to display the Living Image Software User s Manual pdf ee E Y Caliper 4 Luminescent or Fluorescent Imaging Acquire a Luminescent Image a a a ee ee a 5 Acquire a Fluorescent Image With Epi lllumination 8 Acquire a Fluorescent Image With Transillumination 4 11 Acquire a Sequence Using the Imaging Wizard 4 14 Manually Set Up an Image Sequence 2 00 2 eee ees 21 Manually Saving Image Data 2 eee a 25 Exporting Image Data 434646 te ee i eee eee ER ew eH ERS ew HSS 26 The IVIS Imaging System is ready to acquire images after the system i
314. tral data describing the autofluorescent subtraction technique using a background filter The graph shows the excitation and emission spectrum of PKH26 and the autofluorescent excitation spectrum of mouse tissue Also included are the spectral passbands for the blue shifted background filter DsRed Bko the primary excitation filter DsRed and the emission filter used with this dye Y Caliper ifeSciences Appendix G Planar Spectral Imaging Planar Spectral Imaging Theory 2 2 0 eee eee ee ee es 275 OMICS Frores g 6448 BG wee ECE EEE KEHOE HEH ew ee eS 277 Luciferase Spectrum 1 we ee irisada ridera d 277 An Example of Planar Spectral Imaging 5 502 884 278 Optimizing the Precision of Planar Spectral Analysis 281 The unique spectral signatures of the luciferase emission spectrum and the optical properties of tissue enable the Living Image software to determine the depth and intensity of light sources inside a living animal The planar spectral imaging algorithm relies on a diffusion model of light propagation in tissue and assumes a point source of light embedded in a flat surface approximation of the mouse The algorithm is designed to provide a fast and robust method to approximate source location and brightness The analysis requires two or more single view images at wavelengths between 560 and 660 nm The Diffuse Tomography DLIT algorithm is a more complete and accurate model It anal
315. ts 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 gha 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 160 Copies or pastes voxels or a source surface so that DLIT or GADLIT and FLIT reconstructions can be displayed on one surface For more details see page 163 Enables you to save the 3D view to a graphic file for example jpg Data Preview Window The Data Preview window shows the image data that the algorithm automatically selects for reconstruction In special cases you may want to include or exclude particular data from this default selection There are two ways to do this e Change the Threshold value Applying a Threshold value excludes or includes some pixels from the reconstruction The software computes the minimum and maximum pixel values of an image based on an histogram of pixel intensities If Threshold 0 5 then pixels with intensity less than 0 5 of the maximum intensity value are excluded from the reconstruction The Threshold can be edited for individual images The Data Preview window is updated when you change the Threshold value e Region selection Use the p
316. ty in a user specified area of the image that is considered background Note Using this type of ROI is optional If the animal has significant autoluminescence or autofluorescence you can determine a background corrected signal in a measurement ROI by subtracting an average background ROI from a measurement ROI e Manual e Free draw Circle or square Subject ROI Identifies a subject animal in an image Note Using this type of ROI is optional It provides a convenient way to automatically associate link a measurement and average background ROI for background corrected ROI measurements when there is significant autoluminescence or autofluorescence e Manual e Automatic e Free draw Square 8 2 Quick Guide Drawing Measurement ROls on an Image or Sequence These steps provide a quick guide on how to apply a measurement ROI to an image or image sequence For more details about measurement ROIs see page 60 1 Open an image or sequence and click ROI Tools in the Tool Palette 2 In the ROI tools select Measurement ROI from the Type drop down list 3 Click the Contour button _ For an image or sequence select Auto All from the drop down list For kinetic data select Kinetic ROI The software automatically draws measurement ROIs on all images The ROI label shows the total intensity in the ROI If you are working with a sequence open an image to view the intensity label dei oe al Pretec o
317. uence editor Figure 4 22 Nn d Repeat step 4 to step 5 for each image in the sequence To set a time delay between each acquisition enter a time minutes in the Delay box in the sequence editor OO To save the sequence setup information xsq a In the sequence editor click the Save button bed feSciences y Caliper p i Chapter 4 Luminescent or Fluorescent Imaging 23 b In the dialog box that appears select a destination directory enter a file name and click Save Reuse option a IS Aa Eim Snar Fire aa Ereaga ey ri IESE C iep epia Sega Tiimi Hape Bobes Haih Pema brag foo Enigeen Imaan u POY Hade i Gn Fiai iii DJ Yes c nai 2 DI wee Mein Bic Emy han x E gt i DI ae Heda Bisi GEF Fin C 1 55 4 DI Hairs Bii 20 im Es i m g Aiba Hair Bici bJ hio z 1 Eien Babu 6 DE Hein Bich ey hn E iie Pes See Serene T bert ei S m ep Lasien z r Oo A 7 PEPE pa i Fi Pom hae atiect hone S Troecshaw MM a j Co enter at Segrar m Lacerta x panava GE upate Ch imean 2 ace Figure 4 22 Control panel and sequence editor with image settings Each row in the sequence editor specifies the acquisition parameters for one image in the sequence Table 4 5 Sequence Editor Item Description Starts the Imaging Wizard Imaging Wizard T Displays a dialog box that enables you to select and open a sequence setup xsq sequenceinfo txt or
318. uence is active Color Table Rainbow Click the drop down arrow to select a color table for the image data For more details on color tables see Pseudocolor Images page 249 Reverse Choose this option to reverse the selected color table Logarithmic Scale Choose this option to apply a log scale to the relationship between numerical data and the color range in the color table A log scale increases the range of meaningful numerical data that can be displayed ifeSchences Caliper Y p Chapter 7 Working With Data 81 Magnifying or Panning in the Image Window To incrementally zoom in or out on an image Click the 4 or button Alternately right click the image and select Zoom In or Zoom Out on the shortcut menu To magnify a selected area in an image 1 Click the button Alternatively right click the image and select Area Zoom on the shortcut menu 2 When the pointer becomes a draw a rectangle around the area that you want to magnify The selected area is magnified when you release the mouse button To reset the magnification remove magnification Click the 4 button Alternately right click the image and select Reset Zoom on the shortcut menu To pan the image window Panning helps you view different areas of a magnified image If the image has not been magnified you cannot pan the image 1 Click the edi button 2 When the pointer becomes a click and hold the pointer whil
319. und on the propagation of light through tissue see Diffusion Model of Light Propagation Through Tissue page 276 In such cases high levels of binning may be appropriate up to 10 or 16 depending on the CCD of the IVIS Imaging System If signal levels are high enough that sensitivity is not an issue then it is better to image at a lower binning level two or four in order to maintain a higher degree of spatial resolution For application specific questions regarding the appropriate binning level please contact Caliper Corporation The IVIS System Control panel provides several binning options The actual binning numbers associated with these settings depends on the CCD chip and type of image Table C 1 These choices should satisfy most user needs However if you want to manually control binning you can specify Manual Binning in the Living Image Tools Preference Camera Settings box 248 Living Image Software User s Manual Table C 1 Binning settings EEV ROPER SiTe Andor Medium Lumin Bin 8 Bin 5 Bin 4 Bin 4 Small high resolution Lumin Bin 4 Bin 2 Bin 2 Bin 2 Large high sensitivity Lumin Bin 16 Bin 10 Bin 8 Bin 8 Medium Photo Bin 4 Bin 2 Bin 2 Bin 2 Small high resolution Photo Bin 2 Bin 1 Bin 1 Bin 1 You can also apply soft binning after an image is acquired Conceptually soft binning is the same as hardware binning groups of pixels are summed and a smaller lower resolution image is produced However in soft bin
320. urements LM Fitting Click to begin the point source fitting Tool Palette Analyze Params Properties Results Select other starting values for the optical properties here Figure 13 10 Point Source Fitting tools Properties tab 130 Living Image Software User s Manual Table 13 3 Properties tab Item Description Tissue Properties M ake a Selection from this drop down list to specify starting values for the parameters other than the defaults Note Selecting a tissue property automatically updates MuaEm MusEm MuaEx and MusEx in the Params tab Internal medium index The internal medium index of the tissue selected from the Tissue of refraction Properties drop down list You can also enter a user specified value Tool Palette Analyze Params Properties Results Error Estimation Starting Chiz 1 24e6 09 Ending Chi 2 2 446 07 Point Source Fitting Results Parameters Fitted walu Mua Erm fcr 5 500 5 660 13 570 0 030 x location of the source fmm 13 405 Y location of the source mm 6 8697 z location of the source fmm 8 522 Photon Density Maps W Export Results Save Results Mame LMIFIT_1 BLIT 3D Reconstruction gt GADBLIT 3D Reconstruction gt 3D Tools gt Spectral Unmixing Figure 13 11 Point Source Fitting tools Results tab Table 13 4 Results tab Item Description Error Estimation Starting ChiSqure Error between the measured and simulated photo
321. use a click and drag operation to move the tag then f click the mouse to set the tag location A line between the pixel and the tag identifies the location associated with the tag 7 7 Creating a Transillumination Overview The transillumination overview tool combines the images of a FLIT sequence a fluorescence sequence acquired in transillumination mode into a single image All of the individual fluorescent signals are stacked over one photograph and the intensity is summed One overview is created per filter pair If two filter pairs were used during acquisition then two overview images will be created All transillumination locations are displayed simultaneously a tool tip displays the transillumination position when you mouse over a transillumination point An overview image is displayed in photon units and can be analyzed using the tools in the Tool Palette NOTE If you choose the Raster Scan option in the Transillumination Setup box the overview image is automatically generated For more details see page 28 1 Load a sequence that was acquired in fluorescence transillumination mode 2 Select Tools Transillumination Overview for lt name gt _SEQ on the menu bar The overview appears YP Caliper il Per ne Chapter 7 Working With Data 91 Overview WIO0HO70H142 707 004 Te Units Radiant Display Overlay F Trarelumination Location Into j i frerd Filorescance K C335 eae es SP bea 1S
322. uto 1 from the drop down list The create tool appears on the image y Caliper F Ber ie Chapter 8 Working With ROI Tools 63 A IL A00047414550 7 00 SEE a Figure 8 10 ROI create tool 3 Use the ring amp to move the create tool to the area where you want to draw the ROI then click Create The ROI appears on the image and the ROI label displays the intensity signal 4 To draw another ROI on the image repeat step 2 to step 3 For information on how to save ROIs see page 77 Drawing an ROI Using the Free Draw Method 1 Open an image and in the ROI tools select the type of ROI that you want to draw from the Type drop down list 2 Click an ROI shape button Circle O Square amp or Contour and select Free Draw from the drop down list In this example the Contour shape was selected for the free draw method The ROI shapes that are available depend on the type of ROI selected 3 If you selected O or 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 64 Living Image Software User s Manual EN H Gh Ei k Liat aun App be all e 1L770050624145507_005 fx E Too Pakette gt liige Adjat 5 F i l e i i ali A eet hoe det 1 jars Pets Wi Piare WH I Pa IROI i 1 14e l
323. voxels Adjust the voxel display by moving the Threshold slider ian Drawing styles for the source surface see Display Source Surface Shading styles for the source surface See Display Source Surface Click to open the color palette from which you can select a display color for the source surface LJ Opacity Adjusts the source surface opacity 168 Living Image Software User s Manual Table 15 10 Source tools continued Item Description Display Voxels Choose this option to display the sources reconstructed using DLIT Threshold Choose this option to apply a minimum threshold intensity to the voxel DLIT ELIT only splay Gradation Use this slider to set a threshold for the percentage voxel intensity above DLIT FLIT only which voxels are opaque and below which voxels will gradually face to transparent The percentage voxel intensity is the percentage relative to the maximum intensity Voxel size The 3D grid spacing size for interpolation of the reconstructed source Smoothing The smoothing box filter size Display voxels as The voxel display mode cubes spheres points or texture Color Table Color scheme for voxel display Reverse Choose this option to apply the colors of the selected color table in reverse order to the photon density scale For example the BlackRed color table represents the source intensity photons sec from low to high using a color sc
324. w appears Figure 5 6 12 If this is the first image of the session you are prompted to enable the autosave function 30 Living Image Software User s Manual Living Image 4 0 This can be changed anytime From the Acquisition menu a Go you want to enable auto saving of acquired data For this session a J Figure 5 4 Autosave prompt 13 To enable autosave click Yes in the prompt and choose a folder in the dialog box that appears If autosave is enabled all images acquired during the session are automatically saved to a user selected folder You can choose a different folder at any time select Acquisition Auto Save on the menu bar The acquisition proceeds and the control panel warns you that X ray radiation is being produced Figure 5 5 The image window and Tool Palette appear when acquisition is completed Figure 5 6 14 If you need to stop the acquisition click Stop in the control panel mT E WIS Acquisition Control Panel Frito Fort ate Fiber Frevdon Fira Er in O a X ray energy warning Click to stop the acquisition iret Figure 5 5 Control panel during X ray image acquisition al C li Y a Iper Chapter 5 X Ray Imaging 31 feSciences i ik a ae E S ar T en i Lith Causa m hani bea al Likes Tari gie Cea te Dori Faer mage adria Gighirata Tool palette _ Check the image min and os max in the color scale to a determine
325. w data view information about the data and load the data To start the browser 1 Click the Browse button 3 Alternately select File gt Browse on the menu bar 2 In the dialog box that appears select the folder of interest and click OK The Living Image Browser appears Figure 7 1 72 Living Image Software User s Manual E E Osta from Chaney a E his aes i Bets netic ia D Taos g E D aTi ee _teep a BTANA SE Cet tae Eini Ahe Reraton tisi inm iam wap aE B E migr aE vai MRE wr aa aiii p Ea a T a T ne my Bi bee eer Sarre sjun EET EO zarran Sens a M reremen p ee a nur i hanana Coreg aes Piei after Life beriani Data fh ADi aei Living Image browser Figure 7 1 Opening the Living Image Browser zs tea Se Jep ona a E NOTE The next time you start the Living Image software and open the Browse For Folder box the software automatically returns to the last folder visited The Living Image browser displays the selected data along with the user ID label information and camera configuration information ME image SEQ image sequence DEM kinetic data or image exported as DICOM file 3 To expand a sequence click the sign next to E 4 To view data properties right click an item and select Properties on the shortcut menu 5 To open data do one of the following e Double click the data row e Right click the data name and select Load on the shortcut m
326. whether the 4 Signal of interest is above ont the noise level and below CCD saturation Figure 5 6 Overlay fluorescent image on X ray image in the image window It may be necessary to use the Image Adjust tools to optimize the overlay display Use the Opacity control to adjust the appearance of the overlay For more details on adjusting image appearance see page 79 Table 5 2 Image window Item Description Units Select the measurement units for the image display from this drop down list The available units depend on the type of image data For more details on measurement units see page 247 Display Select the image type for example X ray that you want to display from this drop down list For more details on the different types of image displays see Table 2 2 page 8 Note If the acquisition included more than two imaging modes for example luminescent X ray and photograph additional drop down lists appear so you can conveniently choose any two images to overlay Dipi eriy ka E eeren mi EE Ay x Info Click to display or hide the image label information ps Opens a dialog box that enables you to export the active view as a graphic file 32 Living Image Software User s Manual Table 5 2 Image window continued Item Description wy Takes a Snapshot that is displayed with the data in the Living Image Browser Pike Hie oa fees Drp HALS ER IH Enpe pi Speed a imt ee nd eh irer apn Living Im
327. yed in the pseudocolor image You can change the appearance of the image data without affecting the underlying numeric pixel values For example you apply a different color table to the data or adjust the range of numeric values associated with the color table Measurements that quantify pixel data produce the same results independent of the appearance of the pseudocolor display A pseudocolor image can be converted to an RGB color code and saved as an RGB image The RGB image looks like a pseudocolor image but does not include the numerical information derived from the light detected in each pixel Therefore the amount of light in an RGB image cannot be quantified In the overlay display mode the pseudocolor luminescent or fluorescent image is displayed on the associated grayscale photographic image Figure D 1 Pixels in the luminescent or fluorescent image that are less than the minimum color table setting are not displayed As a result the lowest intensity color in the table is transparent and this enables you to view the underlying photographic image in regions where the luminescent light emission is low Y Caliper a p Appendix D Image Data Display amp Measurement 251 ifeSchences D 2 Quantifying Image Data The Living Image software can quantify and display scientific image data several types of measurements Table D 1 Data display units Data Display Description Recommended For Counts An uncalibrated measurement of the Im
328. you do not manually stop the pump the pump continues to run after acquisition ends until the specified infusion volume is reached NOTE The information in the infusion pump control panel is saved in the click info file During acquisition if you start infusion then manually stop and restart the infusion only the last actual start and stop is saved to the click info file not the start stop settings in the panel ey N O Y Caliper Appendix IVIS Syringe Injection System 291 1 2 Tracking Infusion in the Maximum vs Time Graph During kinetic acquisition the blue shaded region in the Max vs Time graph indicates the infusion period During acquisition if you start infusion then manually stop and restart infusion only the last actual start and stop is recorded in the Maximum vs Time graph The graph stops recording infusion when acquisition stops even though the pump may not be stopped ti sm i a a a a a8 qp i phd i T i mi Pr k f LT l Time secs 3 Time secs 25 45 Figure 1 4 Tracking infusion 1 3 Closing the Infusion Pump Control Panel 1 Close the kinetic control panel 2 Click Acquisition gt Infusion Pump Setup on the menu bar The check mark is removed and the panel closes 292 Living Image Software User s Manual This page intentionally blank Y Caliper ifeSchences Appendix J Menu Commands Tool Bar amp
329. yzes images of surface light emission to produce a three dimensional 3D reconstruction of the luminescent light sources in a subject For more details on DLIT analysis see Chapter 14 page 171 and Appendix H page 283 G 1 Planar Spectral Imaging Theory An image acquired on an IVIS Imaging System is a diffuse projection on the surface of the animal from the luminescent sources located deeper inside Information about the depth of the luminescent cells can help quantify the source brightness and provide information on the location of the cells The Living Image software uses spectroscopic information from a single view image to estimate the depth of the luminescent cells The method takes advantage of the fact that firefly luciferase bioluminescence is emitted from 500 to 700 nm a region of the spectrum where there are major contrasts in tissue optical properties Figure G 1 In this portion of the spectrum tissue absorption drops off dramatically between 500 580 nm green yellow wavelengths and 600 750 nm red wavelengths due mainly to the presence hemoglobin As a result the luminescent signal observed on the surface of the animal is dependent on both the wavelength and the thickness of the tissue through which it travels The depth and absolute photon flux of a single point source can be determined from two or more images acquired at different wavelengths using relatively simple analytical expressions derived from the diffusion model o

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Living Image® Software - NYU Langone Medical Center