Living Image® Software User’s Manual Version 4.0 © 2002-2010 Caliper Corporation. All rights reserved. PN 126545 Rev 01 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: Contents 1 Welcome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.1 What’s New In Living Image 4.0 Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1.2 About This Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1.3 Contacting Caliper Technical Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 2 Overview of Imaging & Image Analysis . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.1 Workflow Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5 2.2 Imaging Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 2.3 About Image Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10 2.4 Image Display & Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11 3 Getting Started . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 3.1 Starting the Living Image Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 3.2 Initializing the IVIS Imaging System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 3.3 Checking the System Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17 3.4 About the IVIS Acquisition Control Panel & Auto Exposure Feature . . . . . . . . . . . . . . . 18 3.5 Tracking System& User Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19 4 Luminescent or Fluorescent Imaging . . . . . . . . . . . . . . . . . . . . . . . . 21 4.1 Acquire a Luminescent Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21 4.2 Acquire a Fluorescent Image With Epi-Illumination . . . . . . . . . . . . . . . . . . . . . . . . 24 4.3 Acquire a Fluorescent Image With Transillumination . . . . . . . . . . . . . . . . . . . . . . . . .28 4.4 Acquire a Sequence Using the Imaging Wizard . . . . . . . . . . . . . . . . . . . . . . . . . . . .32 4.5 Manually Set Up an Image Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38 4.6 High Resolution Imaging With the Optical Zoom Lens Attachment . . . . . . . . . . . . . . . . .43 4.7 Manually Saving Image Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44 4.8 Exporting Image Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 5 X-Ray Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 5.1 Acquire an X-Ray Image . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47 5.2 Acquire an Image Sequence That Includes X-Ray Images . . . . . . . . . . . . . . . . . . . . . 52 5.3 High Resolution Imaging With the Optical Zoom Lens Attachment . . . . . . . . . . . . . . . . 59 5.4 Measuring Relative Density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61 6 Kinetic Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 6.1 Kinetic Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 6.2 High Resolution Imaging With the Optical Zoom Lens Attachment . . . . . . . . . . . . . . . . 70 6.3 Viewing & Editing Data in the Kinetic Acquisition Window . . . . . . . . . . . . . . . . . . . . 72 6.4 Saving Kinetic Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74 6.5 Playing Kinetic Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 7 Working With Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 7.1 Opening Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 7.2 About the Image Window & Tool Palette . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 ii Living Image Software User’s Manual 7.3 Viewing Image Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 7.4 Adjusting Image Appearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89 7.5 Correcting Image Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 7.6 Viewing Intensity Data & Making Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . .93 7.7 Creating a Transillumination Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100 7.8 Overlaying Multiple Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 7.9 Rendering Intensity Data in Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 7.10 Exporting or Printing Images . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 7.11 Editing an Image Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 7.12 Creating an Image Sequence from Individual Images . . . . . . . . . . . . . . . . . . . . . . . 108 8 Working With ROI Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 8.1 About ROIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 8.2 Quick Guide: Drawing Measurement ROIs on an Image or Sequence . . . . . . . . . . . . . . . 112 8.3 ROI Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114 8.4 Measurement ROIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116 8.5 Subject ROIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 8.6 Measuring Background-Corrected Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 8.7 Kinetic ROIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122 8.8 Managing ROIs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125 8.9 Managing the ROI Measurements Table . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136 9 Image Math . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 9.1 Creating a New Image Using Image Math . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 9.2 Subtracting Tissue Autofluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146 10 Planar Spectral Image Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 10.1 Image Sequence Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 10.2 Planar Spectral Image Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149 10.3 Viewing Graphical Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152 10.4 Managing Planar Spectral Imaging Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154 11 Spectral Unmixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 11.1 Image Sequence Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 11.2 Performing Spectral Unmixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 11.3 Spectra Window . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162 11.4 Spectral Unmixing Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164 11.5 Spectral Unmixing Options . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165 12 Reconstructing a 3D Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 12.1 Generating a Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170 12.2 Managing Surfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 12.3 Export or Import a Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 Contents iii 13 Point Source Fitting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 13.1 Image Sequence Requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 13.2 Displaying the Point Source Fitting Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 13.3 Point Source Fitting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178 13.4 Checking the Point Source Fitting Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180 13.5 Exporting Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181 13.6 Managing Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182 13.7 Point Source Fitting Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 14 3D Quantification Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 14.1 Preparing & Imaging the Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187 14.2 Creating a Quantification Database . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188 14.3 Managing Quantification Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 15 3D Reconstruction of Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193 15.1 Reconstructing Luminescent Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195 15.2 Reconstructing Fluorescent Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 15.3 3D Reconstruction Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204 15.4 Checking the Reconstruction Quality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207 15.5 Measuring Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 15.6 Displaying Luminescent & Fluorescent Sources on One Surface . . . . . . . . . . . . . . . . . 212 15.7 Exporting a 3D Scene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 15.8 3D Tools Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 15.9 3D Tools - Surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 15.10 3D Tools - Source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216 15.11 3D Tools - Registration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 15.12 3D Tools - Animate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 15.13 DLIT/FLIT Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 Appendix A IVIS Acquisition Control Panel . . . . . . . . . . . . . . . . . . . . . . . 231 A.1 Control Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 A.2 Manually Setting the Focus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 236 Appendix B Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237 B.1 General Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238 B.2 User Preferences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 B.3 Acquisition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241 B.4 Theme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242 B.5 Tissue Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245 Appendix C Detection Sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 C.1 CCD Detection Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247 C.2 Binning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248 C.3 Smoothing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250 iv Living Image Software User’s Manual Appendix D Image Data Display & Measurement . . . . . . . . . . . . . . . . . . . . 251 D.1 Image Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251 D.2 Quantifying Image Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253 D.3 Flat Fielding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 D.4 Cosmic Ray Corrections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256 Appendix E Luminescent Background Sources & Corrections . . . . . . . . . . . . 257 E.1 Electronic Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257 E.2 Background Light On the Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258 E.3 Background Light From the Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 260 Appendix F Fluorescent Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 F.1 Description and Theory of Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263 F.2 Filter Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 F.3 Working with Fluorescent Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267 F.4 Image Data Display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 268 F.5 Fluorescent Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 269 F.6 Subtracting Instrument Fluorescent Background . . . . . . . . . . . . . . . . . . . . . . . . . . 274 F.7 Adaptive Background Subtraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 F.8 Subtracting Tissue Autofluorescence Using Background Filters . . . . . . . . . . . . . . . . . . 275 Appendix G Planar Spectral Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . 277 G.1 Planar Spectral Imaging Theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277 G.2 Optical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 G.3 Luciferase Spectrum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 279 G.4 An Example of Planar Spectral Imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280 G.5 Optimizing the Precision of Planar Spectral Analysis . . . . . . . . . . . . . . . . . . . . . . . . 283 Appendix H DLIT & FLIT Reconstruction of Sources . . . . . . . . . . . . . . . . . . 285 H.1 Determining Surface Topography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285 H.2 Converting Light Emission to a Photon Density Map . . . . . . . . . . . . . . . . . . . . . . . 286 H.3 Defining the Linear Relationship Between a Source and Photon Density or NTF Efficiency . . . 287 H.4 Determining the Best Approximate Solution to the Linear System . . . . . . . . . . . . . . . . . 288 H.5 Source & Tissue Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 288 Appendix I IVIS Syringe Injection System . . . . . . . . . . . . . . . . . . . . . . . . 291 I.1 Controlling the Infusion Pump . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 291 I.2 Tracking Infusion in the Maximum vs. Time Graph . . . . . . . . . . . . . . . . . . . . . . . . . 293 I.3 Closing the Infusion Pump Control Panel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293 Appendix J Menu Commands, Tool Bar, & Shortcuts . . . . . . . . . . . . . . . . . 295 Welcome 1 What’s New In Living Image 4.0 Software . . . . . . . . . . . . . . . . . . . . . . 1 About This Manual . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Contacting Caliper Technical Support . . . . . . . . . . . . . . . . . . . . . . . . 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 47 with a luminescence, fluorescence, or photographic image. Determine relative density in an x-ray image using ROIs 61 Fluorescence Imaging NTF Efficiency image available for fluorescent acquisition with transillumination 28 Kinetic Imaging Acquire ventral kinetic data using the Ventral Imaging Chamber 65 Close Up/High Resolution Imaging Optical Zoom Lens attachment enables close up/high resolution imaging (luminescent, 43 fluorescent, X-ray, or kinetic imaging) on the IVIS Lumina, IVIS Lumina XR, and IVIS Kinetic 59 imaging systems 70 3D Surfaces Select a particular region of the subject to reconstruct 170 Export a 3D surface or a user-selected portion of the surface in DICOM format. Multi-frame 175 export option supports 3D CT reconstruction in third party software. Expanded export and import capabilities for reconstructed 3D surfaces (XMH, DXF, STL, IV) 175 3D Sources Living Image software automatically selects the data to include in the reconstruction based 199 on the Threshold% value Select particular data for the reconstruction 199 Quantify the number of cells in a luminescent source or dye molecules in a fluorescent source 197 Display luminescent or fluorescent sources on one surface 212 Export source voxels or source surfaces 212 Spectral Unmixing A spectral unmixing wizard for image sequence setup 159 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 162 to the plot (from the Living Image source library or the spectrum associated with an ROI) View user-selected probes in the composite view 161 Apply signal thresholds 159 ROIs Automatically draw ROIs on all images in the Sequence View window. 112 2 Living Image Software User’s Manual New Feature or Updated Tool Page General Activity log tracks session duration for each user and IVIS statistics (for example, the 238 number and type of acquired images, fluorescent lamp usage, X-ray tube usage). 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. NOTE 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 appropriate 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 . Numbered steps explain how to carry 1. To start the Living Image software, click the icon on out a procedure. the desktop. Document names are italicized. Living Image Software User’s Guide Note information NOTE A note presents pertinent details on a topic. or Note: Notes may also appear in this format. Caution information ! CAUTION CAUTION! A caution note warns you that your actions may have nonreversible consequences or may cause loss of data. Important information ! IMPORTANT ALERT! Important information advises you of actions that are essential to the correct performance of the instrument or software. Chapter 1 | Welcome 3 Living Image Help There are several ways to obtain help on the software features: • To view a tooltip about a button function, put the mouse cursor over the button. • To view a brief description about an item in the user interface, click the 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). 1.3 Contacting Caliper Technical Support If you need technical support, please contact Caliper at: Telephone: 1.877.LabChip (877.522.2447) Toll Free in the United States 1.508.435.9761 E-mail: 4 Living Image Software User’s Manual [This page intentionally blank.]