Living Image 4.4 Software Release Notes New Features and Improvements Contents

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1 Contents 1. Purpose New Features Cherenkov Imaging Mode Updates for IVIS Lumina Series III System Illumination Module Lumina III Filters Expanded Probe Database in the Imaging Wizard FLIT algorithm improvements D Data Visualization Improvements Longitudinal Study window Tool Palette reorganization and simplification DyCE Trans-illumination imaging Acquisition improvements Browser displays saturated images Perform Smoothing and Binning on a sequence Volume Slice Viewer access shortcuts D ROI enhancements Support for Mac OS X Support for high-speed I/O board with NI-DAQmx driver Improvements Known Issues in Living Image System Requirements Video Card Requirements... 13

2 1. Purpose This document provides a brief overview of new features and improvements in Living Image 4.4. This release serves as an update for Living Image version on IVIS Lumina, Kinetic, Lumina XR, Spectrum and Spectrum CT instruments. Major features include the introduction of the Cherenkov imaging mode in the Wizard, integration of the entire PerkinElmer catalogue of fluorescent agents, support for IVIS Lumina Series III, FLIT algorithm improvements, and tool palette simplification. Living Image 4.4 release also provides improvements to the existing software features and bug fixes. 2. New Features 2.1. Cherenkov Imaging Mode The Living Image 4.4 Imaging Wizard now offers a Cherenkov Imaging Mode which guides the user through optimal camera parameter settings for high signal-to-noise when imaging the Cherenkov light emitted from radionuclide-injected animal subjects. One can easily set up a bio-distribution scan of radiopharmaceuticals using the Cherenkov DyCE mode. The user can choose from a selection of radionuclides at acquisition time, so that the data may be decay-corrected if necessary at analysis time, such as for kinetic curves from DyCE analysis. (Note: The Cherenkov emission spectrum is the same for all radionuclides. One can choose Spectral Unmixing in Cherenkov mode to unmix radionuclides from other light-emitting probes of very different spectra, but one cannot spectrally unmix to distinguish Cherenkov signal between radionuclides). Figure 1: Imaging Wizard workflow for Cherenkov mode

3 2.2. Updates for IVIS Lumina Series III System Illumination Module The IVIS Lumina III imaging system contains a new 150W extended range tungsten halogen lamp. This new illumination module gives extended excitation power into the near infrared wavelengths. Figure 2 shows the power increase for the IVIS Lumina III systems at wavelengths greater than 700nm. This power increase will yield higher signal-to-noise ratios for fluorescent probes that are specifically designed to be excited at NIR wavelengths where the auto fluorescence from animal tissue is low. The new illumination module is also specifically designed to handle the extra heat caused by the transmission of additional IR light which includes using specially designed high temperature fiber optic cables. Figure 2: Comparison of the spectral output for the Lumina III and the Lumina II illumination modules Lumina III Filters The Lumina III has a newly designed 22 position excitation filter wheel which is filled with 19 narrow band excitation filters and one block for bioluminescent imaging. The excitation filters have a 20nm band pass and span the wavelength region from nm. The exciters were designed specifically to do spectral unmixing on the excitation side (Table 1). The emission filters were also redesigned to span the wavelength region of nm using only one filter wheel assembly thereby eliminating the need for the user to change emission filter wheels. The new set of seven emission filters have a 40nm band pass and hit all major emission peaks for the newly expanded probe database (see section 2.3).

4 Lumina LT Lumina III Excitation Filters Emission Filters Excitation Filters Emission Filters Center l Bandwidth Center l Bandwidth Center l Bandwidth Center l Bandwidth Table 1: Description of the fluorescent excitation and emission filters that are currently in the Lumina Series III instruments. Note: the Lumina LT has a different set of excitation and emission filters Expanded Probe Database in the Imaging Wizard Living Image now offers an expanded probe database used by the Imaging Wizard. Table 2 is an alphabetical list of the 99 fluorescent reporters that are currently in the software. The database is organized in a tree-like structure in order to simplify the selection of the desired probe (see Figure 3). Each probe is categorized as an agent, dye, protein or quantum dot. If the user is unsure where there probe is located there is also an alphabetical listing. If the probe being used is not located in the database, the user can input the peak excitation and emission wavelengths into the software in order to determine filter selection. Frequently used probes will be saved in the Recently Used list.

5 1. 2-DG BoneProbe IntegriSense QDot AlexaFluor CatB 680 FAST 52. IntegriSense ReninSense 680 FAST 3. AlexaFluor CatB 750 FAST 53. IntegriSense RJ-2-DG AlexaFluor CatK 680 FAST 54. irfp 79. Rodamine Red 5. AlexaFluor COX mcherry 80. Superhance AlexaFluor Cy3 56. MMPSense 645 FAST 81. tdtomato 7. AlexaFluor Cy5 57. MMPSense Texas Red 8. AlexaFluor Cy MMPSense 750 FAST 83. TLectinSense AlexaFluor Cy7 59. mplum 84. TransferiSense AlexaFluor DADO 60. Neutrophil Elastase 680 FAST 85. TurboFP AlexaFluor DiD 61. OsteoSense 680 EX 86. TurboFP AlexaFluor DiR 62. OsteoSense 750 EX 87. TurboGFP 13. AlexaFluor DsRed (RFP) 63. OsteoSense TurboRFP 14. AlexaFluor Doxorubicin 64. PKH TurboYFP 15. AlexaFluor egfp 65. ProSense Vivo Tag AminoSPARK FITC 66. ProSense 750 EX 91. VivoTag AminoSPARK FolateRSense ProSense 750 FAST 92. VivoTag AngioSense 680 EX 43. GastroSense PSA 750 FAST 93. VivoTag AngioSense 750 EX 44. Genhance Qdot VivoTag-S AngioSPARK Genhance Qdot VivoTrack 680 (dye) 21. AngioSPARK GFP 71. QDot XLCF Annexin Vivo HER2Sense QDot XLCF Bacterial Detection HypoxiSense QDot XLCF BacteriSense ICG 74. QDot YFP 25. BombesinRSense Integin QDot 705 Table 2: List of the expanded reporter library that has been incorporated into the Living Image 4.4 software Figure 3: Screenshot of the Imaging Wizard showing the organization of the tree-like structure for the expanded probe database.

6 2.4. FLIT algorithm improvements Artifacts caused by excitation beam coupling to tissue observed in FLIT reconstructions analyzed by LI4.3.1 and prior versions have been greatly reduced. The reduction of these artifacts allows for greatly improved interpretation of 3D fluorescence tomographic reconstructions D Data Visualization Improvements For CT volume, Living Image 4.4 offers improved contrast between tissues, higher dynamic range of the scene, and more realistic lighting on the tissue interfaces. For DLIT and FLIT 3D source data, voxels are now rendered with higher visual contrast, better depth rendering and improved spatial registration accuracy with CT volume data. Rendering settings for source voxels are optimized for provided color palettes and Jet is introduced as the new default color palette Longitudinal Study window The 3D voxel display minimum and maximum color scale values can be manually set for improved flexibility in data visualization. The maximum color scale value is limited to be 100 max(v i ), where i=1,..,n for N total voxels of intensity v i. The minimum color scale value is limited to be 0.

7 2.7. Tool Palette reorganization and simplification The Tool Palette has been reorganized for improved ease of use. The Image Information palette has been retired and its tools incorporated into the Image Adjust section, which now also contains Filtering tools. Additionally, when the Tool Palette is docked in the main application window, it automatically adjusts to display selected tools in full view DyCE Trans-illumination imaging DyCE image acquisition and analysis is now available for transillumination fluorescence imaging with single-point illumination, allowing for increased sensitivity of dynamic fluorescence imaging to deep sources. Fluorescence DyCE data acquired with transillumination excitation will be analyzed with DyCE in NTF Efficiency units by default. Users will have the choice to analyze the data in Radiant Efficiency units as well Acquisition improvements Living Image 4.4 software includes the following enhancements to the data acquisition workflow: Ability to close and save a sequence dataset while another sequence acquisition is in progress Trans-illumination Setup window restricts point selection to rectangle (applies to IVIS Spectrum and Spectrum CT) Mask Grid Points to Subject option is applied by default in Trans-illumination Setup Window (applies to IVIS Spectrum and Spectrum CT) A warning caption appears on image thumbnail if loading trans-illumination image data containing saturated pixels (applies to IVIS Spectrum and Spectrum CT) Imaging chamber door can be opened earlier during a CT scan while CT image reconstruction is in progress (applies to IVIS Spectrum CT) DyCE sequence setup limits max number of images to 200 DyCE time interval begins at the start of first luminescent exposure after the photograph Browser displays saturated images The Living Image Browser has been improved to indicate images with saturated pixels. A red icon will alert the user if optical data sequence contains any images with saturated pixels, those images are not recommended for analysis.

8 2.11. Perform Smoothing and Binning on a sequence Smoothing and Binning image operations can now be applied to the entire sequence, as opposed to previously applicable to individual image only. The tools are available in the Image Adjust tool palette for sequence and single image modes. Smoothing computes the average signal of a specified number of pixels (for example, 5 x 5) and replaces the original signal with the average signal. Binning specifies the number of pixels in the image data that are grouped together to form a larger pixel Volume Slice Viewer access shortcuts Within the 3D View tab, simply clicking on a 2D plane will now automatically switch the tool palette to the Slice tab to enable quicker adjustment of the grayscale ranges. Double-clicking the Coronal, Sagittal, or Transaxial window pane now automatically launches the Volume Slice Viewer. Then, switch to single slice view or choose to render thick slice, adjust slice thickness, scroll, and zoom in on individual slice features.

9 D ROI enhancements To simplify manipulation of an ROI in 3D space, Living Image 4.4 includes the following enhancements to the 3D ROI tool: ROI position Instead of defaulting the center of the image, new 3D ROIs are now placed at the intersection of slice planes. ROI Size - Larger default size for 3D ROI. ROI handles - Larger ROI handles for easier manipulation. ROI translational adjustment - Double-click the ROI in the perspective view to open 3D ROI Properties dialog box for finer adjustments. ROI angular adjustment - Angle parameters in the ROI Properties dialog box provide another way to rotate the 3D ROI in the x, y, or z plane. Measurement units for CT images - The units for thresholding a Spectrum CT image are now in HU, enabling easier comparison between animal and across time points Support for Mac OS X 10.8 Living Image 4.4 analysis module is compatible with the Mac OS X 10.8 (code-named Mountain Lion ). The Living Image 4.4 Installation and Licensing Guide describes the necessary steps to install and license Living Image application on the Mac OS X 10.8.

10 2.15. Support for high-speed I/O board with NI-DAQmx driver Living Image 4.4 provides transparent support for the National Instruments 6535-series high-speed I/O board for use on new or upgraded IVIS Spectrum systems. Systems running with this new board require the installation of both the NI-DAQmx software library (version or higher) and the Microsoft.NET Framework 4.0. Installation of NI-DAQmx does not require the removal of the older NIDAQ libraries. The Living Image software will automatically determine if the new software driver is present and which I/O driver to use. The new board must be installed in a PCI Express (PCIx) slot. The software will automatically determine its location in the computer s backplane. 3. Improvements The following table is a summary of the improvements. ID LI-5765 LI-5795 LI-5774 LI-2067 LI-5622 LI-5619 LI-5537 LI-5519 LI-5454 LI-5348 LI-5328 LI-5323 LI-5321 LI-5310 LI-5309 LI-5306 LI-5299 LI-5285 LI-5281 LI-5279 LI-5263 LI-5262 LI-5215 LI-5616 Summary New Large Animal imaging mode on Lumina XR Support for IVIS Spectrum LT instrument Limit Filter Pair mode in the Imaging Wizard to only one pair Allow binning/smoothing operation for spectral unmixing results Support for Lumina III with removable emission filter wheels Pre-defined filter selection table for Spectral Unmixing scan on the IVIS Spectrum Prompt user earlier to acquire new CT reference images Decay-correct data for Cerenkov data in Spectral Unmixing/DyCE Allow analysis of NTF Efficiency DyCE images Warn user against 3D reconstruction acquisition when FOV D is selected Support manual co-registration with Bruker MR Tool Palette scrolling improvement 'Info' tab is available in 3D View window Display unit and min/max for the color bar in Unmixing tab Brightness controls for "Image Cube" in Spectral Unmixing Improvements to volume masking in surface topography Improved usability in of histogram tool Display Em Gain in the Browser for kinetic data Ability to switch between auto and manual exposure in the DyCE Sequence Setup table Recognize DyCE dataset if partially loaded New volume color table 'Jet' for 3D reconstruction sources Improve accessibility to X-ray MIP image New default display settings in surface & CT for 3D dataset acquired on Spectrum CT Convert traditional NI-DAQ I/O to NI-DAQmx I/O

11 4. Known Issues in Living Image 4.4 Living Image 4.4 Software Release Notes 4.1 LI-5089 High Resolution CT scan can sometimes cause application crash or computer freeze In the case of a Living Image crash or computer freeze at the time of high resolution CT scan, please restart the computer. If the problem persists, power cycle the IVIS Spectrum CT. 4.2 LI-5206 Tool Palette disappears or 3D tools missing in the tool palette This issue may affect Spectrum CT acquisition computer after acquisition of a 3D sequence. If the tool palette is missing or loads an incomplete set of tools, please save, close and reload the dataset. Docking the tool palette will alleviate these symptoms. To dock the tool palette in the main window, drag the palette to the right of left size of the window and release, or choose the default option in User Preferences dialog. 4.3 LI-5771 Quantum/ Spectrum co-registration with carbon bed Registration of a Quantum uct image with the structured light surface from an IVIS Spectrum is facilitated by a hardware bed with a custom designed fiducial. In some cases, unexpected deflection in the hardware bed makes it algorithmically challenging to detect the fiducial. Until a complete software solution is implemented, a modified hardware bed will be available upon request D settings on computers with dual graphic cards If your computer (mostly laptops) is equipped with dual graphic cards, please follow the next figure to default the high-performance graphics card for the Living Image software. Otherwise, Living Image 3D viewer, especially with the 3D Multi-Modality tool, may not function correctly when running on low-end integrated graphics hardware. Here is an example of a laptop with both Intel integrated graphics and NVIDIA graphics. Open the NVIDIA control panel and manage 3D settings. In the program settings, add Living Image executable (livingimage.exe) to customize and then select the preferred graphics processor to High-performance NVIDIA processor.

12 5. System Requirements PC: Windows 7 32-bit / Windows XP * 32-bit 2GHz Core 2 Duo or higher processor recommended 4GB RAM * Please note that future revisions of Living Image will discontinue support of Windows XP on the analysis module Windows 7 64-bit 2GHz Quad Core (i5, i7) processors 8GB RAM recommended for IVIS Spectrum CT data analysis Mac: Mac OS X 10.6 to GHz Core 2 Duo or higher processor recommended 4GB RAM or higher recommended for IVIS Spectrum CT data analysis * for analysis module only; for 3D Multi-Modality support Mac OS X 10.6 to 10.7 is required on Mac computers equipped with ATI Radeon video card.

13 Note: Mac PowerPC platform is not supported in Living Image 4.4 Please note that support for Mac OS X 10.5 has been discontinued in Living Image Video Card Requirements 3D Multi-Modality tools require that the graphics processing unit (GPU) meet the minimum specifications shown below. If the appropriate license is not installed or the GPU does not meet these specifications, the 3D Multi-Modality tools will not appear in the tool palette. Minimum GPU specifications Specification OpenGL Version Requirement* OpenGL Extension Requirement* Graphics Card Memory: Description OpenGL 2.0 and above GL-EXT-Texture3D Recommended: 1GB (Dedicated) Consumer Graphics Cards (Desktop/ Mobile, Windows/Mac) Workstation Graphics Cards (Desktop/ Mobile, Windows/Mac) Supported: NVIDIA GeForce 8 Series and above (8, 9, 100, 200, 300 and 400 series) ATI Radeon HD 4000 Series and above (4000 and 5000 series) Recommended: Desktop - NVIDIA GeForce GT 240 and above Mobile - NVIDIA GeForce GT 230M and above Supported: NVIDIA Quadro NVS Series and Above (NVS & FX series) ATI FireGL V5600 and Above (FireGL, FirePro & CrossFire series) Recommended: Desktop - Quadro FX 1800 and above Mobile - Quadro FX 880M and above *If these specifications are not met, the 3D Multi-Modality tools will not appear in the tool palette.