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IVIS Imaging System Discovery at the Speed of Light TM Jack Yang Product Manager 2010.09.02 1 Genomics, Proteomics and Functional Study 2 Non-invasive in vivo imaging Continued monitor Low variation 3R (Replacement, Reduction, Refinement) Drug Discovry Today (2002) 7:125 3 Total solution for in vivo imaging IVIS Optical Imaging Quantum FX Micro CT Vevo HR Ultrasound Cellvizio In vivo microscopy IVIS Optical Imaging Quantum FX Micro CT Vevo HR Ultrasound Cellvizio In vivo microscopy Multi-type of in vivo imaging microPET microCT microSPECT Optical Imaging Ultrasound Optical bioluminescence Optical fluorescence microMRI GENE & DEVELOPMENT 2003 17:545 6 Advantage of optical imaging 1.High throughput 2.No radiation 3.Easy to operation 4.Dual function (fluorescence and bioluminescence) 5.Functional analysis 7 Basic Method 8 Reporter Molecules Luciferase, Fluorescent Protein Fluorescent dyes Quantum dots ATP and O2 required for luciferase Label Cells Label Bacteria Label Genes 9 Imaging Principle Imaging system Autofluorescence Autoluminescence Luminescent or Fluorescent source 10 An “Imaging” Window of Opportunity Fluorophore Excitation 11 Spectra Emission Luciferase Spectra Demonstration of Light Transmission Tissue is not transparent - Light absorbance depends on wavelength Doyle et al, 2004 Nature Medicine The source of light luciferin Bioluminescence +ATP, Mg2+, O2 Luciferase Renilla Light + Oxyluciferin +AMP, PPi, CO2 Excitation Wavelength GFP Quantum dot excited Emission state Wavelength NIR dye Fluorescence ground state 13 Bioluminescence and fluorescence Bioluminescence Just light out No background Fluorescence Light in and Light out Autofluorescence interference 14 Quantified images Luminance detection Fluorescence detection NATURE MEDICINE (2004) 10: 1257 絕對定量功能 Absolute Quantification reference Journal of Biomedical Optics, (2001) ® Living Image Software Region of Interest (ROI) ROI 4=2.344x105 Absolute Calibrated Data in: photons s-1 cm-2 sr -1 17 Calibrated Physical Units • Living Image® automatically compensates for device settings: Exposure time, f/stop, Binning, and Field of View. • Calibrated units are Photons per Second, representing the flux radiating omni-directionally from a user defined region. 2 sec exposure, f/stop 1, Small binning ~5000 counts peak 10 sec exposure, f/stop 1, Small binning ~25000 counts peak 2.82 x 108 photons/sec 2.82 x 108 photons/sec BIOLUMINESCENCE SENSITIVITY Cooled (-90C) camera with large CCD chip area for high sensitivity light detection Precision filters Most sensitive system available Resolves multiple bioluminescent reporters Detects down to 3 cells in vivo: Low f-number and large diameter lens Gives high sensitivity and uniform light collection Rabinovich et al, PNAS,2008 In vivo imaging of s.c. implanted T cells transduced with optimized firefly luciferase. 19 19 FLUORESCENCE SENSITIVITY In Vivo Quantitation of Doxorubicin Using Spectral Unmixing Non-unmixed image Non-unmixed image Unmixed image 64 128 64 32 32 16 8 4.E-06 R² = 0.9671 Fluorescence Dox (ng) 256 Unmixed image 2.E-06 0.E+00 0 64 128 192 256 Doxorubicin (ng) Spectral Unmixing enables detection of as little as 8 ng of Doxorubicin in vivo IVIS enables direct correlation between Doxorubicin signal and concentration both in vitro and in vivo Simple to use Unmixing software significantly improves signal to background ratio at low concentrations 20 20 Longitudinal Non-Invasive Monitoring of Disease (and Treatment) 21 Untreated 9L-Glioma (Rat) -2 0 4 10 14 x 103 12 10 8 6 4 2 Source: Brian Ross, Ph.D., University of Michigan Medical Center 22 Longitudinal Assessment of Tumor Growth and Response to Anti-Angiogenic Therapy 105 U87 glioma cells implanted 14 days prior Control Anti-angiogenesis inhibitors (week 4) Szentirmai et al, 2006 23 Tumor Induced Damage Monitoring Cell Model: MDA-MB-231 (breast cancer cell line) J Clinic Invest Massauge group, 2005 24 Brain Tumor Therapy • Intra-cranial glioma model • • U87MG-Luc Cells Anti-angiogenesis therapy • Test a non-viral delivery system (Transposon-sleep beauty, SB) • Test anti-tumor effects of statin-AE and sFlt-1 Mol Therapy 2005 Cell Trafficking, Survival and Engraftment Patterns In Vivo 26 Cell Transplantation and Trafficking Patterns Stem Cell Foci Formation and Hematopoiesis Transplantation of 250 Luc+ HSC into Lethally Irradiated Hosts Cao et al, Stem Cells,2004 27 Ultrasound Guided Delivery of Image-guided cardiac cell delivery Bioluminescent Cardiomyoblasts 心成肌細胞 Image-guided cardiac cell delivery: Sprague-Dawley rats (n = 11) were imaged using high-resolution ultrasound, and stably transfected cardiomyoblasts (plasmid-CMV-firefly luciferase) were injected into the anterior cardiac wall under ultrasound guidance (parasternal long-axis view), using a 28-gauge needle. After injection, bioluminescence imaging was performed using a cooled charged-coupled camera. The signal detected was positively correlated with the amount of cells transplanted (R(2) = 0.94, P = 0.03). Optical signal could be followed longitudinally using bioluminescence imaging. Rodriguez-Porcel & Gambhir et al, 2005 Stroke Repairing Yellew: Infarcted site White: Injection site Neuron Progenitor Cell Tracing Stroke Kim et al, 2004 29 Neurotropic Viral Infection • Viral Model: Sindbis Virus (Alphavirus) • 103 CFU virus inoculated at right hind foot J Virol Cook & Griffin, 2003 30 CUBICIN (Cubist Pharmaceuticals) Cubicin (Daptomycin) is an FDA approved drug for infections caused by Staphylococcus aureus Mouse Infection Model: Peritonitis Model Mice inoculated with lethal dose of a bioluminescent S. aureus (MRSA) Spread of infection monitored in real time using an IVIS system. 5 mice per treatment imaged hourly and non- invasively – no need to sac at each time point. A 3 log difference in viable bacteria demonstrated within 4 hours. Fewer animals used per group and per time point Mortin et al., Antimicrobial Agents and Chemotherapy, May 2007, p. 1787–1794 in vivo fluorescence imaging of subcutaneous U87MG glioblastoma RGD-Cy5.5 Fluorescence probe Cancer research (2004) 64: 8009 Genetic Components of Disease Expression, Pathways and Therapy 33 TRANSGENICS (LPTA) Vascular Endothelial Cell Growth Factor Receptor VEGFR2 Pr luc Day 14 CHROMOSOME VEGF induction of VEGFR2 promoter causes luciferase expression VEGF - secreted during angiogenesis VEGFR2 - surface receptor for VEGF Transgenic VEGFR2-luc Embryos in a Non- Tg Mother 34 Reduction of VEGFR2 Expression During Development 1 Week 2 Weeks 60 4 Weeks 6 Weeks 5000 1000 1000 4000 800 800 3000 600 600 400 400 200 200 55 50 x103 45 40 2000 35 1000 30 Signal Intensity 100,000 Signal Intensity 10,000 Signal Intensity 200 Zhang et al. (Xenogen), 2004 Signal Intensity <100 Induction of VEGFR-2 Expression During Wound Healing Day 0 Day 4 Day 6 Day 11 Zhang et al. (Xenogen), 2004 Day 18 Day 24 Monitoring Angiogenesis During Tumor Development Tumor growth and development imaged by GFP fluorescence Angiogenesis imaged by VEGFR2-luc expressing bioluminescence VEGFR2-luc KI mice implanted with 106 LL/2-GFP tumor cells and imaged at day18 NFĸB Induction by LPS Control 0 4 7 24 Zhang, Xenogen & Rune Blomhoff, Oslo Peyer’s patches LPS Effect of Dexamethasone and EGCG on iNOS Expression Zhang et al. (Xenogen), 2003 Protein-protein Interaction Rapamycin-Induced Association of FRB and FKBP PNAS Piwnica-Worms group, 2004 Bioware® Luciferase Cell Lines Luciferase expression tumor cell lines 1. Breast Cancer: MCF-7, MDA-MB 2. Colorectal cancer: HT-29, LoVo-6 3. Lung cancer: A549 4. Melanoma: B16 5. Prostate cancer : PC3M, LNCaP 6. Cervical: HeLa 7. Brain tumor: U-87 8. Renal Tumor: ACHN Microorganisms Gram Negative Bacteria 1. Escherichia coli 2. Hemophilus influenzae 3. Pseudomonas aeruginosa 4. Samonella typhimurium 5. Shigella dysenteriae 6. Yersinia enterocolitica Gram Positive Bacteria 1. Listeria monocytogenes 2. Staphylococcus aureus 3. Streptococcus pneumoniae Fungi Candida albicans Infectious Disease Application Drug Discovery LPTA® Luciferase Animal Model Light Producing Transgenic Animal Animal Model Application Ho-1 Oxidative Stress Cyp450 Drug Metabolism iNOS Sepsis Epx Asthma IL-2 Innate immunity Kap Androgen VEGF Angiogenesis Ins2 Diabetes Kdr Wound healing Stem Cell/Adoptive Transfer Tracking stem cells Engraftment Differentiation Transplant biology Donor cells from LPTAs β-actin-luc GAPDH-luc Reporter specific (e.g., Rip-luc) Tracking immune response Lymphocytes Eosinophils Neutrophils IVIS® Spectrum Imaging System - Hardware • Customized for in vivo imaging • Highly sensitive camera with a large dynamic range Standard Images are Composed of Two Images Photographic + Luminescent = Overlay Alignment Light Projector • Allows rapid and reproducible positioning of subjects. • Size changes with Field of View setting Field-of-View (typical) FOV D: 21.5 cm 1.5x FOV B: 6.5 cm 5x FOV C: 12.5 cm 2.5x FOV A: 4.0 cm 8.7x Living Image Control Panel Controls Sensitivity Imaging Wizard Select for assistance in setting up bioluminescence or fluorescence sequences • Good labeling practices are necessary for effective data browsing Image Labeling Image Cataloging & Browsing Tools User defined information Quantification • Tool palette for adjusting scale/opacity etc. • Region of interest (ROI) tools to measure surface intensities Regions of Interest Tools ROI shapes available: • Square • Circle • Contour • Grid ROI’s can be created: • Manually • Automatically • Free Draw Important to be consistent with ROI selections Measurement Table • Measurement table displays information about each ROI • Table is user configurable and can be exported to a spreadsheet Calibrated Physical Units • Living Image® automatically compensates for device settings: Exposure time, f/stop, Binning, and Field of View. • Calibrated units are Photons per Second, representing the flux radiating omni-directionally from a user defined region. 2 sec exposure, f/stop 1, Small binning ~5000 counts peak 10 sec exposure, f/stop 1, Small binning ~25000 counts peak 2.82 x 108 photons/sec 2.82 x 108 photons/sec Fluorescence Fluorophore Excitation Wavelength excited state ground state Emission Wavelength IVIS® Fluorescence Imaging •18 Emission filters (computer controlled) Emission filter wheel •10 Excitation filters (computer controlled) •150 Watt Tungsten/Halogen lamp Excitation filter wheel (computer controlled intensity) •Low Auto Fluorescence optics and fibers Trans-illumination Light source Optical switch Excitation and Emission Filters 100 Transmission % 80 10 Excitation filters – 35 nm bandwidth 710 - 760 nm 60 810 - 875 nm 40 20 0 400 615 - 665 nm 440 480 695 - 770 nm 520 560 600 640 680 720 Wavelength (nm) 500 - 550 nm 445 - 490 nm 575 - 650 nm 515 - 575 nm 18 Emission filters – 20 nm bandwidth 760 What is Spectral Unmixing? • Calculates concentrations of different fluorescent components • Requires images acquired at multiple wavelengths to perform the spectral analysis • Emission or excitation scan • Quantitative and qualitative results In vitro Spectral Unmixing Example: Dyes in a Dish XF 680 Top and bottom: XF680 and XF750 mixture: 1:1 Left middle: XF680 only Right middle: XF750 only 1 x1014 molecules per spot Ex640, Em700-820nm Composite XF 750 Image Overlay Capabilities Bioluminescent Tumor DHE Image Overlay 3D Tomography (Luminescence) Spectral scans with Emission filters allows for absolute quantification and source localization 3D Tomography (Fluorescence) Spatial scans with trans-illumination light source allows for absolute quantification and source localization In vivo dual modality tomography Fluorescence Imaging Tomography - FLIT Bioluminescence Imaging Tomography - DLIT Mouse Left Tumor Depth [mm] Mouse Right Tumor Depth [mm] DLIT 5.8 2.9 FLIT -- 3.1 Utilize Well Plate Quantification to Determine Cell Number or pMol of Reporter • Dilute your cells or dye and image • Select Well Plate Quantification from Tools menu • Enter cell number or concentration per well • Save as a library • Choose library when reconstructing Utilize Well Plate Quantification to Determine Cell Number or pMol of Reporter 2D 3D 5.44e5 cells vs. 1.253e8 photons/sec 2.93e4 cells vs. 1.79e7 photons/sec Automatic Mouse Atlas Registration in LI4.0 Unregistered Coregistered Total solution for in vivo imaging IVIS Optical Imaging Quantum FX Micro CT Vevo HR Ultrasound Cellvizio In vivo microscopy Quantum Micro CT High resolution CT for longitudinal studies New, flat-panel detector technology and a micro-focus X-ray tube provides high resolution imaging for small animals with radiation doses low enough to enable longitudinal studies. High throughput and ease of use supports stand-alone CT use or as a complement to IVIS Spectrum. Whole animal imaging 200mm dynamic scan for 3D images High resolution 20µ pixel for whole animal imaging 10µ pixel for specimen imaging High speed imaging 17 second scan time delivers low radiation Volume rendering Dedicated processing of 3D images for 45 second reconstruction Co-registration with IVIS Spectrum Cone X-ray beam for high speed imaging This CT exposes a cone-shaped X-ray beam on the 2D detector. The revolving arm rotates 360° around the sample to acquire full 3D data. This provides 17-second high-speed imaging. High-resolution micro-focused X-ray source The 90 kV (maximum X-ray output) and 5 micrometer-focused X-ray source create highresolution images that can be studied at the 20 micrometer Quantum Micro CT High resolution CT for longitudinal studies The small format instrument is designed to fit the space constraints and high throughput workflow of a small-animal imaging laboratory. Like all IVIS instruments the system is easy for biologists to use without the need for a dedicated operator Fluoroscopy mode Real-time imaging enables precise animal positioning Purpose-built for small animal imaging Integrated animal handling Compatible with XGI-8 anesthesia Animal transfer bed for IVIS Spectrum High speed imaging 17 second scan for high throughput High speed analysis 3D processing board for 45 second results Intelligent co-registration workflow to facilitate 3D fluorescence and bioluminescence analysis using IVIS Spectrum Quantum Micro CT High resolution CT for longitudinal studies Imaging modalities: • Fluoroscopy allow real-time imaging and positioning • 17 second scan for data collection • 45 second processing time for 3D reconstruction • Display tools for soft tissue and bone contrast Quantum Micro CT High resolution CT for longitudinal studies Low X-ray dosage supports longitudinal studies Day 0 Day 7 Day 10 Day 14 High resolution images show bone growth below implant 3D reconstruction Data courtesy of Matsumoto Dental University Graduate School of Oral Medicine Quantum Micro CT High resolution CT for longitudinal studies Soft tissue imaging and tumor measurement using contrast agent and 3D reconstruction. Quantum Micro CT High resolution CT for longitudinal studies Soft tissue imaging and tumor measurement using contrast agent and 3D reconstruction. Animal tumor model system • 2 x 106 PSN-1 cells • Balb/c mice • Imaged day 15 Imaging parameters • 17 second can time • 20µ resolution • 5~8 mGy dosage Tumor volume results: • CT volume 17.78 mm3 • Excised volume 15.82 mm3 • Multiple time points measured Analytical software options: • Metabolic mapping to show body fat • 3D alignment to compare bone growth pattern • Bone mineral density analysis tools Quantum Micro CT High resolution CT for longitudinal studies Micro-Ultrasound: Clarity Conventional clinical ultrasound (human fetus) 20cm 3 - 15 MHz 3cm Melon Coffee Bean Micro-ultrasound (mouse fetus) 30 – 80 MHz High-frequency = High-resolution How Does Micro-Ultrasound Work? What Can the Vevo® Do For Me? Image Guided Injections No need for invasive surgery Cardiotoxicity studies For cardiovascular evaluations Gene therapy Gene delivery through sonoporation Tumor dynamics: see it all MicroMarkerTM Microbubbles Phospholipid Layer 2-3 μm C2F10 / N2 Core Microbubble Applications Targeted MicroMarkerTM Microbubbles Cell Surface Marker VEGFR-2 / PCAM / VCAM / PSelectin / etc. User-selected antibody Biotin Streptavidin Olive KP, et al. Science 2009;324(5933):1400-1 Lacefield, et al. Cancer Res 2007;67(6):2830-9 Shaked Y, et al. Science 2006;313(5794):1785-7 The Cellvizio® LAB Solution What is the Cellvizio solution? • In vivo confocal microscope • Unique miniaturized fibered microprobes • Sophisticated real-time and postacquisition software The World’s Smallest Microscope Laser Scanning Unit • 488 or 660 nm laser source • High-speed confocal microscope • Single pixel detector (APD) Blue LSU 488 Microprobes™ • Tens of thousands of fiber optics bundled together • Ultra miniaturized optical objective [ 500 - 700 ] Red LSU 660 [ 680 - 1000 ] • 300μm diameter Cells and Vessels Imaged Deep within the Brain Capillaries in the cortex. IV FITC-Albumin FOV ø 300 µ m GFP olfactory glomerulus. FOV ø 300 µ m Neurons of a transgenic mouse expressing Clomeleon. FOV 300 x 420 µ m CamKinase-GFP neurons in the striatum. FOV 160 x 120 µ m GFP stem cells in living mouse brain. FOV 300 x 420 µ m Reaching multiple regions in the Deep Brain Hippocampus Alzheimer Accumbens Addictions VTA Addictions Substantia Nigra Parkinson Conscious in vivo in situ imaging in the hippocampus Mobile implant: S300 microprobe hippocampus U. Maskos (Institut Pasteur) et al, SFN 2008 Neuron Precursor Migration “In vivo imaging of migrating neurons in the mammalian forebrain" Sensory Neurons Glomeruli Periglomerular cell Mitral cell Proliferation Granule cell Migration RMS OB Time Lapse Movie of Neuron precursors in the olfactory bulb labeled with CellTracker Green. - FOV 400 x 280 µ m Integration Lledo and Saghatelyan (2005) Trends in neurosciences Vol.28 No.5 Calcium Imaging Electrical stimulation (thalamus) Chemical Stimulation (bicuculine, Hippocampus) Calcium Imaging Changes in intracellular calcium in the hippocampus in response to bicuculline-induced epileptiform activity. A) Time course of the fluorescence intensity of Oregon Green BAPTA-1 in cells shown in (B) (C) and (D) corresponding electroencephalogram (EEG) Vincent et al. (2006). EMBO 7(11), 1154–1161 Peripheral Nerve Experimental Set-Up • Label: Nerve labeling or use of • • • transgenic mice with fluorescent nervous tissue Access: External or minimal incision (2mm) of the skin to access nervous tissue (nerve, sensory fibers, neuromuscular junction) Record: Real time acquisition of tissue condition. Scan the Microprobe along nerve or across tissue Analyze: E.g. Measure the length of outgrowth or nerve fiber density Thy1-YFP - Saphenous nerve D0, H0: Intact Saphenous Nerve D0, H1: Crushed Nerve D1 post crush: Degenerated Nerve D4 post crush: Regenerated Nerve Thy1-YFP mouse FOV 400 x 280 m Longitudinal imaging of neuroregeneration… with single fiber resolution Courtesy of Igor Charvet and Paolo Meda, Medical Research Center, Geneva, Switzerland • • • • Angiogenesis – Experimental Set-Up Label: IV injection of FITCDextran (500 kDa) or AngioSense Access: Minimal incision of the skin to access subcutaneous tumor Record: Acquisition of tumoral vascularization sequences Analyze: Analysis of the images to measure the functional capillary density Courtesy of Descartes Image, O. Clément, Necker School of Medicine, Paris, France Tumor Angiogenesis Longitudinal analysis of tumor vascularization in PyMT mice 9 weeks Vessel Vessel Vessel Vessel Length: 1847 Area: 23428 Volume: 289900 Diameter: 12.69 +/- 6.09 10 weeks Vessel Vessel Vessel Vessel Length: 3347 Area: 51830 Volume: 801566 Diameter: 14.89 +/- 6.98 11 weeks Vessel Vessel Vessel Vessel Length: 3729 Area: 65460 Volume: 1212684 Diameter: 16.80 +/- 9.16 Increased vascular density during tumor development in abdominal mammary gland Imaging mouse colonic tumors with Cellvizio® LAB Cellvizio®LAB ProFlex™ MiniZ microprobe (0.94mm) Vascular architecture Normal mucosa Adenoma Thanks for your attention