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In Vivo Optical Imaging from the Whole Animal to the Cellular Level

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  • 1. In Vivo Optical Imaging from theWhole Animal to the CellularLevelAntonio SanchezAbhishek Trikha
  • 2. UVP: An Imaging CompanySmall Animal (in vivo, ex vivo) Endoscopy /Microscopy (in vivo, intravital) In vitro
  • 3. Publications Referencing in vivo Over Two Decades, 1990-2011 3000 2500Publications 2000 1500 1000 500 0 1990-1995 1996-2000 2000-2005 2005-2011
  • 4. In Vivo ImagingNon-destructiveRepeated experimentationLocalized process in time and space
  • 5. 6-8 week old mouseSubcutaneous injection Primary Fluorescent Area (mm ) 2HCT116 fibrosarcoma cancer cell 400Dual color (GFP nuclei, RFP cytoplasm) 300Weekly measurements 200 100 Regression 95% confidence r = .89, p < 0.05 0 0 2000 4000 6000 8000 3 Primary Tumor Volume (mm )
  • 6. Technical issues SolutionsAbsorbance Observe superficially Move to higher wavelengthScattering High intensity light source Increase exposure time High sensitivity cameraAutofluorescence Move to higher wavelength Optimize filter selectionMotion artifact High sensitivity camera Immobilize tissueDim signal High intensity light source Increase exposure time
  • 7. Hemoglobin is the major 2.2 area 1absorber in animal tissue intensity (A.U.) 1.7 area 2 1.2 0.7 0.2 450 500 550 600 650 wavelength (nm) Autofluorescence from endogenous molecules
  • 8. Use of Fluorescent Genetic Reporters Fast imaging (milliseconds) No exogenous substrate needed Relatively inexpensive Fluorescence + optical imaging = highthroughput and versatility for in vivo studies
  • 9. Fluorescent Proteins: Tools forImagingThe use of fluorescent proteins for imaging is revolutionizing in vivo biologyGreen fluorescent protein (GFP) can be genetically linked with almost any proteinPermanent and heritable label in live cells to study protein function and locationWith multiple colors (CFP/GFP/RFP), many processes can be visualized simultaneously in cells
  • 10. Marine originStabilityMutations
  • 11. SelectFluorescentProteins Chudakov D M et al. Physiol Rev 2010;90:1103-1163
  • 12. Day 10 14 28 17 24 Genetic engineering of human MIA-PaCa-2 pancreatic cancer cells Primary Primary to express RFP Primary Primary Primary Genetic vector with Red Fluorescent Protein (RFP)) Diffuse Metastasis Metastasis Metastases MetastasesReal time whole body Surgical Orthotopicimaging of tumor Tumor Implantationgrowth and metastasis of MIA-PaCa-2-RFP
  • 13. Alexa Fluor 488 Early detection of orthotopic pancreatic cancer with Alexa750 conjugated antibody Mack, GS. Nature Biotechnology. 28(3) 2010 Qdot
  • 14. Why Near-IR? Near- Light Penetration in 1mm Mouse Liver Tissue Avoid skin 0.02 autofluroescence 0.016 (~650nm) Transmission Efficiency Near IR Near IR Deep penetration. 0.012 IFP RFP: (3X)2 penetration 0.008 RFP depth of GFP NIR: near (8X)2 0.004 GFP penetration depth! 0 350 450 550 650 750 Wavelength (nm)
  • 15. iBox In Vivo Systems: Whole Animal to Cells iBox Explorer iBox Scientia iBox SpectraMicro: organs to cells Macro: 1 to 5 mice Rapid screening
  • 16. iBox Explorer iBox ExplorerImaging MicroscopeImaging Microscope Select Science Product Highlight
  • 17. BioLiteFiberoptics Coaxial Side
  • 18. BioLite8 Excitation filter capability
  • 19. Emission Filters-Explorer Darkroom Filters- Changeable filters
  • 20. Dual Excitation Light Path
  • 21. iBox Explorer Imaging head and fibers Fiberoptics CoaxialRetractableOrange Filter Viewer for Fiberoptics enhanced Side Lighting sample viewing Stage
  • 22. Excitation and Emission Filters
  • 23. BioLite Operation- Operation-Full Automation/Presets VisionWorks Software Panel Adjustable IntensitySetting Intensity %1 02 123 254 405 756 100
  • 24. Emission Filter Selection
  • 25. X-Y-Z Control Magnification FOV (mm2) 2.5 5.8 4.5 3.2 8.8 1.7 16.5 0.9
  • 26. Bookmarks-Bookmarks- Recalling Position On- On-The-The-Fly
  • 27. MOUSE SKIN-FLAP MODEL SKIN-Schematic diagram of the skin flapmodel in live mice for imagingintravascular trafficking .An arc-shaped incision was madein the abdominal skin, and then theskin flap was spread and fixed on a flatstand with pins.HT-1080 cells were injected into theepigastrica cranialis vein through acatheter. Hoffman RM. Methods Mol Biol. 2007;411:121-9.
  • 28. In Vivo Image Through Skin- Skin- Flap GFP-Tagged Human Fibrosarcoma Cell 16.5x 8.8x FOV= 1700 um
  • 29. Measurement of Individual Tumor Cells
  • 30. Spatial Calibration
  • 31. Pixels to Micrometers
  • 32. Calculation of Single Tumor Cell Diameter
  • 33. Magnify Tumors to View Single Cells 4.5x0.5x Human colon cancer HT-29 GFP and 13 days post tumor tissue implantation
  • 34. Injected LLC Cells Individual Cells 16.5x2.5xInjected Lewis Lung Carcinoma Cells
  • 35. Histology of mouse thyroidstained with cancer specificantibody conjugated withAlexa488. Both imageswere captured with iBoxExplorer.
  • 36. Current Applications Future Applications• Fluorescent protein tagged cells • Microfluidics• Fluorophore tagged cells • Nanotechnology• Fluorophore tagged antibodies • Drug distribution• Organ imaging • Microwell assays• Tissue imaging • Microarrays• Cell imaging • HTP in-well assays• Fluorescence imaging • Biomarker assays• White light imaging• Colorimetric imaging
  • 37. iBox® iBox® Scientia Small Animal Imaging System High sensitivity cameras/optics ◦ Cooled and ultracooled CCDs Increased resolution ◦ High megapixel CCDs Excitation and emission automation ◦ The BioLite Excitation light engine ◦ 8 excitation filters (400-750nm) ◦ Epi 365nm UV ◦ 5 emission filters (to NIR) Capture and analytical software ◦ Integrates darkroom, camera, lens automation Anesthesia system Temperature controlled imaging surface
  • 38. Tracking Stained Bacteria In-Vivo In- 5x108 stained salmonella, subcutaneous injection Labeled with Molecular Targeting CellVue Red and NIR815 CellVue NIR815 No Treatment CellVue RED Treatment 14 days after injection 20 days after injection CellVue RED
  • 39. Cancer-Cell- Cancer-Cell-Killing Efficacy of UV Light UV-induced cancer cell death was wave-length and dose dependentDiagram of minimal residual cancer (MRC) model and UVC treatmentDose and wave-length dependency Customized UVC pen light of UV-induced cell death for in vivo irradiation Journal of Cellular Biochemistry 110:1439–1446 (2010)

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