In Vivo Optical Imaging from theWhole Animal to the CellularLevelAntonio SanchezAbhishek Trikha
UVP: An Imaging CompanySmall Animal (in vivo, ex vivo)  Endoscopy /Microscopy    (in vivo,   intravital)    In vitro
Publications Referencing in vivo                   Over Two Decades, 1990-2011               3000               2500Public...
In Vivo ImagingNon-destructiveRepeated experimentationLocalized process in time and space
6-8 week old mouseSubcutaneous injection    Primary Fluorescent Area (mm )   2HCT116 fibrosarcoma cancer cell      400Dual...
Technical issues   SolutionsAbsorbance         Observe superficially                   Move to higher wavelengthScattering...
Hemoglobin is the major                        2.2                                                                        ...
Use of Fluorescent Genetic Reporters     Fast imaging (milliseconds)     No exogenous substrate needed     Relatively inex...
Fluorescent Proteins: Tools forImagingThe use of fluorescent proteins for imaging is  revolutionizing in vivo biologyGreen...
Marine originStabilityMutations
SelectFluorescentProteins              Chudakov D M et al. Physiol Rev 2010;90:1103-1163
Day 10     14     28     17     24                                              Genetic engineering of                    ...
Alexa Fluor 488                           Early detection of orthotopic pancreatic                           cancer with A...
Why Near-IR?                        Near-                                                                   Light Penetrat...
iBox In Vivo Systems:               Whole Animal to Cells iBox Explorer           iBox Scientia        iBox SpectraMicro: ...
iBox Explorer   iBox ExplorerImaging MicroscopeImaging Microscope   Select Science Product Highlight
BioLiteFiberoptics  Coaxial     Side
BioLite8 Excitation filter capability
Emission Filters-Explorer Darkroom         Filters-           Changeable filters
Dual Excitation Light Path
iBox Explorer                Imaging head and fibers                                           Fiberoptics                ...
Excitation and Emission Filters
BioLite           Operation-           Operation-Full Automation/Presets                            VisionWorks Software P...
Emission Filter Selection
X-Y-Z Control                Magnification    FOV                                (mm2)                     2.5         5.8...
Bookmarks-Bookmarks- Recalling Position On-                              On-The-The-Fly
MOUSE SKIN-FLAP MODEL                        SKIN-Schematic diagram of the skin flapmodel in live mice for imagingintravas...
In Vivo Image Through Skin-                                      Skin-                            Flap GFP-Tagged Human  F...
Measurement of Individual Tumor            Cells
Spatial Calibration
Pixels to Micrometers
Calculation of Single Tumor Cell           Diameter
Magnify Tumors to View         Single Cells             4.5x0.5x                        Human colon                       ...
Injected LLC Cells                               Individual                               Cells                       16.5...
Histology of mouse thyroidstained with cancer specificantibody conjugated withAlexa488. Both imageswere captured with iBox...
Current Applications                   Future Applications•   Fluorescent protein tagged cells   •   Microfluidics•   Fluo...
iBox®                   iBox® Scientia Small Animal                        Imaging System   High sensitivity cameras/opti...
Tracking Stained Bacteria In-Vivo                               In-   5x108 stained salmonella, subcutaneous injection  ...
Cancer-Cell-   Cancer-Cell-Killing Efficacy of UV Light           UV-induced cancer cell death was wave-length and dose de...
In Vivo Optical Imaging from the Whole Animal to the Cellular Level
In Vivo Optical Imaging from the Whole Animal to the Cellular Level
In Vivo Optical Imaging from the Whole Animal to the Cellular Level
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In Vivo Optical Imaging from the Whole Animal to the Cellular Level

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

  1. 1. In Vivo Optical Imaging from theWhole Animal to the CellularLevelAntonio SanchezAbhishek Trikha
  2. 2. UVP: An Imaging CompanySmall Animal (in vivo, ex vivo) Endoscopy /Microscopy (in vivo, intravital) In vitro
  3. 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. 4. In Vivo ImagingNon-destructiveRepeated experimentationLocalized process in time and space
  5. 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. 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. 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. 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. 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. 10. Marine originStabilityMutations
  11. 11. SelectFluorescentProteins Chudakov D M et al. Physiol Rev 2010;90:1103-1163
  12. 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. 13. Alexa Fluor 488 Early detection of orthotopic pancreatic cancer with Alexa750 conjugated antibody Mack, GS. Nature Biotechnology. 28(3) 2010 Qdot
  14. 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. 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. 16. iBox Explorer iBox ExplorerImaging MicroscopeImaging Microscope Select Science Product Highlight
  17. 17. BioLiteFiberoptics Coaxial Side
  18. 18. BioLite8 Excitation filter capability
  19. 19. Emission Filters-Explorer Darkroom Filters- Changeable filters
  20. 20. Dual Excitation Light Path
  21. 21. iBox Explorer Imaging head and fibers Fiberoptics CoaxialRetractableOrange Filter Viewer for Fiberoptics enhanced Side Lighting sample viewing Stage
  22. 22. Excitation and Emission Filters
  23. 23. BioLite Operation- Operation-Full Automation/Presets VisionWorks Software Panel Adjustable IntensitySetting Intensity %1 02 123 254 405 756 100
  24. 24. Emission Filter Selection
  25. 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. 26. Bookmarks-Bookmarks- Recalling Position On- On-The-The-Fly
  27. 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. 28. In Vivo Image Through Skin- Skin- Flap GFP-Tagged Human Fibrosarcoma Cell 16.5x 8.8x FOV= 1700 um
  29. 29. Measurement of Individual Tumor Cells
  30. 30. Spatial Calibration
  31. 31. Pixels to Micrometers
  32. 32. Calculation of Single Tumor Cell Diameter
  33. 33. Magnify Tumors to View Single Cells 4.5x0.5x Human colon cancer HT-29 GFP and 13 days post tumor tissue implantation
  34. 34. Injected LLC Cells Individual Cells 16.5x2.5xInjected Lewis Lung Carcinoma Cells
  35. 35. Histology of mouse thyroidstained with cancer specificantibody conjugated withAlexa488. Both imageswere captured with iBoxExplorer.
  36. 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. 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. 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. 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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