in vivo optical imaging from the whole animal to the cellular level
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In Vivo Optical Imaging from theIn Vivo Optical Imaging from theWhole Animal to the CellularWhole Animal to the CellularLevelLevel
Antonio SanchezAbhishek TrikhaAntonio SanchezAbhishek Trikha
UVP: An Imaging CompanyUVP: An Imaging Company
Small Animal (invivo, ex vivo)
Endoscopy/Microscopy
(in vivo,intravital)
In vitro
50010001500200025003000
Pub
licat
ions
Publications Referencing in vivoOver Two Decades, 1990-2011
0500
1990-1995 1996-2000 2000-2005 2005-2011
Pub
licat
ions
In Vivo ImagingIn Vivo Imaging
Non-destructive
Repeated experimentation
Localized process in time and space
Non-destructive
Repeated experimentation
Localized process in time and space
6-8 week old mouseSubcutaneous injectionHCT116 fibrosarcoma cancer cellDual color (GFP nuclei, RFP cytoplasm)Weekly measurements
Primary Tumor Volume (mm 3)
Prim
ary
Fluo
resc
ent A
rea
(mm
2 )
0
100
200
300
400
0 2000 4000 6000 8000
Regression95% confidencer = .89, p < 0.05
6-8 week old mouseSubcutaneous injectionHCT116 fibrosarcoma cancer cellDual color (GFP nuclei, RFP cytoplasm)Weekly measurements
Primary Tumor Volume (mm 3)
Prim
ary
Fluo
resc
ent A
rea
(mm
2 )
0
100
200
300
400
0 2000 4000 6000 8000
Regression95% confidencer = .89, p < 0.05
Technical issues Solutions
Absorbance Observe superficiallyMove to higher wavelength
Scattering High intensity light sourceIncrease exposure timeHigh sensitivity camera
Autofluorescence Move to higher wavelengthOptimize filter selection
Autofluorescence Move to higher wavelengthOptimize filter selection
Motion artifact High sensitivity cameraImmobilize tissue
Dim signal High intensity light sourceIncrease exposure time
0.2
0.7
1.2
1.7
2.2
450 500 550 600 650wavelength (nm)
inte
nsity
(A.U
.) area 1area 2
Hemoglobin is the majorabsorber in animal tissue
0.2
0.7
1.2
1.7
2.2
450 500 550 600 650wavelength (nm)
inte
nsity
(A.U
.) area 1area 2
Autofluorescence fromendogenous molecules
Use of Fluorescent Genetic Reporters
Fast imaging (milliseconds)
No exogenous substrate needed
Relatively inexpensive
Fast imaging (milliseconds)
No exogenous substrate needed
Relatively inexpensive
Fluorescence + optical imaging = highthroughput and versatility for in vivo studies
Fluorescent Proteins: Tools forFluorescent Proteins: Tools forImagingImagingThe use of fluorescent proteins for imaging is
revolutionizing in vivo biology
Green fluorescent protein (GFP) can be genetically linkedwith almost any protein
Permanent and heritable label in live cells to study proteinfunction and location
With multiple colors (CFP/GFP/RFP), many processes canbe visualized simultaneously in cells
The use of fluorescent proteins for imaging isrevolutionizing in vivo biology
Green fluorescent protein (GFP) can be genetically linkedwith almost any protein
Permanent and heritable label in live cells to study proteinfunction and location
With multiple colors (CFP/GFP/RFP), many processes canbe visualized simultaneously in cells
Marine origin
Stability
Mutations
Marine origin
Stability
Mutations
SelectFluorescentProteins
Chudakov D M et al. Physiol Rev 2010;90:1103-1163
SelectFluorescentProteins
Genetic engineering ofhuman MIA-PaCa-2pancreatic cancer cellsto express RFP
Genetic vector with RedFluorescent Protein (RFP))
Day 10
Primary
Day 14
Primary
Metastasis
Day 17
Primary
Metastasis
Day 24
PrimaryPrimary
DiffuseMetastases
Day 28
Surgical OrthotopicTumor Implantationof MIA-PaCa-2-RFP
Real time whole bodyimaging of tumorgrowth and metastasis
MetastasisMetastasis
Metastases
DiffuseMetastases
Alexa Fluor 488
Mack, GS. Nature Biotechnology. 28(3) 2010
Early detection of orthotopic pancreaticcancer with Alexa750 conjugated antibody
Alexa Fluor 488
Qdot
Why NearWhy Near--IR?IR?
Avoid skinautofluroescence(~650nm)
Deep penetration. RFP: (3X)2 penetration
depth of GFP NIR: near (8X)2
penetration depth!
Light Penetration in 1mm Mouse Liver Tissue
0
0.004
0.008
0.012
0.016
0.02
350 450 550 650 750
Wavelength (nm)
Tran
smis
sion
Eff
icie
ncy N
ear IR
Avoid skinautofluroescence(~650nm)
Deep penetration. RFP: (3X)2 penetration
depth of GFP NIR: near (8X)2
penetration depth!
Light Penetration in 1mm Mouse Liver Tissue
0
0.004
0.008
0.012
0.016
0.02
350 450 550 650 750
Wavelength (nm)
Tran
smis
sion
Eff
icie
ncy N
ear IR
RFP
IFP
GFP
iBoxiBox In Vivo Systems:In Vivo Systems:Whole Animal to CellsWhole Animal to Cells
iBox ScientiaMacro: 1 to 5 mice
iBox ExplorerMicro: organs to cells
iBox SpectraRapid screening
iBox
ExplorerIm
aging Microscope
Select Science Product Highlight
iBox
ExplorerIm
aging Microscope
BioLiteBioLiteFiberopticsFiberoptics
Coaxial Side
BioLiteBioLite8 Excitation filter capability8 Excitation filter capability
Emission FiltersEmission Filters--Explorer DarkroomExplorer DarkroomChangeable filtersChangeable filters
Dual Excitation Light Path
iBoxiBox ExplorerExplorerImaging head and fibersImaging head and fibers
FiberopticsCoaxial
RetractableOrange Filter
Viewer forenhanced
sampleviewing
FiberopticsSide Lighting
RetractableOrange Filter
Viewer forenhanced
sampleviewing
Stage
ExcitationExcitation and Emission Filtersand Emission Filters
BioLiteBioLiteOperationOperation--Full Automation/PresetsFull Automation/Presets
Setting Intensity %
1 0
2 12
VisionWorks Software PanelAdjustable Intensity
2 12
3 25
4 40
5 75
6 100
Emission Filter SelectionEmission Filter Selection
XX--YY--Z ControlZ Control
Magnification FOV(mm2)
2.5 5.8
4.5 3.2
8.8 1.78.8 1.7
16.5 0.9
BookmarksBookmarks-- Recalling Position OnRecalling Position On--TheThe--FlyFly
MOUSE SKINMOUSE SKIN--FLAP MODELFLAP MODEL
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.
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.
In Vivo Image Through SkinIn Vivo Image Through Skin--FlapFlap
GFP-Tagged HumanFibrosarcoma Cell
8.8x FOV= 1700 um
16.5x
GFP-Tagged HumanFibrosarcoma Cell
8.8x FOV= 1700 um
Measurement of Individual TumorMeasurement of Individual TumorCellsCells
Spatial CalibrationSpatial Calibration
Pixels to MicrometersPixels to Micrometers
Calculation of Single Tumor CellCalculation of Single Tumor CellDiameterDiameter
Magnify Tumors to ViewMagnify Tumors to ViewSingle CellsSingle Cells
Human coloncancer HT-29GFP and 13 dayspost tumor
tissueimplantation
0.5x 4.5xHuman coloncancer HT-29GFP and 13 dayspost tumor
tissueimplantation
Injected LLC CellsInjected LLC Cells
16.5x
IndividualCells
Injected Lewis Lung Carcinoma Cells
2.5x
Histology of mouse thyroidstained with cancer specificantibody conjugated withAlexa488. Both imageswere captured with iBoxExplorer.
Current Applications
• Fluorescent protein tagged cells• Fluorophore tagged cells• Fluorophore tagged antibodies• Organ imaging• Tissue imaging• Cell imaging• Fluorescence imaging• White light imaging• Colorimetric imaging
Future Applications
• Microfluidics• Nanotechnology• Drug distribution• Microwell assays• Microarrays• HTP in-well assays• Biomarker assays
Current Applications
• Fluorescent protein tagged cells• Fluorophore tagged cells• Fluorophore tagged antibodies• Organ imaging• Tissue imaging• Cell imaging• Fluorescence imaging• White light imaging• Colorimetric imaging
Future Applications
• Microfluidics• Nanotechnology• Drug distribution• Microwell assays• Microarrays• HTP in-well assays• Biomarker assays
iBoxiBox®® ScientiaScientia Small AnimalSmall AnimalImaging SystemImaging 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
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
Tracking Stained BacteriaTracking Stained Bacteria InIn--VivoVivo
5x108 stained salmonella, subcutaneous injection Labeled with Molecular Targeting CellVue Red and
NIR815
CellVue NIR815 No Treatment
14 days after injection 20 days after injectionCellVue RED
5x108 stained salmonella, subcutaneous injection Labeled with Molecular Targeting CellVue Red and
NIR815
CellVue RED Treatment
No Treatment
CancerCancer--CellCell--Killing Efficacy ofKilling Efficacy of UVUV LightLight
UV-induced cancer cell death was wave-length and dose dependent
Journal of Cellular Biochemistry 110:1439–1446 (2010)
Customized UVC pen lightfor in vivo irradiation
Diagram of minimal residual cancer (MRC) model and UVC treatment
Dose and wave-length dependencyof UV-induced cell death
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