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

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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