5. 6-8 week old mouse
Subcutaneous injection
Primary Fluorescent Area (mm )
2
HCT116 fibrosarcoma cancer cell 400
Dual color (GFP nuclei, RFP cytoplasm)
300
Weekly 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 Solutions
Absorbance Observe superficially
Move to higher wavelength
Scattering High intensity light source
Increase exposure time
High sensitivity camera
Autofluorescence Move to higher wavelength
Optimize filter selection
Motion artifact High sensitivity camera
Immobilize tissue
Dim signal High intensity light source
Increase exposure time
7. Hemoglobin is the major 2.2
area 1
absorber 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 = high
throughput and versatility for in vivo studies
9. Fluorescent Proteins: Tools for
Imaging
The use of fluorescent proteins for imaging is
revolutionizing in vivo biology
Green fluorescent protein (GFP) can be genetically linked
with almost any protein
Permanent and heritable label in live cells to study protein
function and location
With multiple colors (CFP/GFP/RFP), many processes can
be visualized simultaneously in cells
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
Metastases
Real time whole body Surgical Orthotopic
imaging of tumor Tumor Implantation
growth 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 Spectra
Micro: organs to cells Macro: 1 to 5 mice Rapid screening
16. iBox Explorer
iBox Explorer
Imaging Microscope
Imaging Microscope
Select Science Product Highlight
21. iBox Explorer
Imaging head and fibers
Fiberoptics
Coaxial
Retractable
Orange Filter
Viewer for Fiberoptics
enhanced Side Lighting
sample
viewing
Stage
27. MOUSE SKIN-FLAP MODEL
SKIN-
Schematic diagram of the skin flap
model in live mice for imaging
intravascular trafficking .
An arc-shaped incision was made
in the abdominal skin, and then the
skin flap was spread and fixed on a flat
stand with pins.
HT-1080 cells were injected into the
epigastrica cranialis vein through a
catheter.
Hoffman RM. Methods Mol Biol. 2007;411:121-9.
28.
29. In Vivo Image Through Skin-
Skin-
Flap
GFP-Tagged Human
Fibrosarcoma Cell 16.5x
8.8x
FOV= 1700 um
36. Histology of mouse thyroid
stained with cancer specific
antibody conjugated with
Alexa488. Both images
were captured with iBox
Explorer.
37. 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
38. 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
39. 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
40. Cancer-Cell-
Cancer-Cell-Killing Efficacy of UV Light
UV-induced cancer cell death was wave-length and dose dependent
Diagram of minimal residual cancer (MRC) model and UVC treatment
Dose 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)
