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IVIS Imaging System
Discovery at the Speed of Light
TM
Jack Yang
Product Manager
2010.09.02
1
Genomics, Proteomics and Functional Study
2
Non-invasive in vivo imaging
Continued monitor
Low variation
3R (Replacement, Reduction, Refinement)
Drug Discovry Today (2002) 7:125
3
Total solution for in vivo imaging
IVIS
Optical Imaging
Quantum FX
Micro CT
Vevo
HR Ultrasound
Cellvizio
In vivo microscopy
IVIS
Optical Imaging
Quantum FX
Micro CT
Vevo
HR Ultrasound
Cellvizio
In vivo microscopy
Multi-type of in vivo imaging
microPET
microCT
microSPECT
Optical Imaging
Ultrasound
Optical bioluminescence
Optical fluorescence
microMRI
GENE & DEVELOPMENT 2003 17:545
6
Advantage of optical imaging
1.High throughput
2.No radiation
3.Easy to operation
4.Dual function
(fluorescence and bioluminescence)
5.Functional analysis
7
Basic Method
8
Reporter Molecules
Luciferase,
Fluorescent Protein
Fluorescent dyes
Quantum dots
ATP and O2 required for luciferase
Label Cells
Label Bacteria
Label Genes
9
Imaging Principle
Imaging system
Autofluorescence
Autoluminescence
Luminescent or
Fluorescent source
10
An “Imaging” Window of Opportunity
Fluorophore
Excitation
11
Spectra
Emission
Luciferase Spectra
Demonstration of Light Transmission
Tissue is not transparent - Light absorbance depends on wavelength
Doyle et al, 2004
Nature Medicine
The source of light
luciferin
Bioluminescence
+ATP, Mg2+, O2
Luciferase
Renilla
Light
+
Oxyluciferin
+AMP, PPi, CO2
Excitation
Wavelength
GFP
Quantum dot
excited
Emission
state
Wavelength
NIR dye
Fluorescence
ground
state
13
Bioluminescence and fluorescence
Bioluminescence
Just light out
No background
Fluorescence
Light in and Light out
Autofluorescence interference
14
Quantified images
Luminance detection
Fluorescence detection
NATURE MEDICINE (2004) 10: 1257
絕對定量功能 Absolute Quantification reference
Journal of Biomedical Optics, (2001)
®
Living Image Software
Region of Interest (ROI)
ROI 4=2.344x105
Absolute Calibrated Data in: photons s-1 cm-2 sr -1
17
Calibrated Physical Units
• Living Image® automatically compensates for device settings: Exposure
time, f/stop, Binning, and Field of View.
• Calibrated units are Photons per Second, representing the flux radiating
omni-directionally from a user defined region.
2 sec exposure, f/stop 1, Small binning
~5000 counts peak
10 sec exposure, f/stop 1, Small binning
~25000 counts peak
2.82 x 108 photons/sec
2.82 x 108 photons/sec
BIOLUMINESCENCE SENSITIVITY
Cooled (-90C) camera with large
CCD chip area for high sensitivity
light detection
Precision filters
Most sensitive system available
Resolves multiple bioluminescent reporters
Detects down to 3 cells in vivo:
Low f-number and
large diameter lens
Gives high sensitivity and
uniform light collection
Rabinovich et al, PNAS,2008
In vivo imaging of s.c. implanted T cells
transduced with optimized firefly luciferase.
19
19
FLUORESCENCE SENSITIVITY
In Vivo Quantitation of Doxorubicin Using Spectral Unmixing
Non-unmixed
image
Non-unmixed
image
Unmixed
image
64
128
64
32
32
16
8
4.E-06
R² = 0.9671
Fluorescence
Dox (ng)
256
Unmixed
image
2.E-06
0.E+00
0
64
128
192
256
Doxorubicin (ng)

Spectral Unmixing enables detection of as little as 8 ng of Doxorubicin in vivo

IVIS enables direct correlation between Doxorubicin signal and concentration both in vitro and in vivo

Simple to use Unmixing software significantly improves signal to background ratio at low concentrations
20
20
Longitudinal Non-Invasive Monitoring of
Disease (and Treatment)
21
Untreated 9L-Glioma (Rat)
-2
0
4
10
14 x 103
12
10
8
6
4
2
Source: Brian Ross, Ph.D., University of Michigan Medical Center
22
Longitudinal Assessment of Tumor Growth and Response
to Anti-Angiogenic Therapy
105 U87 glioma cells implanted 14 days prior
Control
Anti-angiogenesis
inhibitors (week 4)
Szentirmai et al, 2006
23
Tumor Induced Damage Monitoring
Cell Model: MDA-MB-231
(breast cancer cell line)
J Clinic Invest
Massauge group, 2005
24
Brain Tumor Therapy
•
Intra-cranial glioma model
•
•
U87MG-Luc Cells
Anti-angiogenesis therapy
• Test a non-viral delivery system
(Transposon-sleep beauty, SB)
• Test anti-tumor effects of statin-AE and
sFlt-1
Mol Therapy 2005
Cell Trafficking, Survival and
Engraftment Patterns In Vivo
26
Cell Transplantation and Trafficking Patterns
Stem Cell Foci Formation and Hematopoiesis
Transplantation of 250 Luc+ HSC into Lethally Irradiated Hosts
Cao et al, Stem Cells,2004
27
Ultrasound Guided Delivery of
Image-guided cardiac cell delivery
Bioluminescent Cardiomyoblasts
心成肌細胞
Image-guided cardiac cell delivery: Sprague-Dawley rats (n = 11) were imaged using high-resolution
ultrasound, and stably transfected cardiomyoblasts (plasmid-CMV-firefly luciferase) were injected into the
anterior cardiac wall under ultrasound guidance (parasternal long-axis view), using a 28-gauge needle. After
injection, bioluminescence imaging was performed using a cooled charged-coupled camera. The signal
detected was positively correlated with the amount of cells transplanted (R(2) = 0.94, P = 0.03). Optical signal
could be followed longitudinally using bioluminescence imaging.
Rodriguez-Porcel & Gambhir et al, 2005
Stroke Repairing
Yellew: Infarcted site
White: Injection site
Neuron Progenitor Cell Tracing
Stroke Kim et al, 2004
29
Neurotropic Viral Infection
•
Viral Model: Sindbis Virus (Alphavirus)
•
103 CFU virus inoculated at right hind foot
J Virol Cook & Griffin, 2003
30
CUBICIN (Cubist Pharmaceuticals)
Cubicin (Daptomycin) is an FDA approved
drug for infections caused by Staphylococcus
aureus
Mouse Infection Model:
Peritonitis Model
 Mice inoculated with lethal dose of a
bioluminescent S. aureus (MRSA)
 Spread of infection monitored in real time using an
IVIS system.
 5 mice per treatment imaged hourly and non-
invasively – no need to sac at each time point.
 A 3 log difference in viable bacteria demonstrated
within 4 hours.
 Fewer animals used per group and per time point
Mortin et al., Antimicrobial Agents and Chemotherapy, May 2007, p. 1787–1794
in vivo fluorescence imaging of subcutaneous
U87MG glioblastoma
RGD-Cy5.5
Fluorescence probe
Cancer research (2004) 64: 8009
Genetic Components of Disease
Expression, Pathways and Therapy
33
TRANSGENICS (LPTA)
Vascular Endothelial Cell Growth Factor Receptor
VEGFR2 Pr
luc
Day 14
CHROMOSOME
VEGF induction of VEGFR2 promoter
causes luciferase expression
VEGF - secreted during angiogenesis
VEGFR2 - surface receptor for VEGF
Transgenic VEGFR2-luc
Embryos in a Non- Tg Mother
34
Reduction of VEGFR2 Expression
During Development
1 Week
2 Weeks
60
4 Weeks
6 Weeks
5000
1000
1000
4000
800
800
3000
600
600
400
400
200
200
55
50
x103
45
40
2000
35
1000
30
Signal Intensity
100,000
Signal Intensity
10,000
Signal Intensity
200
Zhang et al. (Xenogen), 2004
Signal Intensity
<100
Induction of VEGFR-2 Expression
During Wound Healing
Day 0
Day 4
Day 6
Day 11
Zhang et al. (Xenogen), 2004
Day 18
Day 24
Monitoring Angiogenesis During
Tumor Development
Tumor growth and
development imaged by
GFP fluorescence
Angiogenesis imaged by
VEGFR2-luc expressing
bioluminescence
VEGFR2-luc KI mice implanted with 106 LL/2-GFP tumor cells and imaged at day18
NFĸB Induction by LPS
Control
0
4
7
24
Zhang, Xenogen & Rune Blomhoff, Oslo
Peyer’s
patches
LPS
Effect of Dexamethasone and EGCG
on iNOS Expression
Zhang et al. (Xenogen), 2003
Protein-protein Interaction
Rapamycin-Induced Association of FRB and FKBP
PNAS
Piwnica-Worms group, 2004
Bioware® Luciferase Cell Lines
Luciferase expression tumor cell lines
1. Breast Cancer: MCF-7, MDA-MB
2. Colorectal cancer: HT-29, LoVo-6
3. Lung cancer: A549
4. Melanoma: B16
5. Prostate cancer : PC3M, LNCaP
6. Cervical: HeLa
7. Brain tumor: U-87
8. Renal Tumor: ACHN
Microorganisms
Gram Negative Bacteria
1. Escherichia coli
2. Hemophilus influenzae
3. Pseudomonas aeruginosa
4. Samonella typhimurium
5. Shigella dysenteriae
6. Yersinia enterocolitica
Gram Positive Bacteria
1. Listeria monocytogenes
2. Staphylococcus aureus
3. Streptococcus pneumoniae
Fungi
Candida albicans
Infectious Disease Application
Drug Discovery
LPTA® Luciferase Animal Model
Light Producing Transgenic Animal
Animal Model
Application
Ho-1
Oxidative Stress
Cyp450
Drug Metabolism
iNOS
Sepsis
Epx
Asthma
IL-2
Innate immunity
Kap
Androgen
VEGF
Angiogenesis
Ins2
Diabetes
Kdr
Wound healing
Stem Cell/Adoptive Transfer
Tracking stem cells
Engraftment
Differentiation
 Transplant biology
Donor cells from LPTAs
β-actin-luc
GAPDH-luc
Reporter specific (e.g., Rip-luc)
 Tracking immune response
Lymphocytes
Eosinophils
Neutrophils
IVIS® Spectrum Imaging System
- Hardware
• Customized for in vivo
imaging
• Highly sensitive camera
with a large dynamic range
Standard Images are Composed of Two Images
Photographic + Luminescent = Overlay
Alignment Light Projector
•
Allows rapid and
reproducible positioning
of subjects.
•
Size changes with Field
of View setting
Field-of-View (typical)
FOV D: 21.5 cm
1.5x
FOV B: 6.5 cm
5x
FOV C: 12.5 cm
2.5x
FOV A: 4.0 cm
8.7x
Living Image Control Panel
Controls Sensitivity
Imaging Wizard
Select for assistance in setting
up bioluminescence or
fluorescence sequences
• Good labeling practices are
necessary for effective data
browsing
Image Labeling
Image Cataloging & Browsing Tools
User defined information
Quantification
• Tool palette for adjusting scale/opacity etc.
• Region of interest (ROI) tools to measure surface intensities
Regions of Interest Tools
ROI shapes available:
• Square
• Circle
• Contour
• Grid
ROI’s can be created:
• Manually
• Automatically
• Free Draw
Important to be consistent with ROI selections
Measurement Table
• Measurement table displays
information about each ROI
• Table is user configurable and can be
exported to a spreadsheet
Calibrated Physical Units
• Living Image® automatically compensates for device settings: Exposure
time, f/stop, Binning, and Field of View.
• Calibrated units are Photons per Second, representing the flux radiating
omni-directionally from a user defined region.
2 sec exposure, f/stop 1, Small binning
~5000 counts peak
10 sec exposure, f/stop 1, Small binning
~25000 counts peak
2.82 x 108 photons/sec
2.82 x 108 photons/sec
Fluorescence
Fluorophore
Excitation
Wavelength
excited state
ground state
Emission
Wavelength
IVIS® Fluorescence Imaging
•18 Emission filters (computer controlled)
Emission
filter wheel
•10 Excitation filters (computer controlled)
•150 Watt Tungsten/Halogen lamp
Excitation
filter wheel
(computer controlled intensity)
•Low Auto Fluorescence optics and fibers
Trans-illumination
Light source
Optical
switch
Excitation and Emission Filters
100
Transmission %
80
10 Excitation filters –
35 nm bandwidth
710 - 760
nm
60
810 - 875
nm
40
20
0
400
615 - 665
nm
440
480
695 - 770
nm
520
560
600
640
680
720
Wavelength (nm)
500 - 550
nm
445 - 490
nm
575 - 650
nm
515 - 575
nm
18 Emission filters –
20 nm bandwidth
760
What is Spectral Unmixing?
• Calculates concentrations of
different fluorescent components
• Requires images acquired at
multiple wavelengths to perform
the spectral analysis
• Emission or excitation scan
• Quantitative and qualitative
results
In vitro Spectral Unmixing Example:
Dyes in a Dish
XF 680
Top and bottom: XF680 and
XF750 mixture: 1:1
Left middle: XF680 only
Right middle: XF750 only
1 x1014 molecules per spot
Ex640, Em700-820nm
Composite
XF 750
Image Overlay Capabilities
Bioluminescent
Tumor
DHE
Image Overlay
3D Tomography
(Luminescence)
Spectral scans with Emission filters allows for
absolute quantification and source localization
3D Tomography
(Fluorescence)
Spatial scans with trans-illumination light source allows
for absolute quantification and source localization
In vivo dual modality tomography
Fluorescence Imaging
Tomography - FLIT
Bioluminescence Imaging
Tomography - DLIT
Mouse Left
Tumor Depth [mm]
Mouse Right
Tumor Depth [mm]
DLIT
5.8
2.9
FLIT
--
3.1
Utilize Well Plate Quantification to Determine Cell
Number or pMol of Reporter
• Dilute your cells or dye and image
• Select Well Plate Quantification from Tools menu
• Enter cell number or
concentration per well
• Save as a library
• Choose library
when reconstructing
Utilize Well Plate Quantification to Determine Cell
Number or pMol of Reporter
2D
3D
5.44e5 cells
vs.
1.253e8 photons/sec
2.93e4 cells
vs.
1.79e7 photons/sec
Automatic Mouse Atlas Registration in LI4.0
Unregistered
Coregistered
Total solution for in vivo imaging
IVIS
Optical Imaging
Quantum FX
Micro CT
Vevo
HR Ultrasound
Cellvizio
In vivo microscopy
Quantum Micro CT
High resolution CT for longitudinal studies
New, flat-panel detector technology and a micro-focus X-ray tube provides high resolution
imaging for small animals with radiation doses low enough to enable longitudinal studies. High
throughput and ease of use supports stand-alone CT use or as a complement to IVIS Spectrum.
Whole animal imaging

200mm dynamic scan for 3D images
High resolution


20µ pixel for whole animal imaging
10µ pixel for specimen imaging
High speed imaging

17 second scan time delivers low radiation
Volume rendering


Dedicated processing of 3D images for
45 second reconstruction
Co-registration with IVIS Spectrum
Cone X-ray beam for high speed imaging
This CT exposes a cone-shaped X-ray beam on the 2D detector. The revolving arm rotates 360°
around the sample to acquire full 3D data. This provides 17-second high-speed imaging.
High-resolution micro-focused X-ray source
The 90 kV (maximum X-ray output) and 5 micrometer-focused X-ray source create highresolution images that can be studied at the 20 micrometer
Quantum Micro CT
High resolution CT for longitudinal studies
The small format instrument is designed to fit the space constraints and high throughput
workflow of a small-animal imaging laboratory. Like all IVIS instruments the system is easy
for biologists to use without the need for a dedicated operator
Fluoroscopy mode

Real-time imaging enables precise animal positioning
Purpose-built for small animal imaging



Integrated animal handling
Compatible with XGI-8 anesthesia
Animal transfer bed for IVIS Spectrum
High speed imaging

17 second scan for high throughput
High speed analysis


3D processing board for 45 second results
Intelligent co-registration workflow to facilitate 3D
fluorescence and bioluminescence analysis using IVIS
Spectrum
Quantum Micro CT
High resolution CT for longitudinal studies
Imaging modalities:
• Fluoroscopy allow real-time imaging and positioning
• 17 second scan for data collection
• 45 second processing time for 3D reconstruction
• Display tools for soft tissue and bone contrast
Quantum Micro CT
High resolution CT for longitudinal studies
Low X-ray dosage supports longitudinal studies
Day 0
Day 7
Day 10
Day 14
High resolution images show bone growth below implant
3D reconstruction
Data courtesy of Matsumoto Dental University Graduate School of Oral Medicine
Quantum Micro CT
High resolution CT for longitudinal studies
Soft tissue imaging and tumor measurement using contrast agent and 3D
reconstruction.
Quantum Micro CT
High resolution CT for longitudinal studies
Soft tissue imaging and tumor measurement using contrast agent and 3D
reconstruction.
Animal tumor model system
• 2 x 106 PSN-1 cells
• Balb/c mice
• Imaged day 15
Imaging parameters
• 17 second can time
• 20µ resolution
• 5~8 mGy dosage
Tumor volume results:
• CT volume 17.78 mm3
• Excised volume 15.82 mm3
• Multiple time points measured
Analytical software
options:
• Metabolic mapping to
show body fat
• 3D alignment to compare
bone growth pattern
• Bone mineral density
analysis tools
Quantum Micro CT
High resolution CT for longitudinal studies
Micro-Ultrasound: Clarity
Conventional
clinical ultrasound
(human fetus)
20cm
3 - 15 MHz
3cm
Melon
Coffee Bean
Micro-ultrasound
(mouse fetus)
30 – 80 MHz
High-frequency = High-resolution
How Does Micro-Ultrasound Work?
What Can the Vevo® Do For Me?
Image Guided Injections
 No need for invasive surgery
Cardiotoxicity studies
 For cardiovascular evaluations
Gene therapy
 Gene delivery through
sonoporation
Tumor dynamics: see it all
MicroMarkerTM Microbubbles
Phospholipid
Layer
2-3 μm
C2F10 / N2 Core
Microbubble Applications
Targeted MicroMarkerTM Microbubbles
Cell Surface Marker
VEGFR-2 / PCAM / VCAM / PSelectin / etc.
User-selected antibody
Biotin
Streptavidin
Olive KP, et al. Science
2009;324(5933):1400-1
Lacefield, et al. Cancer Res
2007;67(6):2830-9
Shaked Y, et al. Science
2006;313(5794):1785-7
The Cellvizio® LAB Solution
What is the Cellvizio solution?
•
In vivo confocal microscope
•
Unique miniaturized fibered
microprobes
• Sophisticated real-time and postacquisition software
The World’s Smallest Microscope
Laser Scanning Unit
• 488 or 660 nm laser
source
• High-speed confocal
microscope
• Single pixel detector
(APD)
Blue LSU
488
Microprobes™
• Tens of thousands of
fiber optics bundled
together
• Ultra miniaturized
optical objective
[ 500 - 700 ]
Red LSU
660
[ 680 - 1000 ]
• 300μm diameter
Cells and Vessels Imaged Deep within the Brain
Capillaries in the cortex.
IV FITC-Albumin
FOV ø 300 µ m
GFP olfactory glomerulus.
FOV ø 300 µ m
Neurons of a
transgenic mouse
expressing Clomeleon.
FOV 300 x 420 µ m
CamKinase-GFP neurons in
the striatum.
FOV 160 x 120 µ m
GFP stem cells in
living mouse brain.
FOV 300 x 420 µ m
Reaching multiple regions in the Deep
Brain
Hippocampus
Alzheimer
Accumbens
Addictions
VTA
Addictions
Substantia Nigra
Parkinson
Conscious in vivo in situ
imaging in the hippocampus
Mobile implant:
S300 microprobe
hippocampus
U. Maskos (Institut Pasteur) et al, SFN 2008
Neuron Precursor Migration
“In vivo imaging of
migrating neurons in the
mammalian forebrain"
Sensory Neurons
Glomeruli
Periglomerular cell
Mitral cell
Proliferation
Granule cell
Migration
RMS
OB
Time Lapse Movie of Neuron
precursors in the olfactory
bulb labeled with CellTracker
Green.
- FOV 400 x 280 µ m
Integration
Lledo and Saghatelyan (2005) Trends in neurosciences Vol.28 No.5
Calcium Imaging
Electrical
stimulation
(thalamus)
Chemical
Stimulation
(bicuculine,
Hippocampus)
Calcium Imaging
Changes in intracellular calcium
in the hippocampus in response
to bicuculline-induced
epileptiform activity.
A) Time course of the fluorescence
intensity of Oregon Green
BAPTA-1 in cells shown in (B)
(C) and (D) corresponding
electroencephalogram (EEG)
Vincent et al. (2006). EMBO 7(11), 1154–1161
Peripheral Nerve Experimental Set-Up
• Label: Nerve labeling or use of
•
•
•
transgenic mice with fluorescent
nervous tissue
Access: External or minimal incision
(2mm) of the skin to access nervous
tissue (nerve, sensory fibers, neuromuscular junction)
Record: Real time acquisition of
tissue condition. Scan the Microprobe
along nerve or across tissue
Analyze: E.g. Measure the length of
outgrowth or nerve fiber density
Thy1-YFP - Saphenous nerve
D0, H0: Intact Saphenous Nerve
D0, H1: Crushed Nerve
D1 post crush: Degenerated Nerve
D4 post crush: Regenerated Nerve
Thy1-YFP mouse FOV 400 x 280 m
Longitudinal imaging of neuroregeneration…
with single fiber resolution
Courtesy of Igor Charvet and Paolo Meda, Medical Research Center, Geneva, Switzerland
•
•
•
•
Angiogenesis –
Experimental Set-Up
Label: IV injection of FITCDextran (500 kDa) or
AngioSense
Access: Minimal incision of the
skin to access subcutaneous
tumor
Record: Acquisition of tumoral
vascularization sequences
Analyze: Analysis of the images
to measure the functional
capillary density
Courtesy of Descartes Image, O. Clément, Necker School of Medicine, Paris, France
Tumor Angiogenesis
Longitudinal analysis of tumor vascularization in PyMT mice
9 weeks
Vessel
Vessel
Vessel
Vessel
Length: 1847
Area: 23428
Volume: 289900
Diameter: 12.69 +/- 6.09
10 weeks
Vessel
Vessel
Vessel
Vessel
Length: 3347
Area: 51830
Volume: 801566
Diameter: 14.89 +/- 6.98
11 weeks
Vessel
Vessel
Vessel
Vessel
Length: 3729
Area: 65460
Volume: 1212684
Diameter: 16.80 +/- 9.16
Increased vascular density during tumor development in abdominal
mammary gland
Imaging mouse colonic tumors with
Cellvizio® LAB
Cellvizio®LAB ProFlex™ MiniZ microprobe
(0.94mm)
Vascular architecture
Normal mucosa
Adenoma
Thanks for
your
attention