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1
IVIS Imaging System
Discovery at the Speed of Light TM
Jack Yang
Product Manager
2
Non-invasive in vivo imaging
Continued monitor
Low variation
3R (Replacement, Reduction, Refinement)
Drug Discovery Today (2002) 7:125
3
microPET
microCT
microSPECT
microMRI
Optical bioluminescence
Optical fluorescence
Ultrasound
GENE & DEVELOPMENT 2003 17:545
Multi-type of in vivo imaging
Aspect M2
Micro MRI
Quantum FX
Micro CT
Vevo 2100
HR Ultrasound
Vevo LAZR
Photoacustic
5
Genisys4
Micro PET
Cellvizio
In vivo microscopy IVIS
Optical imaging
Maestro
Fluorescence
6
Advantage of optical imaging
1.High throughput
2.No radiation
3.Easy to operation
4.Dual function
(fluorescence and bioluminescence)
5.Functional analysis
IVIS can cover multi-optical imaging
8
Basic Method
9
Reporter Molecules
Luciferase, Fluorescent Protein
Fluorescent dyes
Quantum dots
Label Cells Label Bacteria Label Genes
ATP and O2 required for luciferase
10
Autofluorescence Autoluminescence
Luminescent or Fluorescent source
Imaging system
Imaging Principle
11
An “Imaging” Window of Opportunity
Excitation Emission Fluorophore Spectra Luciferase Spectra
Demonstration of Light Transmission
Tissue is not transparent - Light absorbance depends on wavelength
Doyle et al, 2004
Nature Medicine
13
Bioluminescence
Oxyluciferin
luciferin
+ATP, Mg2+, O2
Light
+
+AMP, PPi, CO2
The source of light
Renilla Luciferase
Fluorescence
GFP
Quantum dot NIR dye
ground
state
Excitation
Wavelength
Emission
Wavelength
excited
state
14
Bioluminescence
Just light out
No background
Fluorescence
Light in and Light out
Autofluorescence interference
Bioluminescence and fluorescence
Quantified images
Luminance detection
Fluorescence detection
NATURE MEDICINE (2004) 10: 1257
Journal of Biomedical Optics, (2001)
絕對定量功能 Absolute Quantification reference
Raw Signal
(Counts)
Exp time: 30 sec 30 sec 60 sec 60 sec 60 sec 60 sec
Binning: small small small small medium medium
Day: 1 2 3 4 5
6
Peak
Counts
1600
800
1200
400
Calibrated Physical Units vs Raw Signal - Example
Calibrated
Signal
(Photons per
second)
Radiance:
Photons per
second
Exp time: 30 sec 30 sec 60 sec 60 sec 60 sec 60 sec
Binning: small small small small medium medium
Day: 1 2 3 4 5
6
Calibrated Physical Units vs Raw Signal - Example
Autoexposure feature available for
bioluminescence and fluorescence
User definable autoexposure settings
Image Wizard - autoexposure
Bioluminescence Imaging Sensitivity
Cooled (-90C) camera with large CCD chip area for high
sensitivity light detection
Low f-number and large diameter lens
Most sensitive system available Resolves multiple bioluminescent
reporters Detects down to even a single cell in vivo
In vivo imaging of s.c. implanted T cells transduced with optimized firefly luciferase (left) and a ‘single’ 4T1 breast cancer
cell (right)
Precision filters
Gives high sensitivity and uniform light collection
Rabinovich et al, PNAS,2008
Single cell (flux: 460-528 photons/sec)
Kim et al, pLOs One et al, 2010
64
32
16
8
256
Do
x (n
g)
128
64
32
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
Non-unmixed image
Unmixed image
Non-unmixed image
Unmixed image
R² = 0.9671
0.E+00
2.E-06
4.E-06
0 64 128 192 256
Flu
orescen
ce
Doxorubicin (ng)
In Vivo Quantitation of Doxorubicin Using Spectral Unmixing
Fluorescence Imaging Sensitivity
Molecular Imaging in Drug Development Your Success is Our Success
IVIS imaging performance is measured by your results: 1000 systems in laboratories worldwide 2,600 peer-reviewed publications
14 drugs in clinical trials (that we know of…we estimate there are a whole lot more)
IVIS Preclinical Imaging •All therapeutic areas
•Mode of action •Efficacy studies
•Pharmacokinetic model •Dosing and treatment
IVIS Bringing Compounds to the Clinic Research using IVIS contributed to 5 FDA approved drugs and 9 drugs currently
in clinical trials.
ABT-888 (Abbott): multiple diverse tumor models
RANKL Inhibitor (Amgen): denosumab and bone metastasis models
Sutent (Pfizer): subcutaneous tumor xenograft
Dasatinib (Bristol-Myers Squibb): chronic myelogenous leukemia
Tasigna (Novartis): leukemia/metastasis model
Cubicin (Cubist Pharmaceuticals): bacterial peritonitis model
CG0070 (Cell Genesys): orthotopic bladder cancer
Panzem (EntreMed Pharmaceuticals): orthotopic gliosarcoma
AEE788 (Novartis): intraperitoneal tumor model
IT-101 (Insert Therapeutics): Ewings sarcoma xenograft
PS-341 (Millennium Pharmaceuticals): intraperitoneal xenograft
Aflibercept (Sanofi-Aventis): orthotopic renal cancer
CHIR-258 (Novartis): orthotopic multiple myeloma model
NPI-0052 (Nereus Pharmaceuticals): subcutaneous tumor model
Peer reviewed references for each example in the IVIS bibliography
24
Longitudinal Non-Invasive Monitoring of Disease (and Treatment)
25
Spontaneous Metastases from Orthotopic Mammary Fat Pad Implant
Brachial
Lymph Node
Representative Mouse #12,
Ventral View Female Nude (Harlan)
In vivo Image
Right Brachial
Lymph Node
Ex vivo Image Histopathology
Micrometastasis Right
Brachial Lymph Node
Exp #078
Female Nude, Harlan
n=7
Images 3 min, 10 bin, FOV 15
-5000
-4000
-3000
-2000
-1000
counts
-1000
- 800
- 600
- 400
- 200
counts
Mouse #12
200x
MDA-MB-231-luc (D3H1), 2x106 cells, mfp
26
Untreated 9L-Glioma (Rat)
Source: Brian Ross, Ph.D., University of Michigan Medical Center
-2 0 4 10
14 x 103
12
10
8
6
4
2
27
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
28
J Clinic Invest
Massauge group, 2005
Tumor Induced Damage Monitoring
Cell Model: MDA-MB-231 (breast cancer cell line)
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
Aflibercept (Sanofi-Aventis)
Mouse Xenograft: Orthotopic Renal Cancer Model
Transfected cells injected into subcapsular space
of left kidney.
Mice sorted according to tumor burden estimated
by luciferase expression using an IVIS system.
IVIS® imaging system used to monitor changes in
tumor burden during 30 days of treatment.
Decrease in luciferase expression measured by
IVIS correlated well with decrease in tumor weight
and with mouse survival
Aflibercept is in Phase II and III clinical trials for multiple indications including one for Renal Cancer
Verheul et al., Clin Cancer Res 2007;13(14) July 15, 2007, p. 4201–4208
32
Cell Trafficking, Survival and Engraftment Patterns In Vivo
33
Cell Transplantation and Trafficking Patterns
Cao et al, Stem Cells,2004
Stem Cell Foci Formation and Hematopoiesis
Transplantation of 250 Luc+ HSC into Lethally Irradiated Hosts
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.
Ultrasound Guided Delivery of
Bioluminescent Cardiomyoblasts
Rodriguez-Porcel & Gambhir et al, 2005
心成肌細胞
Image-guided cardiac cell delivery
35
Stroke Kim et al, 2004
Yellew: Infarcted site
White: Injection site
Neuron Progenitor Cell Tracing
Stroke Repairing
Dynamic tracking of stem cells in an acute liver
failure model
World J Gastroenterol 2012 February 14; 18(6): 507-516
New drug development & in vivo tracking
39
Neurotropic Viral Infection
• Viral Model: Sindbis Virus (Alphavirus)
• 103 CFU virus inoculated at right hind foot
J Virol Cook & Griffin, 2003
Ljubimova et al., Nanomedicine 2008
Multitargeting Drug Delivery Poly (malic acid) nanoconjugates
Polycefin family of natural-derived
polymeric drug-delivery vehicles
Heirarchic conjugation of functional
groups onto the backbone of poly(malic acid),
an alipathic polyester from the microorganism
Physarum polycephalum
Conjugated with AlexaFluor 680
Antibody to transferrin receptor -
overexpressed on tumor cells
Antibody to nucleosome specific 2C5 that
reacts with various cancer cells via tumor cell
surface-bound nucleosomes
NIRF Imaging of Arthritis
Lee et al,Biomaterials,2008
NIRF-HA based gold nanoprobes detect reactive oxygen species (ROS) and
hyaluronidase (HAdase).
in vivo fluorescence imaging of subcutaneous
U87MG glioblastoma
Cancer research (2004) 64: 8009
RGD-Cy5.5
Fluorescence probe
Cubicin (Daptomycin) is an FDA approved drug for infections caused by Staphylococcus aureus
Mortin et al., Antimicrobial Agents and Chemotherapy, May 2007, p. 1787–1794
New drug development 1. Noninvasive assessment of cancer response to
therapy. Nat Med. Mar 2008
2. Preparation of peptide-conjugated quantum dots for
tumor vasculature-targeted imaging. Nature protocols.
2008
3. Magnetically retainable microparticles for drug
delivery to the joint: efficacy studies in an antigen-
induced arthritis model in mice. Arthritis Res Ther. 2009
4. Molecular imaging of phosphorylation events for drug
development. Mol Imaging Biol. May-Jun 2009
5. 2'-Hydroxycinnamaldehyde shows antitumor activity
against oral cancer in vitro and in vivo in a rat tumor
model. Anticancer Res. Feb 2010.
6. Revisiting Old Drugs as Novel Agents for
Retinoblastoma: In vitro and In vivo Antitumor Activity
of Cardenolides. Invest Ophthalmol VisSci. 2009.
45
Genetic Components of Disease Expression, Pathways and Therapy
LPTA® Animal Models
Genetically modified to emit light when a tagged gene is activated
Validated for key therapeutic areas
Accelerates clinical development candidate selection
Biophotonic
Animal Models
Drug Metabolism Toxicology
Oncology Inflammation
Neurology Metabolic Disease
Brain Kidney Pancreas Liver
Rat Insulin Promoter -islet Cells
Pancreatic Islets from RIP-luc Tg Mouse Transplanted
into NOD Host
RIP-luc Streptozotocin Treatment
Decreased Luciferase Expression
10.0
8.0
6.0
4.0
2.0
Day 0 2 7 14 21
Control
STZ
Ph
oto
ns/s
(x10
3) * = P≤0.05
** = P≤0.01
Pancreas Luminometer Assay
0
2
4
6
8
10
12
RL
U/m
g P
rote
in Control
STZ
*
Luciferase Signal
0.0
1.0
2.0
3.0
4.0
5.0
6.0
0 4 9 14 19 Days
Ph
oto
ns/s
(x10
6)
Control (n=10)
STZ (n=9)
*
*
*
* ** **
** **
**
50
TRANSGENICS (LPTA) Vascular Endothelial Cell Growth Factor Receptor
VEGF - secreted during angiogenesis VEGFR2 - surface receptor for VEGF
VEGF induction of VEGFR2 promoter
causes luciferase expression
VEGFR2 Pr luc
CHROMOSOME
Day 14
Transgenic VEGFR2-luc
Embryos in a Non- Tg Mother
Reduction of VEGFR2 Expression During Development
1 Week
Signal Intensity
100,000
2 Weeks
Signal Intensity
10,000
1000
2000
3000
4000
5000
4 Weeks
Signal Intensity
200
6 Weeks
Signal Intensity
<100
200
400
600
800
1000
200
400
600
800
1000 60
35
40
45
50
55
30
x10
3
Zhang et al. (Xenogen), 2004
Induction of VEGFR-2 Expression During Wound Healing
Day 0 Day 4 Day 6 Day 11 Day 18 Day 24
Zhang et al. (Xenogen), 2004
Monitoring Angiogenesis During Tumor Development
VEGFR2-luc KI mice implanted with 106 LL/2-GFP tumor cells and imaged at day18
Tumor growth and
development imaged by
GFP fluorescence
Angiogenesis imaged by
VEGFR2-luc expressing
bioluminescence
NFĸB Induction by LPS
Control LPS
Peyer’s
patches
7
4
0
24
Zhang, Xenogen & Rune Blomhoff, Oslo
Effect of Anti-inflammation Agents on NF-kB Activation
Effect of Dexamethasone and EGCG on iNOS Expression
Zhang et al. (Xenogen), 2003
Lights on Hypoxia Animal Model: HIF-Luc
Hypoxia Inducible Factor (HIF)
Promote adaptation to hypoxia
Ischemic Disease
Proc. Natl. Acad. Sci. USA
Safran, Michal et al. ,2006