2008-05-13 optical imaging nih presentation
TRANSCRIPT
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“The visual representation, characterization, and quantification of
biological processes at the cellular and subcellular levels within
intact living organisms.” (Massoud and Gambhir, 2003)
Combining the targeting technology of molecular biology with the
detection technology of imaging instrumentation
and function
There are a number of drivers in Small Animal Imaging
–
– Integrates both temporal and spatial biodistribution of a
molecular probe
– Value of
for basic biological research
– Can efficiently survey whole animals
– Potential for screening
– Eventually bridge between animal studies and human studies
Translation of in vitro technology to an in vivo technology
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(Massoud and
Gambhir, 2003)
–
Animal Imaging
(Molecular Imaging)
Invasive/Minimally Invasive
(Intravital Imaging)
Non-Invasive
(Whole Animal Imaging)
Microscopy
Fiber Optic
Optical
Other Modalities
Physiological
Microscopes
Fluorescence
Planar Imaging
Bioluminescnce
Fluorescence
Tomography
Fluorescence
Planar Imaging
MRI
CT
PET
SPECT
UltraSound
Current industry focus on the instrument….dearth of reagents and applications
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Ultrasound mm 50 m
X-ray Computed Tomography No Limit 50 m
Magnetic Resonance Imaging No Limit 10 - 100 m
Positron Emission
Tomography No Limit 1 – 2 mm
Single Photon Emission
Computed Tomography No Limit 1 – 2 mm
Bioluminescence Imagingcm Several mm
Fluorescence Tomography 5 – 6 cm 1 – 2 mm
Fluorescence Imaging (Planar) < 1 cm 1 – 2 mm
0 – 150 m 2.5 m
Adapted from Weissleder (2002) Nature Reviews Cancer 2:1-8
Confocal Microscopy addresses the resolution limitation of whole animal imaging
High sensitivity, low cost, & ease of use take Optical Imaging to the benchtop
Combining Imaging Modalities Enables Animal Physiology in light of Anatomy
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Hemodynamic parameters (H2
15O,
15O-
butanol, 11
CO, 13
NH3…)
Substrate metabolism (18
F-FDG, 15
O2,
11C-
palmitic acid…)
Protein synthesis (11
C-leucine, 11
C-methionine,
11C-tyrosine)
Enzymatic activity (11
C-deprenyl, 18
F-
deoxyuracil…)
Drugs (11
C-cocaine, 13
N-cisplatin, 18
F-
fluorouracil…)
Receptor affinity (11
C-raclopride, 11
C-
carfentanil, 11
C-scopalamine)
Neurotransmitter biochemistry (18
F-
fluorodopa, 11
C-ephedrine…)
Gene expression (18
F-penciclovir, 18
F-
antisense oligonucleotides…)
MINItrace PET Tracer Production System
11C, 13N, 15O, 18F
Limited Repertoire, Radionucleotides and Requires Access to Cyclotron
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7
8
Average selling price
Micro-PET without cyclotron $600,000
Micro-SPECT/CT without cyclotron $500,000
Micro-CT $243,000
Micro-MRI $1,000,000
Frost and Sullivan, 2004
Dr. Bradley E. Patt. President and Co-Founder, Gamma MedicalTM
, Inc.
…
.”
Mr. Alexander Tokman, General Manager, Genomics and Molecular Imaging at GE Healthcare.
C. Sur (Merck and Co., Inc.) Fifth Inter-Institute Workshop on Optical Diagnostics Imaging from Bench to Bedside at the National
Institutes of Health. 25-27 September 2006
9 Goal: High Content In Vivo Imaging
Where
External image of bone
metastasis From Hoffman (2002). Green Fluorescent Protein Imaging of Tumour Growth,
Metastasis, and Angiogenesis in mouse models. The Lancet Oncology.
3:546-556
Functional Activity
Real-time imaging of protease
inhibition From Mahmood and Weissleder (2003). Near-Infrared Optical Imaging of Proteases
in Cancer. Molecular Cancer Therapeutics. 2: 489-496.
When
Near-infrared images after injection
with endostatin-Cy5.5
From Hassan and Klaunberg. (2004) Biomedical Applications of Fluorescence Imaging In
Vivo. Comparative Medicine. 54(6): 635-644
Why
Disease is multifactorial
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High Content In Vivo Imaging: More Information Per Experiment
Imaging of multiple targets with a disease process Imaging targets in atherothrombosis
Processes of atherogenesis ranging from pre-lesional to
advanced plaque Choudhury, Fuster and Fayad (2004) Nature Reviews Drug Discovery 3: 913-925
Profiling proteases within normal and
cancer cells Affinity labeling of papain family proteases using fluorescence
activity-based probes From Greenbaum et al (2002). Chemical Approaches for functional Probing the Proteome.
Molecular and Cellular Proteomics 1:60-68.
Multiplex with:
1. Labeled antibody
2. Intravascular
marker (blood flow)
3. Interstitial marker
(capillary leak)
Antibody Localization Massoud and Gambhir (2003). Molecular Imaging in Living Subjects: Seeing
Fundamental Biological Processes in a new light. Genes & Development 17: 545-580.
Disease model validation Visualization of angiogenesis in live tumor tissue
GFP-expressing blood vessels visualized in the RFP-
expressing mouse melanoma Yan M, Li L, Jiang P, Moossa AR, Penman S, and Hoffman RM (2003) PNAS 100 (24):
14259-14262
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Near Infrared Fluorescent Dyes Allow Higher Sensitivity
Near Infrared 770 – 1400 nm
Absorption coefficient as function of wavelength for water and tissue
Blue Green Red
Near IR
Plot of the peak intensity as a function of source depth
At 1 cm, attenuation factor is: -Blue spectral region: 10-10 -Near IR spectral: 10-2
Troy, Jekic-McMullen, Sambucetti and Rice (2004) Molecular Imaging 3(1):9-23.
Optical microscopy does not have the same limitations
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Color Selection ♦ Brightness ♦ Photostability
Dyes for Whole Animal
Optical Imaging
Dyes for Cellular
Optical Imaging
Bone
Liver
Kidneys
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An athymic nu/nu mouse was injected with 106 LS174T Human Colorectal Adenocarcinoma cells (ATCC CL-188) subcutaneously. When the tumor mass reached one centimeter in diameter, 50 g of AlexaFluor750-labeled Anti-CEA antibody was injected IV into the tail vein. The image was obtained 24 hours post injection.
Over-Derivatization Increases Clearance, Reducing Specific Localization
Left: CEA+ LS174T tumor bearing nu/nu mouse Right: CEA- SW620 tumor bearing nu/nu mouse Imaged with CRi Maestro Imaging System (Ex: 740nm; Em: 790-950 nm)
Degree of Labeling: Fluorophores per antibody High Medium Low
Effect of Degree of Antibody Labeling
Anti-CEA Antibody-AlexaFluor® 750
0
20
40
60
80
100
120
140
160
0 10 20 30 40 50
Time (hrs)
Liv
er
Flu
ore
scen
ce
DOL 1.1
DOL 2.4
DOL 3.9
DOL 6.0
14
40 min
120 min
tumor
tumor
Fluorescent Glucose: Potential Tumor Metabolic Marker
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655 605 585 565 525 nm
25nm
Size of the nanocrystal determines the color
Size is tunable from ~2-10 nm (±3%)
Size distribution determines the spectral width
Highly fluorescent, nanometer-size, single crystals of semiconductor materials - semiconductors “shrunk” to the size of a protein yield optical properties
~6nm ~2nm
Bright, narrow spectrum enable multispectral applications
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Core Nanocrystal (CdSe) - Size determines color
Inorganic Shell (ZnS) - Electronic & chemical barrier - Improves brightness and stability
Organic Coating - Provides water solubility &
functional groups for conjugation to Abs, oligonucleotides, proteins, or small molecules
Biomolecules -Covalently attached to polymer shell
- Immuoglobulins - Streptavidin, Protein A - Receptor ligands - Oligonucleotides
-Available in Innovator’s Toolkit
15 - 18 nm
Approximately the size of IgM or Ferritin -require different fixation methods (see web for protocols)
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400 500 600 700 800 900
Wavelength (nm)
525
605
655
705
800
565
Minimal (<5%) cross-talk using 20nm bandpass filters
Simplified signal un-mixing >> simplified multiplex labeling
Well-separated narrow spectra enable multiplexing
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Non-toxic
Provide analysis of phenotype, metabolism, proliferation,
differentiation
Quantum dots remain within cell
Are passed to daughter cells for 6-8 generations typically
Are ideal tools for studying cell-cell interactions
Are ideal tools for tracking cell fate in living systems
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In-vivo Vascular Imaging
• Venous injection at increasing resolution
• Bright signal allows highly detailed vascular
analysis
• Red colors allow deeper, higher resolution
imaging than dyes
• Long circulation times allows detailed
vascular imaging
QTracker® 800 labeling vasculature
nu/nu mouse
LS174T xenograft
Ex: 465nm Em: 740-950nm
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BSA: Capillary Leak QTracker 800: Vasculature
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5 min 1 hour 2 hours
Qtracker® 655 non-targeted quantum dots
Bovine Serum Albumin (BSA), Alexa Fluor® 750 conjugate
Qtracker® for Blood flow, BSA for Capillary Permeability
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Anti-CEA-AlexaFluor® 680
Qtracker® 800 non-targeted quantum dots
Composite
Combining Blood Flow with Targeting
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A multicolored mixture of FluoSpheres® fluorescent microspheres imaged through red, green, and blue filter sets. The three fluorescent images were then overlaid onto a differential interference contrast (DIC) image.
A double-labeled microsphere from the FocalCheck DoubleGreen Fluorescent Microsphere Kit. The bead was imaged as a z-series using a Carl Zeiss LSM 510 META system. The two green-fluorescent dyes were separated by spectral unmixing, and one of the dyes was pseudocolored red. In this composite image, the complete z-series is shown prior to software rendering. Rendering fills in the missing information between the slices by interpolation to create a solid object.
Cat # Product Name
S31201 SAIVI 715 injectable contrast agent *0.1 m microspheres
S31203 SAIVI 715 injectable contrast agent *2 m microspheres
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Imaging of 0.1 m and 2 m Fluorescent Microspheres in an arthritic model 100 L of 1% 0.1 m fluorescent microspheres were injected
Inflammation was modeled by inducing polyarticular collagen-induced arthritis (CIA) in 4-6 week old female Balb/c mice. Antibody-mediated CIA was induced
by intravenous injection of 2 mg Artrogen-CIA Monoclonal Antibody Blend (Chemicon). Three days after antibody treatment, each mouse received 50 g
Lipopolysaccharide (LPS; Chemicon) intraperitoneally. Seven days after the initial injection, the mice had recovered from the LPS toxicity and symptoms of
arthritis were observed.
Accumulation 0.1 m Fluorescent Microspheres At
Site of Inflammation
0
1
2
3
4
5
6
0 5 10 15 20 25 30
Time (Days)
Flu
ore
scen
ce (
X 1
06)
Accumulation 2 m Fluorescent Microspheres At
Site of Inflammation
0
1
2
3
4
5
6
0 5 10 15
Time (Days)
Flu
ore
scen
ce (
x10
6)
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Fluorescent Microspheres Non-Targeted Quantum Dots
26 Quantum Dots coated with Surface 1 appear limited to Kupffer cells
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Mouse leg bone
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Front leg sternum
Bronchiolar epithelium
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Five-color lymphatic drainage imaging of mice injected
with five different distinct G6-(Bz-DTPA)119-(NIR)4-(Bz-
DTPA-111In)1 nanoprobes.. Five primary draining lymph
nodes were simultaneously visualized with different
colors through the skin. Kobayashi H, Koyama Y, Barrett T, Hama Y,
Regino CAS, Shin IS, Jang B-S, Le N, Paik CH, Choyke PL, and Urano Y.
(2007). Multimodal Nanoprobes for Radionuclide and Five-Color Near-
Infrared Optical Lymphatic Imaging. ACS Nano. 1 (4): 258-264.
Radionuclide Optical Radionuclide Optical
Post-mortem
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What’s Next ?
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Invitrogen Has Expertise In Designing Labeled Substrates
Optimal In vivo Functional Probe:
• Localize to point of interest
• Enzyme recognizes probe as a
substrate
• Fluorescent product concentrates
in locality of target
• Fluorogenic substrate
• Product entrapment
• Fluorescent product remain in
locality of target
• Signal amplification NIR fluorescence imaging using
a cathepsin B-activatable probe Weissleder and Ntziachristos (2003)
Nature Medicine 9(1):123-128.
Fluorogenic Protease Substrates
Activity-Based Probes
Intramolecularly
quenched substrate
Protease
Fluorescent cleavage
products
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In-situ gelatinolytic activity in 10 µm coronal brain sections detected using
DQ gelatin. Gelatinolytic activity is associated with induction of cortical spreading depression on
one side of the cortex (CSD) and not the other (nCSD). C shows the region marked by a square in
A at higher magnification. D and E show localized gelatinolytic activity in blood vessels (J Clin
Investigation 113:1447–1455 (2004))
3 hrs 24 hrs
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Z-DEVD-R110
Nonfluorescent
Caspase 3 Caspase 3
Rhodamine 110
Fluorescent
O NH
CO
O
HN Asp Val Glu Asp CBZAspValGluAspCBZ
O
C
H2
N
O
O-
NH2
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0
50000
100000
150000
200000
250000
300000
0 50 100 150 200 250 300
Time (minutes)
Flu
ore
scen
ce (
485/5
25 n
m)
0.00
0.26
0.52
1.04
2.08
4.17
8.33
16.67
[Ac-DEVD-CHO] (nM) Inhibition of staurosporine-induced (t=0) caspase 3 activity in HeLa cells
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OCH
OCH2
CH2
O P
O
O
OCH2
CH2
NH
CCH3
(CH2
)14
O
C
O
(CH2
)4
H3
C
H3
C
F F
NB
N C (CH2
)5
NH
O
NO2
O2
N
HOCH
OCH2
CH2
O P
O
O
OCH2
CH2
NH
CCH3
(CH2
)14
O
C (CH2
)5
NH
O
NO2
O2
NC
O
(CH2
)4
H3
C
H3
C
F F
NB
N
OH
Fluorescent Fatty Acid
Phospholipase A2
cleavage
Intramolecularly Quenched Substrate
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Imaging of enzymatic activity in contrast to substrate distribution
Science 292:1385–1388 (2001)
PED6 (D23739)
Phospholipase A2-activity
dependent probe
BODIPY PC
Phospholipase-independent
lipid marker
Unquenched probe demonstrates
uptake through swallowing
gall bladder
pharynx
gall bladder
intestine
Atorvastatin (ATR) inhibits processing (absorption) of PED6 (fat soluble) (F) but not of BODIPY FL-C5 (water soluble, short chain fatty acid) (G)
Phospholipase A2
(CH2)14 C O
O
O
N
B
N
H3CFF
(CH2)4 C
O
H3C
CH2
CH
CH2 O P O
O
O-
CH2CH2NH
CH3
C
O
(CH2)5NH2
(CH2)14 C O
O
O
N
B
N
H3CFF
(CH2)4 C
O
H3C
CH2
CH
CH2 O P O
O
O-
CH2CH2NH
CH3
C
O
(CH2)5 NH
O2N
NO2
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530/550-BODIPY DCG-04
Molecular Probes’ dyes have been used as Activity-Based Probes In vivo
In vivo profiling of cathepsin activity
during RIP-TAG tumorigenesis
BODIPY530/550-DCG-01 (161 g, 150
nmoles) injected IV (tail vein). Following 1 – 2
hours, animals were fixed, the pancreas
isolated Joyce et al. (2004) Cathepsin Cysteine Proteases Are Effectors
of Invasive Growth And Angiogenesis During Multistage
Tumorignesis. Cancer Cell 5:443-453
DCG-04 signal (A,C,E,G) and
DAPI/DCG-04 merged islets
A, B: Normal islets
C, D: Dyslastic islets
E, F: Tumors
G, H: Invasive tumor fronts
Competition experiments on tumor lysates demonstrating specificity of the DCG-04 probe. Incubation of equally loaded tumor lysates with a broad-spectrum inhibitor, JPM-OEt, abolishes activity in the 30-40 kDa range, whereas incubation with MB-074, a cathepsin B-specific inhibitor, abolishes cathepsin B activity (*) Cat B
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From: Blum G, von Degenfeld GV, Merchant MJ, Blau HM and Bogyo M (2007). Noninvasive optical imaging of cysteine protease activity using
fluorescently quenched activity-based probes. Nature Chemical Biology. 3 (10): 668-677.
QB137: Quenched
QB123: Nonquenched
- Cat B - Cat L - Cat L
Tumor Liver Kidney Spleen Brain Signal to Background 137 123 137 123 137 123 137 123 137 123
GB123
GB137
Tumor
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•Phospholipidosis LipidTOX™
phospholipid stains
No Chloroquine
Detection of Phospholipidosis and Steatosis in HepG2 Cells
No CsA
10 M Chloroquine
30 M CsA
LipidTOX™ Detection Kits for “Pre-Lethal” Cytotoxoicity Screening
•Steatosis LipidTOX™
neutral lipid stains
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Color change upon Ca2+
release
+Ca2+ HEK 293T cells
Owl Monkey Kidney Cells 20 µM ATP
Owl Monkey Kidney Cells Stimulated with ATP Photographed with Olympus Flow View 1000
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Organelle Lights™ Mito-GFP reagent 100 X
Nikon
Organelle Lights™ ER-GFP reagent 63 X
Zeiss Axiovert
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A viable bovine pulmonary artery endothelial cell
incubated with the ratiometric mitochondrial potential
indicator, JC-9. In live cells, JC-9 exists either as a
green-fluorescent monomer at depolarized
membrane potentials, or as a red-fluorescent J-
aggregate at hyperpolarized membrane potentials. Imaging the Brain. Imaging in Alzheimer’s disease
models. Three-color in vivo multiphoton image showing
a plaque (blue, stained with a vital amyloid dye)
surrounded by brain vasculature (red, filled with
fluorescent dextran)l and neurites labeled with
fluorescent protein. Misgeld T, and Kerschensteiner M. (2006).
In vivo imaging of the diseased nervous system. Nature Reviews
Neuroscience. 7: 449-463.
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Imaging of Ca2+ waves in
gastrulating zebrafish embryos
detected by microinjected f-
aequorin (recombinant aequorin
reconstituted with the
coelentrazine f luminophore).
The images are pseudocolored
to represent Ca2+-dependent
luminescent flux. The sequences
depict three different spatial
wave types that are represented
scehmatically at the end. Gilland E,
Miller AL, Karplus E, Baker R, and Webb SE.
(1999) Imaging of multicellular large-scale
rhythmic calcium waves during zebrafish
gastrulation. Proc. Natl. Acad. Sci. USA. 96:
157-161.
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Monitoring reporter gene expression from a fusion vector Fusion of a PET reporter gene (tk) and an optical bioluminescence reporter gene (rl ) rl - renilla luciferase Tk – thymidine kinase FHBG – 9-4-[18F]fluoro-3-hydroxymethylbutyl)guanine
Imaging serial increase in rl gene expression over time in tumors stably expressing the tk20rl fusion Ray, Wu and Gambhir (2003). Cancer Research 93: 1160-1165
Time course of luciferase signal following intraperitoneal injection of luciferin Burgos, Rosol, Moats, Vhankaldyyan, Kohn, Nelson, Jr, and Laug (2003) Biotechniques 34: 1184-1188
Fluorogenic Reporter Systems Are in Progress
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Immunohisto-
chemistry
Cellular
Imaging
In Vivo
Imaging
CRI Instrument:
Spectral Deconvolution
Validation
Discovery
Verification
Workflow Integration
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Larry Greenfield
Louis Leong
Birte Aggeler
Hee Chol Kang
Yi-Zhen Hu
Iain Johnson
Julie Nyhus
Matthew Shallice
Tom Steinberg
Yu-Zhong Zhang