small animal optical imaging
TRANSCRIPT
Small Animal In Vivo Imaging
(SAIVI)
Lawrence Greenfield, M.D., Ph.D.
Summary
• 25 years experience in developing fluorescent molecules
• 45 chemists available with expertise in organic and inorganic
dyes, ligands, and enzyme substrates
• Established catalog of reagents for in vitro imaging including
fluorescent molecules, antibodies and reporters
• Now applying our expertise and tools to enable animal imaging
• Looking to understand key characteristics required to make
effective animal imaging reagents
Applying Our Expertise To Enable Animal Imaging
What is Molecular Imaging?
• “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 to image and monitor
both cellular and animal physiology and function in vivo
• There are a number of drivers in Small Animal Imaging
– In vivo ≠in vitro
– Integrates both temporal and spatial biodistribution of a molecular probe
– Value of integrating molecular events with cellular and animal physiology
in vivo for basic biological research
– Can efficiently survey whole animals
– Potential for rapid in vivo screening
– Eventually bridge between animal studies and human studies
Translation of in vitro technology to an in vivo technology
Small Animal Imaging Modality Overview
• “The visual representation, characterization, and quantification of biological processes at the
cellular and subcellular levels within intact living organisms.” (Massoud and Gambhir, 2003) – Combines targeting technology of molecular biology with the detection technology of imaging instrumentation to image and
monitor both cellular function and animal physiology in vivo
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
High sensitivity, low cost, & ease of use take Optical Imaging to the benchtop
Comparison of Small Animal In Vivo Imaging
FT
Fluorescence Tomography
FI
Fluorescence Imaging (Planar)
BLI
Bioluminescence Imaging
SPECT Single Photon Emission
Computed Tomography
PET Positron Emission
Tomography
US
Ultrasound
CT
X-ray Computed Tomography
MRI
Magnetic Resonance Imaging
Modality Metabolism Physiology Anatomy Molecular Cost Sensitivity Depth
No limit
No limit
Resolution
50 mm mm
10 – 100 mm
50 mm
1 – 2 mm No limit
No limit 1 – 2 mm
Several mm cm
< 1 cm 1 – 2 mm
1 – 2 mm 5-6 cm
Adapted from Weissleder (2002) Nature Reviews Cancer 2:1-8
Biological Imaging Using PET
• Hemodynamic parameters (H215O, 15O-butanol, 11CO,
13NH3…)
• Substrate metabolism (18F-FDG, 15O2, 11C-palmitic
acid…)
• Protein synthesis (11C-leucine, 11C-methionine, 11C-
tyrosine)
• Enzymatic activity (11C-deprenyl, 18F-deoxyuracil…)
• Drugs (11C-cocaine, 13N-cisplatin, 18F-fluorouracil…)
• Receptor affinity (11C-raclopride, 11C-carfentanil, 11C-
scopalamine)
• Neurotransmitter biochemistry (18F-fluorodopa, 11C-
ephedrine…)
• Gene expression (18F-penciclovir, 18F-antisense
oligonucleotides…)
MINItrace PET Tracer Production System
11C, 13N, 15O, 18F
Limited Repertoire, Radionucleotides and Requires Access to Cyclotron
Current Status
Optical Imaging At Benchtop
Price comparison
Average selling price
Optical Imaging $115,000
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
“The number of small animal imaging labs being built all over the world is
staggering. Many of these centers are built to accommodate multiple
imaging modalities.
Dr. Bradley E. Patt. President and Co-Founder, Gamma MedicalTM
, Inc.
Multi-modality imaging is the key and GE has introduced the ExploreTM
product family that includes small animal CT, PET, and optical scanners.
… .”
Mr. Alexander Tokman, General Manager, Genomics and Molecular Imaging at GE
Healthcare.
Instrumentation
CRI Maestro CRI Nuance
Goal: High Content In Vivo Imaging
Objectives
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
Background: The Power of Multiplexing
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
Background: Near Infrared Dyes
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.
Color Selection ♦ Brightness ♦ Photostability
Number of Scans
Photostability/Internal Quenching
AF 488
AF 546
Fluorescein
Cy3
Comparison of photobleaching rates on
cytoskeleton of bovine pulmonary artery.
A Label is not a label …..
Protein Tumor T1/2b
(h)
TBC
(ml/h/kg)
[35S]260F9 No 180 0.38
[35S]260F9 Yes 61 1.1
111In-260F9 No 50 2.4
111In-260F9 Yes 44 2.6
Summary of Pharmacokinetic Parameters
Female Balb/C nude (nu/nu) mice with and without MX-1
tumors were injected with indicated radioactive proteins. The
r2 values ranged between 0.99 and 1.00. (Taken from
Greenfield & Dovey (1992). Antibody, Immunoconjugates
and Radiopharmaceuticals 5 (1): 73-59)
Labeling for Detection Labeling for Imaging
Effect Of Degree Of Derivatization
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 mg 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)
Accumulation AlexaFluor750-Labeled Anti-CEA
0
5
10
15
20
25
30
35
40
0 20 40 60 80 100 120 140 160 180
Time After Injection (Hours)
Sig
na
l:B
ack
gro
un
d
Degree of Labeling: Fluorophores per antibody High Medium Low
Effect of Degree of Derivatization
Effect of Degree of Antibody Labeling
Anti-CEA Antibody-AlexaFluor® 750
0
50
100
150
200
250
300
350
400
0 10 20 30 40 50
Time (hrs)
Tu
mo
r F
luo
rescen
ce
DOL 1.1
DOL 2.4
DOL 3.9
DOL 6.0
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
Effect of Degree of Antibody Labeling
Anti-CEA Antibody-AlexaFluor® 750
0
1
2
3
4
5
6
7
0 10 20 30 40 50
Time (hrs)
Tu
mo
r/L
iver
Flu
ore
scen
ce
DOL 1.1
DOL 2.4
DOL 3.9
DOL 6.0
DOL 1.1 DOL 2.4
DOL 3.9 DOL 6.0
Labeling For Optimal Tumor Localization
Robust Labeling of Antibodies
Mouse Polyclonal IgG: Alexa Fluor® 750
(n=3)
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
0 1 2 3
mg IgG
DO
L
Mouse Polyclonal IgG: Alexa Fluor® 680
(n=3)
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
0 1 2 3 4 5
mg IgG
DO
L
• Labeling at level optimal for
imaging
• Simple to use
No optimization required
Requires less material
Requires less preparation
• Reproducible labeling
Monoclonal Antibodies
Polyclonal Antibodies
AlexaFluor®488
AlexaFluor® 680
AlexaFluor® 750
AlexaFluor® 790
• Faster
• 1.5 hrs 5 hours
Contrast Reagent Characterization
Binding Curves for Anti CD3 label at two DOLS with AF488
0
20
40
60
80
100
120
140
1.00E-11 1.00E-10 1.00E-09 1.00E-08 1.00E-07
Antibody Concentration (M)
Sig
nal
of
4.3
DO
L
0
10
20
30
40
50
60
70
80
90
Sig
nal
of
2.7
DO
L
Anti CD3- 4.3 DOL
Anti CD3- 2.7 DOL
Anti CD3- 4.3 DOL Anti CD3- 2.7 DOL
One site binding (hyperbola)
Best-fit values
BMAX 125.7 87.04
KD 1.41E-09 1.07E-09
Rapid labeling kit applicable to flow cytometry
Binding Characterized by flow cytometry
Potential Tumor Marker
40 min
120 min
tumor
tumor
Potential Tumor Metabolic Marker
What Are Quantum Dots?
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
Quantum dot Conjugates are Engineered
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
Optimized for performance
Approximately the size of IgM or Ferritin -require different fixation methods (see web for protocols)
Emission Spectra of Quantum dot Conjugates
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
Quantum Dots with CRI Instrumentation
Qtracker® Cell Labeling Kits
• 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
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® Non-targeted contrast
QTracker® 800 labeling vasculature
nu/nu mouse
LS174T xenograft
Ex: 465nm Em: 740-950nm
Real Time Physiology
BSA: Capillary Leak QTracker 800: Vasculature
Monitoring Tumor Blood Physiology
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
Multiplexing with the CRi Maestro
Anti-CEA-AlexaFluor® 680
Qtracker® 800 non-targeted quantum dots
Composite
Combining Blood Flow with Targeting
Imaging: 7 Days Post Injection
Su
rfa
ce
2
Su
rfa
ce
4
Su
rfa
ce
2
Front leg
Lungs
Su
rfa
ce
5
Gross
Su
rfa
ce
3
Su
rfa
ce
2
Gross
Su
rfa
ce
4
Su
rfa
ce
5
Liver: Surface 1
Liver: Surface 1 : Objective – 100X
•
•
Lung: Surface 1
Lung: Surface 5: Objective – 20X
Bronchiolar epithelium
Fluorescent Microspheres
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 mm microspheres
S31203 SAIVI 715 injectable contrast agent *2 mm microspheres
SAIVI 715 Injectable Contrast Agent Microspheres
Imaging of 0.1 mm and 2 mm Fluorescent Microspheres in an arthritic model 100 mL of 1% 0.1 mm 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 mg 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 mm 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 mm Fluorescent Microspheres At
Site of Inflammation
0
1
2
3
4
5
6
0 5 10 15
Time (Days)F
luo
rescen
ce (
x10
6)
Following blood flow
What’s Next ?
Bone imaging
Bone imaging
Control Bone imaging reagent
Supine
(tummy up)
Leg bones
Liver
Sternum
Spine
Lymph node
Prone
(tummy down)
Bone Imaging Reagent
spleen kidneys
spine
liver lungs
skin
leg
kidneys spleen
spine
liver
lungs
skin
intestine
Control Bone imaging reagent
Orientation
Bone Imaging Reagent found here
Bone imaging reagent --- Histologic Identification
Post injection of bone imaging reagent
Mouse leg bone
Bisphosphonate Labels
N+
O
HN O
S
-OO
O
S
O-
O
O
OH
PPO
O-
N
S
O-
O
O
SO
O
O-
Pamidronate-IRDye78PAM78
O-
O
O-
O-
Zaheer A., Lenkinski RE, Mahmood A, Jones AG, Cantley LC, and Frangioni JV. (2001) In vivo near-infrared fluorescence imaging of osteoblastic activity. Nature Biotechnology 19: 1148-1154.
Zaheer A, Murshed M, De Grand AM, Morgan TG, Karsenty G, and Frangioni JV. (2006). Optical Imaging of Hydroxyapatite in the Calcified Vasculature of Transgenic Animals. Arterioscler Thromb Vasc Biol. 25(6): 1132-1136.
Multiplex Capabilities
Probing Functional Activity
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
Activity Probes
Activity Probes As Enabling Technology
Flow Cytometry
Imaging
Zymography
(Histopathology)
Proteomics
High Content Screening
Confidential Information
Intramolecularly
quenched substrate
Protease
Fluorescent cleavage
products
Protease Detection: DQ Substrates
In-situ MMP-9 zymography
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
Fluorogenic aminopeptidase substrates
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
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
Real-time detection of caspase 3 activity
[Ac-DEVD-CHO] (nM) Inhibition of staurosporine-induced (t=0) caspase 3 activity in HeLa cells
PED6 phospholipase A2 substrate
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
PED6: In vivo Imaging of Lipid Metabolism
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
Activity-Based Probes In Vivo
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 mg, 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
Activity Probes: Caspase
Signal (SS Induced Jurkats) to Noise (Uninduced Jurkats) as
a Function of FAM-VAD-FMK Incubation Time
0
5
10
15
20
25
30
35
40
0 20 40 60 80 100 120 140
FAM-VAD-FMK Incubation Time (minutes)
Ra
tio
of
Ind
uc
ed
to
Un
ind
uc
ed
Sig
na
l
• Apoptosis induced for 4 hours with staurosporine
• Cells resuspended in fresh media with labeled
caspase probe for indicated times
•Phospholipidosis LipidTOX™
phospholipid stains
No Chloroquine
Detection of Phospholipidosis and Steatosis in HepG2 Cells
No CsA
10 mM Chloroquine
30 mM CsA
LipidTOX™ Detection Kits for “Pre-Lethal” Cytotoxoicity Screening
•Steatosis LipidTOX™
neutral lipid stains
Calcium flux in porcine stem cells
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
Multiplexing with Premo™ Organelle Lights
Organelle Lights™ Mito-GFP reagent 100 X
Nikon
Organelle Lights™ ER-GFP reagent 63 X
Zeiss Axiovert
In vivo imaging of gene expression
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
Whole Animal Imaging Systems
eXplore Optix System Advanced Research Technology Inc
Maestro™ Cambridge Research and Instrumentation, Inc
FMT – Fluorescent Molecular Tomography System ViSen Medical
Kodak Xenogen
Cell~Vizio MaunaKea
Our Goal is Quantification
Pharmacokinetics BSA Alexa Fluor 750
0.00
0.20
0.40
0.60
0.80
1.00
1.20
0 20 40 60 80 100 120
Time (min)
Flu
ore
scen
ce (
no
rmali
zed
6 m
in)
Tumor Interstitial
Body Interstitial
Tumor: Rectangle
Leg
Clearance of Dye-Modulator By Kidneys
0
100
200
300
400
500
600
700
800
0 50 100 150
Time (Minutes)
To
tal
Flu
ore
scen
ce
Nontumor-Bearing
Tumor-Bearing
From Pretty Pictures Pharmacokinetics / Pharmacodynamics
Work with instrumentation and Software for Quantification
0 min
95 min
35 min
Current SAIVITM
Imaging Agent Evaluation
Applications for biochemical assays with orthogonal dye pairs?
NON-TARGETED
0.1 µm microspheres pooling
in sites of Inflammation
INDUCED ARTHRITIS
TARGETED CONTRAST
Alexa Fluor® 750 Anti – CEA
24 hours Ex:687/Em:740
COLON CANCER
VASCULAR IMAGING
QTracker® 800 Labeling
Vasculature
PASSIVELY TARGETED
CONTRAST
VASCULAR PERMEABILITY
Fluorescent BSA
Custom • Conjugation • Small Molecule Synthesis
Integrated Solutions
Immunohisto-
chemistry
Cellular
Imaging
In Vivo
Imaging
CRI Instrument:
Spectral Deconvolution
Validation
Discovery
Verification
Workflow Integration
Acknowledgements
Larry Greenfield
Louis Leong
Birte Aggeler
Hee Chol Kang
Yi-Zhen Hu
Iain Johnson
Julie Nyhus
Matthew Shallice
Tom Steinberg
Yu-Zhong Zhang