2

“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

3

(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

4

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

5

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

6

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

10

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

11

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

12

Color Selection ♦ Brightness ♦ Photostability

Dyes for Whole Animal

Optical Imaging

Dyes for Cellular

Optical Imaging

Bone

Liver

Kidneys

13

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

15

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

16

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)

17

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

18

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

19

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

20

BSA: Capillary Leak QTracker 800: Vasculature

21

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

22

Anti-CEA-AlexaFluor® 680

Qtracker® 800 non-targeted quantum dots

Composite

Combining Blood Flow with Targeting

23

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

24

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)

25

Fluorescent Microspheres Non-Targeted Quantum Dots

26 Quantum Dots coated with Surface 1 appear limited to Kupffer cells

27

Mouse leg bone

28

Front leg sternum

Bronchiolar epithelium

29

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

30

What’s Next ?

31

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

33

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

34

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

35

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

36

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

37

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

38

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

39

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

40

•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

41

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

42

Organelle Lights™ Mito-GFP reagent 100 X

Nikon

Organelle Lights™ ER-GFP reagent 63 X

Zeiss Axiovert

43

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.

44

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.

45

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

46

Immunohisto-

chemistry

Cellular

Imaging

In Vivo

Imaging

CRI Instrument:

Spectral Deconvolution

Validation

Discovery

Verification

Workflow Integration

47

Larry Greenfield

Louis Leong

Birte Aggeler

Hee Chol Kang

Yi-Zhen Hu

Iain Johnson

Julie Nyhus

Matthew Shallice

Tom Steinberg

Yu-Zhong Zhang