2. 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. 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. 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 Imaging
cm 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. 5
 Hemodynamic parameters (H2
15O, 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

6. 6

7. 7

8. 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. 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. 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. 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. 12
Color Selection ♦ Brightness ♦ Photostability
Dyes for Whole Animal
Optical Imaging
Dyes for Cellular
Optical Imaging
Bone
Liver
Kidneys

13. 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-CEAAntibody-AlexaFluor® 750
0
20
40
60
80
100
120
140
160
0 10 20 30 40 50
Time (hrs)
LiverFluorescence
DOL 1.1
DOL 2.4
DOL 3.9
DOL 6.0

14. 14
40 min
120 min
tumor
tumor
Fluorescent Glucose: Potential Tumor Metabolic Marker

15. 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. 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. 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. 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. 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. 20
BSA: Capillary Leak QTracker 800: Vasculature

21. 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. 22
Anti-CEA-AlexaFluor® 680
Qtracker® 800 non-targeted quantum dots
Composite
Combining Blood Flow with Targeting

23. 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. 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)
Fluorescence(X10
6
)
Accumulation 2 m Fluorescent Microspheres At
Site of Inflammation
0
1
2
3
4
5
6
0 5 10 15
Time (Days)
Fluorescence(x10
6
)

25. 25
Fluorescent Microspheres Non-Targeted Quantum Dots

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

27. 27
Mouse leg bone

28. 28
Front leg sternum
Bronchiolar epithelium

29. 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. 30
What’s Next ?

31. 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

32. Intramolecularly
quenched substrate
Protease
Fluorescent cleavage
products

33. 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. 34
Z-DEVD-R110
Nonfluorescent
Caspase 3Caspase 3
Rhodamine 110
Fluorescent
O NH
C
O
O
HN Asp Val Glu Asp CBZAspValGluAspCBZ
O
C
H
2
N
O
O
-
NH
2

35. 35
0
50000
100000
150000
200000
250000
300000
0 50 100 150 200 250 300
Time (minutes)
Fluorescence(485/525nm)
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. 36
OCH
OCH
2
CH
2
O P
O
O

OCH
2
CH
2
NH
CCH
3
(CH
2
)
14
O
C
O
(CH
2
)
4
H
3
C
H
3
C
F F
N
B
N C (CH
2
)
5
NH
O
NO
2
O
2
N
HOCH
OCH
2
CH
2
O P
O
O

OCH
2
CH
2
NH
CCH
3
(CH
2
)
14
O
C (CH
2
)
5
NH
O
NO
2
O
2
N
C
O
(CH
2
)
4
H
3
C
H
3
C
F F
N
B
N
OH
Fluorescent Fatty Acid
Phospholipase A2
cleavage
Intramolecularly Quenched Substrate

37. 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
H3C
FF
(CH2)4 C
O
H3C
CH2
CH
CH2 O P O
O
O-
CH2CH2NH
CH3
C
O
(CH2)5 NH2
(CH2)14 C O
O
O
N
B
N
H3C
FF
(CH2)4 C
O
H3C
CH2
CH
CH2 O P O
O
O-
CH2CH2NH
CH3
C
O
(CH2)5 NH
O2N
NO2

38. 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. 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 BrainSignal to Background
137 123 137 123 137 123 137 123 137 123
GB123
GB137
Tumor

40. 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. 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. 42
Organelle Lights™ Mito-GFP reagent
100 X
Nikon
Organelle Lights™ ER-GFP reagent
63 X
Zeiss Axiovert

43. 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. 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. 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. 46
Immunohisto-
chemistry
Cellular
Imaging
In Vivo
Imaging
CRI Instrument:
Spectral Deconvolution
Validation
Discovery
Verification
Workflow Integration

47. 47
Larry Greenfield
Louis Leong
Birte Aggeler
Hee Chol Kang
Yi-Zhen Hu
Iain Johnson
Julie Nyhus
Matthew Shallice
Tom Steinberg
Yu-Zhong Zhang