1st Faculty of Medicine, Charles University in Prague Center for Advanced Preclinical Imaging (CAPI)
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI)
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Optical ImagingOI
ADVANTAGES DISADVANTAGES
• High sensitivity • Limited penetration depth
• Easy to handle • „NO“ quantification
• Established methods • Nude mice only (fluorescence)
• Cheap
• Functional Imaging
• No ionizing radiation
• in vivo – ex vivo
Optical Imaging is based on the detection of weak light by a highly sensitive
and high resolution CCD camera
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Optical Imagingreporter genes
• reporter gene produces a protein product which is able to signal where, when, and in what quantity it is being translated
• Reporter genes are inherently vital i.e. keep your cells alive (selection process)
• Reporter genes allow to image structure and function
• Attach a reporter to your gene of interest.
– Fusion Gene is your gene of interest fused to your reporter gene
– Fusion Protein is the expression of your Fusion Gene
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Optical Imagingfusion genes
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Optical Imagingreporter genes
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Optical Imagingreporter genes
• Bioluminescence reporter genes
– Enzymes from fireflies catalyze a reaction that produces light
– Reporter genes come in many colors
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
AbsorptionBiological (NIR) window
• Best light transmission in tissue in
near infra-red (NIR) (700-900 nm)
• Fluorophores, fluorescent proteins
or bioluminiscence that emitting light
at NIR wavelength preferred for in
vivo imaging
• Most optical systems measure the
intensity emitted at the surface of
the animal
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
AbsorptionHemoglobin
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Scattering
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
ScatteringRaman scattering
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Raman ScatteringLevel diagram
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Raman ScatteringBasic mechanism
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Raman SpectroscopySpectrum of oral bacteria
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Raman ScatteringImaging Applications
• Chemical analysis of tissue, in vitro AND in vivo (breast, artery, blood)
• Disease classification
topical review: Hanlon et al., “Prospects for in
vivo Raman spectroscopy,” Phys. Med. Biol. 45,
R1-R59 (2000)
• High-resolution, molecularly specific microscopy
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Illumination
Epi-Illumination Transillumination
Planar imaging implementations:
(a) Epi-illumination shines light from the same side that back-emitted light is
collected.
(b) Transillumination shines light though the volume of interest and collects
transmitted light in the opposite side.
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Advantages
Fast imaging technique
Good for near surface structures
Sensitivity dependend on
absorption and background
fluorescence
Disadvantages
Not quantitative
Sensitivity varies with wave
length
Fluorescence reflectance imaging Epi-Illumination
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
FluorescenceFluorescence reflectance imaging (Epi-Illumination)
In vivo epi-illumination imaging of cathepsin activity from a nude female
mouse with two HT1080 tumors implanted subcutaneously
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
FluorescenceEpi-Illumination vs Transillumination
Transillumination images yield similar nonlinear dependencies to epi-illumination images.
A notable feature of transillumination though is that the volume of interest is entirely
sampled because light propagates through it.
Top row:
MMTV/neu transgenic mouse;
spontaneous tumors
a) Epi illumination (excitation WL)
b) Transillumination (excitation WL)
c) Transillumination fluorescence
d) Corrected (fluorescence/excit. WL)
Bottom Row:
Postmortem image of fluorescent tube
inserted in oesophagus
e) Epi illumination (excitation WL)
f) Transillumination (excitation WL)
g) Transillumination fluorescence
h) Corrected (fluorescence/excit. WL)
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Tomographic Implementations
Tomographic implementations
a) Reflectance arrangements – all sources
and detectors are on the same side
b) Limited projection angle arrangement
c) Complete projection angle arrangement
(360°)
d) typical diffuse propagation pattern in
highly scattering media for a source and
a detector as indicated by the two
arrows. Photons propagate along
curved paths and a single source can
cover, in principle, the entire volume.
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
360° tomographic imaging
Complete projection tomographic imaging
(a) Cross section of a solid phantom experimentally imaged.
(b) tomographic image using 72 projections.
(c) Tomographic image using a single projection.
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
TomographyFMT – Fluorescence-mediated Molecular Tomography
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
In vivo FMT of 9L
gliosarkomas in
mice brain
TomographyFMT – Fluorescence-mediated Molecular Tomography
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
TomographyFMT – Fluorescence-mediated Molecular Tomography
Tomographic imaging of fluorescent proteins and corresponding X-ray CT from a nude
mouse implanted with GFP-expressing lung tumors, obtained 10 days post
implantation.
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Fluorescence Spectroscopy
Ref. Mycek and Pogue, Handbook of Biomedical Fluorescence
0
2
4
6
8
10
300 350 400 450 500 550 600 650 700
Flu
ore
sc
en
ce
In
ten
sit
y [
a.u
.]
Fluorescence emission wavelength [nm]
Porphyrins (Hp)
FlavinsElastin
Collagen
NADH
Tryptophan
Pyridoxine
BMajor biological fluorophores:
• structural proteins: collagen and
elastin crosslinks
• coenzymes for cellular energy
metabolism (electron acceptors):
• flavin adenine dinucleotide (FAD)
• nicotinamide adenine dinucleotide,
reduced form (NADH)
• aromatic amino acids: side groups
on proteins
• porphyrins: precursors to heme
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Instrumentation
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
InstrumentationPlatform design
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
InstrumentationPlatform design
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Instrumentation
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Optical Imaging
Optical Imaging
Fluorescence imaging
Non-Fluorescence-
based optical imaging
Microscopic fluorescence
imaging
Macroscopic fluorescence
imaging
bioluminescence imaging
optical coherence tomography
photoacoustic microscopy
tissue spectroscopy
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Luminiscence
www.answers.com/topic/glowing-tobacco-plant-jpg
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Optical ImagingNon-fluorescence based OI: luminescence imaging
luciferase–luciferin pairs
firefly (Photinus pyralis) luciferase–
luciferin
Renilla reniformis luciferase–
coelenterazine
Gaussia princeps luciferase–
coelenterazine
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Optical ImagingLuminiscence
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Optical ImagingLuminiscence
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Luminiscencereporter genes
• A method to image molecular interactions.
• Break luciferase into two pieces, make two fusion genes.
• If fusion genes interact, luciferase pieces can interact
• Interaction can be visualized by luciferin injection
Luker et al., 2004
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Fluorescence
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Fluorescence Imaging
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Absorption
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
AbsorptionElectronic transitions
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
AbsorptionVibrational transitions
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
AbsorptionRotational Transitions
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
AbsorptionHow to talk about absorption
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
AbsorptionFluorescence
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Optical ImagingFluorescence
Green Fluorescence Protein (GFP)
Excitation
e.g. ~480 nm
Emission
e.g. ~520 nm
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
FluorescenceFluorescence-dyes
genetik.fu-berlin.de/institut/en_GFP_fly3.jpg
GFPdriven by the Engrailed promoter
www.beverlytang.com/photos/gfp_fish.jpg
GFP Fish Commercially Available
bioephemera.com/wp-content/uploads/2007/04/albagreen.jpg
ALBAThe GFP bunny
Katushka vs. DsRedon muscle actin promoter
http://www.hhmi.org/sites/default/files/News/2007/redprotein20070826_287.jpg
www.answers.com/topic/glowing-tobacco-plant-jpg
•In vivo imaging is possible.
•Water the plant with a solution
containing luciferin and it begins to
glow.
Transgenic Luciferase Tobacco plant
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
Tracking Stem Cell Survival
1 day 4 days
Time after implantationMesenchymal stem cells
derived from human bone marrow
1st Faculty of Medicine, Charles University in Prague
Center for Advanced Preclinical Imaging (CAPI) [email protected]
THANK YOU FOR YOUR ATTENTION