1st Faculty of Medicine, Charles University in Prague ... · Optical Imaging reporter genes •...

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)

Center for Advanced Preclinical Imaging (CAPI) s.eigner@seznam.cz

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

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

Optical Imagingfusion genes

• Bioluminescence reporter genes

– Enzymes from fireflies catalyze a reaction that produces light

– Reporter genes come in many colors

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

AbsorptionHemoglobin

Scattering

ScatteringRaman scattering

Raman ScatteringLevel diagram

Raman ScatteringBasic mechanism

Raman SpectroscopySpectrum of oral bacteria

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

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.

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

FluorescenceFluorescence reflectance imaging (Epi-Illumination)

In vivo epi-illumination imaging of cathepsin activity from a nude female

mouse with two HT1080 tumors implanted subcutaneously

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)

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.

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.

TomographyFMT – Fluorescence-mediated Molecular Tomography

In vivo FMT of 9L

gliosarkomas in

mice brain

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.

Fluorescence Spectroscopy

Ref. Mycek and Pogue, Handbook of Biomedical Fluorescence

300 350 400 450 500 550 600 650 700

Fluorescence emission wavelength [nm]

Porphyrins (Hp)

FlavinsElastin

Collagen

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

Instrumentation

InstrumentationPlatform design

Instrumentation

Optical Imaging

Fluorescence imaging

Non-Fluorescence-

based optical imaging

Microscopic fluorescence

imaging

Macroscopic fluorescence

imaging

bioluminescence imaging

optical coherence tomography

photoacoustic microscopy

tissue spectroscopy

Luminiscence

www.answers.com/topic/glowing-tobacco-plant-jpg

Optical ImagingNon-fluorescence based OI: luminescence imaging

luciferase–luciferin pairs

firefly (Photinus pyralis) luciferase–

luciferin

Renilla reniformis luciferase–

coelenterazine

Gaussia princeps luciferase–

coelenterazine

Optical ImagingLuminiscence

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

Fluorescence

Fluorescence Imaging

Absorption

AbsorptionElectronic transitions

AbsorptionVibrational transitions

AbsorptionRotational Transitions

AbsorptionHow to talk about absorption

AbsorptionFluorescence

Optical ImagingFluorescence

Green Fluorescence Protein (GFP)

Excitation

e.g. ~480 nm

Emission

e.g. ~520 nm

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

Transgenic Luciferase Tobacco plant

Tracking Stem Cell Survival

1 day 4 days

Time after implantationMesenchymal stem cells

derived from human bone marrow

THANK YOU FOR YOUR ATTENTION

1st Faculty of Medicine, Charles University in Prague ... · Optical Imaging reporter genes •...

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