biomedicinsk optik ljusutbredning i vävnad · biomedicinsk optik ljusutbredning i vÄvnad...

2014-02-19

Biophotonics@Lunduniversity 1

Biomedicinsk OptikLjusutbredning i vävnadSTEFAN ANDERSSON-ENGELS

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w w w . a t o m i c . p h y s i c s . l u . s e / b i o p h o t o n i c s

Biomedicinsk Optik

STEFAN ANDERSSON-ENGELS

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Biomedicinsk Optik

INNEHÅLL

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► Introduktion och definitioner

► Ljusutredning i vävnad

► Diagnostiska Tillämpningar

► Behandlingstillämpningar

Biomedicinsk Optik

LJUSUTBREDNING I VÄVNAD

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ina Reistad 2012-10-22

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Computer Lab in MatLab

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Absorption spectra

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Absorption of light in tissue is due to chromophores (= molecules absorbing light). The absorption probability is a material property that varies with the wavelength of light.

tissue_abs.m

(5% blood, 75% water and 15% fat)

665 715 765 815 865 915 965 10150

20

40

60

80

100Tissue absorption vs water concentration

Wavelength (nm)

Abs

orpt

ion

Coe

ffici

ent (

m-1

)

Sat=65%

Sat=75%

Sat=85%

400 500 600 700 800 900 10000

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000Tissue absorption vs oxygen saturation

Wavelength (nm)

Abs

orpt

ion

Coe

ffici

ent (

m-1

)

Sat=65%Sat=75%Sat=85%

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Scattering spectra

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Scattering of light in tissue is due to changes in the refractive index on the microscopic scale. The scattering probability is a material property that varies with the wavelength of light.

tissue_sca.m

(a_Rayleigh = 400, a_Mie = 1900)

450 500 550 600 650 700 750 800 850 900 950 1000 10500

500

1000

1500

2000

2500

3000Tissue scattering for Rayleigh and Mie scattering

Wavelength (nm)

Red

uced

Sca

tterin

g C

oeffi

cien

t (m

-1)

Total scattering

Rayleigh scattering

Mie scattering

400 500 600 700 800 900 10000

500

1000

1500

2000

2500Tissue scattering for different sizes of scatterers

Wavelength (nm)

Red

uced

Sca

tterin

g C

oeffi

cien

t (m

-1)

b_Mie=1

b_Mie=1.5

b_Mie=2

Effective attenuation spectra

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The effective attenuation of light in tissue is due to both absorption and scattering properties, and thus is a material property that varies with the wavelength of light.

tissue_mueff.m

400 600 800 1000 12000

500

1000

1500

2000

2500Tissue absorption

Wavelength (nm)

Abs

orpt

ion

Coe

ffici

ent (

m-1

)

Blood Conc=5%

Blood Sat=65%

Water Conc=70%

Lipid Conc=15%

400 600 800 1000 12000

500

1000

1500

2000

2500Tissue scattering

Wavelength (nm)

Red

uced

Sca

tterin

g C

oeffi

cien

t (m

-1)

Rayleigh strength = 500

Mie strength = 1000

b-parameter = 1

400 600 800 1000 12000

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000Tissue scattering

Wavelength (nm)

Effe

ctiv

e A

ttenu

atio

n C

oeffi

cien

t (m

-1)

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Fluence rate in an infinite medium

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The fluence rate at a distance from a source depends on the optical properties (= wavelength-dependent) and the distance from the source.

CWinfinite.m

(5% blood, 60% oxygen saturation, 65% water and 15% fat,a_Rayleigh = 500, a_Mie = 1000, b_Mie = 1)

Fluence rate as a function of wavelength and radial distance from point source

Radial distance (cm)

Wav

elen

gth

(nm

)

0.005 0.01 0.015 0.02

400

600

800

1000

1200

1400

500 600 700 800 900 1000 1100 1200 13000

5

10

15

20

25

Wavelength (nm)

Flu

ence

Rat

e (W

m-2

)

10 mm15 mm20 mm

Fluence rate at various distances

Fluence rate in a semi-infinite medium

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The fluence rate at a distance from a source depends on the optical properties (= wavelength-dependent), the distance from the source and the tissue boundary.

CWsemi.m

(5% blood, 65% oxygen saturation and 15% fat,a_Rayleigh = 500, a_Mie = 1000, b_Mie = 1)

400 500 600 700 800 900 1000 1100 1200 13000

2

4

6

8

10

12Fluence rate spectrum as a function of wavelength at position (rho,z)

Wavelength (nm)

Flu

ence

Rat

e (W

m-2

)

60% H2O

70% H2O

80% H2O

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Diffuse Reflectance from a semi-infinite medium

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The diffuse reflectance at a distance from a source depends on the optical properties (= wavelength-dependent), the distance from the source and the tissue boundary.

CWsemi.m

(5% blood, 65% oxygen saturation and 15% fat,a_Rayleigh = 500, a_Mie = 1000, b_Mie = 1)

400 500 600 700 800 900 1000 1100 1200 13000

2

4

6

8

10

12Fluence rate spectrum as a function of wavelength at position (rho,z)

Wavelength (nm)

Flu

ence

Rat

e (W

m-2

)

60% H2O

70% H2O

80% H2O

Diffuse reflectance

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Reflektionsdiagnostik

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Prediction and measurement of the light dose

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Aim: Understand the importance of optical measurements and dosimetry

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Tissue Optics success – Pulse oximetri

15► Examples from internet

Time-resolved depth profile

Pulsed source

Time-resolved Detector

Fiber-opticbeamsplitter

Tissue

Computer

Amplifier Bandpass filter

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Vågor - interferens

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The OCT setup

Broadbandsource

Detector

Fiber-opticbeamsplitter

Tissue

Scanningreference mirror

Computer

Amplifier Bandpass filter

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Kommersiella OCT system

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Courtesy Peter Andersen, DTU, Denmark

Humphrey® Optical Coherence Tomography Scanner vid Herlevssjukhuset.

Mätning av ögonbotten

Courtesy Peter Andersen, DTU,

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Fluorescencsdiagnostik

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Outline

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Fluorescence principles

Sensitivity concerns and tissue optics

3D fluorescence imaging

Clinical applications

Endoscopy

Discrimination of urinary bladder malignancies

Fluorescence guided brain tumour resection

Preclinical imaging and contrast agents

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What is fluorescence?

Molecularenergy

Absorptionof light

Fluorescence emission

One fluorophore – 109 photons/sec

Influenced by a varying external EM field

Fluorescence emission

Broad emission

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Property Definition SignificanceFluorescenceexcitation spectrum

A plot of excitation wavelength versus fluorescence intensity generated by a fluorophore.

Use excitation light as close to the peak of the excitation spectrum of the fluorophore as possible.

Absorption spectrum

A plot of wavelength versus absorbance of a chromophore or fluorophore.

The absorption spectrum of a fluorophore is usually very similar to the fluorescence excitation spectrum.

Fluorescenceemission spectrum

A plot of emission wavelength versus fluorescence intensity generated by a fluorophore.

Fluorescence emission spectroscopy is the most straightforward basis for distinguishing several fluorophores (incl. tissue autofluorescence).

Extinction coefficient (EC)

Capacity for light absorption at a specific wavelength.

Fluorescence signal (“brightness”) is proportional to the product of the extinction coefficient (at the relevant excitation wavelength) and the fluorescence quantum yield.

Fluorescence quantum yield (QY)

Number of fluorescence photons emitted per excitation photon absorbed.

See “Extinction coefficient”.

Fluorescencelifetime

Time from a short excitation pulse till the fluorescence emission has dropped to 1/e

May be important to distinguish different fluorophores. Is also a parameter that can vary drastically depending on the microenvironment.

Quenching Loss of fluorescence signal due to short-range interactions between the fluorophoreand the local molecular environment

Loss of fluorescence is reversible to the extent that the causative molecular interactions can be controlled. Important in FörsterResonance Energy Transfer (FRET).

Photobleaching Destruction of the excited fluorophore due to photosensitized generation of reactive oxygen species (ROS), particularly singlet oxygen (1O2).

Loss of fluorescence signal is irreversible if the bleached fluorophore population is not replenished (e.g., via diffusion). Extent of photobleaching is dependent on the duration and intensity of exposure to excitation light.

Spectroscopic properties of fluorescent dyes

Outline

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Endoscopy




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Fluorescence sensitivity2014-02-19

27Image from: http://en.wikipedia.org/wiki/Fluorescence

Signal-to-noise is determined by:Number of excitation photons emitted on the sampleFraction of these excitation photons reaching a fluorophoreExtintion coefficient of fluorophore for the excitation light usedFluorescence quantum yieldFraction of emitted fluorescence photons reaching the detectorDetector noise and sensitivity at emission wavelength

Signal-to-background also depends on:Background light intensity (room light, unfiltered excitation light or other fluorophores)

Wavelength matters!

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Excitation wavelength versus Effective attenuation spectra

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400 600 800 1000 1200

Tissue scattering

Wavelength (nm)

Tissue attenuationThin tissue diagnostics –use light with shallow penetration

Deep tissue diagnostics –use light with deep penetration anda geometry suppressing shallow light

Selection of fluorophore

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Select fluorophore, detection geometry and excitation wavelengthwisely for the application of interest

Superficial lesion Deeply located lesion

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Autofluorescence as background

Adapted from Adams et al, JBO 12 (2007)

Mouse with no injection – pure autofluorescence

Excitation @ 690 nm

Autofluorescence is strongly wavelength dependent

Excitation @ 780 nm

Fluorescence: Tissue autofluorescence Protoporphyrin IX

J. Johansson, Dissertation thesis, LTH (1993)., af Klinteberg et al. (1999)

337 nm excitation

400 500 600 700

Carotene

NADHElastin

Collagen

Wavelength (nm)

Flu

ores

cenc

e in

tens

ity [

a.u.

] 405 nm excitation

500 550 600 650 700 750

Wavelength (nm)

Protoporphyrin IX

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Haematoporphyrin derivative (HpD), (Photofrin) 630 nm -aminolevulinic acid (ALA) 635 nmMesotetrahydroxyphenychlorin (mTHPC), (Foscan) 652 nm

Tin Etiopurpurin (Pyrlytin) 660 nm675 nmPhthalocyanins

Lutetium texaphyrin (Lutrin) 732 nm

Bacteriochlorophyll (Tookad) 760 nm

RED Absorption

Peak

Hypericin (HY), 590 nm

Tumour localising agents - photosensitisers (PDT)tumour markers (LIF)

Outline

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Endoscopy




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Ill-conditioned reconstruction Tomography

X-Raysource

Imageplate

Rotation

Object

Rotation

Object

Lightsource Detector

Linear image reconstruction Non-linear image reconstruction

Diffuse Optical Tomography

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Rotation

Object

Lightsource

Detector

Fluorescence-mediated tomography

Fluorescence tomography

Sensitivity maps-

Jacobian matrix

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Outline

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Endoscopy




Diagnostics and therapy

Courtesy Ingrid Wang

Tumour marker(lokally or

systemically)

... ... ...

Wait a few hours

Fluorescencediagnostics

Photodynamic therapy (PDT)

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White-light image (A1) 5-ALA-induced PPIX fluorescence image (A2)

of a patient with squamous cell carcinoma.

Hautmann et al. Respir. Res., 8 (2007)

Fluorescence angiography

Fluorescein angiography before and after PDT of abnormal blood vessels at the retina, choroidalneovascularization (CNV)

Hikichi et al., RETINA 21 (2001)

In-vivo PpIX fluorescence in bronchial tree

normaltissue

infiltrationzone

solid tumor

Stummer et al. Lancet Oncol. (2006)

Fluorescence-guided brain tumour resections

Fluorescence Diagnostics2014-02-19

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Fluorescence Endoscopy

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Clinical endoscopic fluorescence measurements

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Multi colourimaging system

500 600 700Wavelength (nm)

Flu

ores

cen

ce I

nte

nsi

ty

TumourNormal

D AB RG

RG

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Multi colour fluorescence imaging system

SpectraPhos ABAndersson-Engels et al. Appl Opt (1994)Svanberg et al. Acta Radiol. (1998)

Multicolour fluorescence imagingBCC below the ear

Svanberg et al. Acta Radiol. (1998)

0

1

2

3

4

5

450 500 550 600 650 700 750

Flu

ore

scen

ce I

nte

nsi

ty (

a.u

.)

Wavelength (nm)

D

A

TAE

TAE

nBCC

nBCC

sBCC

Fc =A — k1D

k2D

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White light image

Digitally processed image

Multicoulour fluorscence

imaging

Svanberg et al. Acta Radiol. (1998)

Fluorescence lifetime spectroscopy

Sun et al., Opt Lett (2008)

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Time-gated fluorescence imaging

Andersson-Engels et al., LSM (2000)

Pulsed laser

Photodiode

Gate and Delay

ICCD

Sample

Fluorescence lifetime imaging

Requejo-Isidro et al., Opt Lett (2004)

Endoscopicautofluorescencelife-time image of lamb kidney in vitro

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Urinary bladder tumour diagnostics2014-02-19

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350 400 450 500 550 600 650 700 7500

20

40

60

80

100

UV IR

Wavelength [nm]

Flu

ore

scen

ce i

nte

nsi

ty

Excitation Emission

Filtered RGB-colour imaging

Courtesy Herbert Stepp

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Instill solution (8 mM, 50 ml), 1 hour waiting, fluorescence cystoscopy

Bladder cancer detection (h-ALA)


Applications in UrologyBladder cancer delineation


Photodynamic Detection (PDD)

Blue Light Fluorescent tumour marker+Fluorescence diagnostics

Normal white light examination Blue light examination

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Brain tumour resections2014-02-19

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Malignant glioma:

• Derived from glial cells: astrocytes and oligodendrocytes

• 5-10 cases per 100,000 per year, 2-3% of cancer deaths

• Ca. 50% of primary brain tumors

• Growth with diffuse infiltration of functional brain

• Very bad prognosis: survival ca. 2 years for grade III,

<1 year for grade IV (glioblastoma multiforme)

• Surgery, radiotherapy, chemotherapy as standard therapy

• The more complete the surgery, the longer the survival

• Resection of safety margin not possible

• Tumor borders (even of bulk tumor) are difficult to identify

Fluorescence guided resection

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Tumor selectivity

normaltissue

infiltrationzone

solid tumor


Courtesy Herbert Stepp, Munich

Tumor selectivity

normaltissue

infiltrationzone

solid tumor

Fluorescence @ 635nm / Remission @ 450 nm

From spectra exited @ 380-440nmobtained with a multifiber detector

19 patients

Stummer et al., JNeurosurg, 2000Courtesy Herbert Stepp, Munich


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NEvents

Median [months]95% CI

6 month rate[%]

95% CI

Log rank

41.0

[32.8 ; 49.2]

139135 (97.1%)4 (2.9%)5.1[3.4;6.0]

FLWL

131126 (96.2%)5 (3.8%)3.6[3.2;4.4]

21.1

[14.0 ; 28.2]

p = 0.0078

Patients at risk:139131

10485

5928

2813

167

85

42

00

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Time [months]0 3 6 9 12 15 18 21

| | ||

|||

|

Censored

Kaplan-Meier estimates (Full Analysis Set)

Cu

mu

lati

ve d

istr

ibu

tio

n

Progression-free survival

Phase III study – primary efficacy variables



Phase III study – secondary efficacy variables

WL FL

Mo

nth

s

0

2

4

6

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10

12

14

16

18

20

13.515.2

+12.6%

WL FL

Mon

ths

0

2

4

6

8

10

12

14

16

18

11.5

13.8

+20.0%

p = 0.1245

hazard ratio: 0.81 [ 0.62 ; 1.06 ]

p = 0.0577

hazard ratio: 0.73 [ 0.53 ; 1.01 ]

Overall survival Overall survival > 55 yrs



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Astrocytoma

PpIX fluorescence5 mm

-2000

-1000

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2000

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4000

5000

6000

7000

440 490 540 590 640 690

15 mm

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60000

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440 490 540 590 640 690

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30000

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50000

60000

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60000

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90000

400 450 500 550 600 650 700

Autofluorescence

Collaboration with Karin Wårdell and Patrik Sturnegk et al.

Outline

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Endoscopy




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Motivation –Preclinical needs of ”deep” in vivo imaging

Need to efficiently predict drug function in the early phase of drug development• Reduce the number of animal used in pre-clinical

research• Optical molecular imaging provide cost effective

tools• Today the fluorescence-based techniques are

limited by• Autofluorescence background• Resolution• Poor light penetration

Modality Resolution Sensitivity Molecular Imaging ?

X-ray, CT m mM no

MRI m mM Probably not

PET mm fM, pM Yes!

Gamma scint. SPECT

mm fM, pM Yes!

Ultrasound mm mM No

Bioluminescence mm pM Yes, preclinical

Endoscopy mm mM Yes

Fluorescence mm <pM Yes!

Photoacoustic mm nM? Yes

Diagnostic imaging with contrast

Multimodality to get the best of each!!

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Small Animal Imaging Techniques

Adapted from Massound et al, Genes & Developmet 17 (2003)

microPET microCT microSPECT

Fluorescence

microMRI

Bioluminescence

Fluorescence imaging of animals

GFP Mouse

Hoffman, Nature Protocols (2006)

Sharma Am. J. Physiol. (2007)

Animal models widely used in biomedical research

More than 90% of animals used are mice

Non-invasive imaging studies very valuable tool

Allow non-invasive longitudinal and dynamic studies

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Why fluorescence?

Adapted from Houston et al, JBO 10 (2005)

90 uCi 111-Indium, IRDye800CW, tail vein,imaging 24 hrs later

About 109 photons/sec/fluorophore

15 minutes 800 ms

Light attenuation

High absorption and scattering

Penetration at most a few centimeters – no whole body imaging

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Alexa fluorophores

http://www.invitrogen.com

IRDye800IRDye 800CW is the dye of choice for protein/antibody labeling applications and for nucleic acid applications requiring high labeling density.

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Smart probes

Klohs et al., Basic Research in Cardiology, 2008

Enzymatic cleavage turns on fluorescence

Klohs et al., Basic Research in Cardiology, 2008

Activatable probe• In a polymer the dye molecules are arranged in close proximity to

each other thereby quenching the fluorescence. • Using target specific enzymes to cleave them provides a

fluorescence signal.

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Bioluminescence

The firefly luciferase enzyme encoded in a gene is used to infect cells and thus createLuciferase photon transgenic animals.

Luciferase is an enzyme catalysing a reaction between D-luciferin, oxygen and ATP yielding light.

The expression of luciferase can be trackedby the administration of D-luciferin

The oxidation of D-luciferin produces light at 500-560 nm.

Luciferase

photonD-luciferin +ATP + O2

Gene reporter– A molecular species that is a product of gene expression that “reports” gene expression in vivo and may or may not provide a signal for imaging (luciferase)

Gene probe– An exogenous molecular species that probes the gene reporter and provides an imaging signal (D-luciferin)

One oxidation of D-luciferin one photon

Bioluminescence on the net

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Experimental arrangement

Bioluminescence imaging Fluorescence imaging

Laser

D-luciferin Fluorophore

Green Fluorescence Protein - GFP

Aequorea victoria – a jellyfish in the Northern Pacific Ocean

What is GFP?

A small naturally occuring protein which is highly fluorescent.GFP consists of 238 amino acids, linked together in a longchain. This chain folds up into the shape of a beer can. Insidethe beer can structure the amino acids 65, 66 and 67 form thechemical group that absorbs UV and blue light, and fluorescesgreen.

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Osamu ShimomuraMarine Biological Laboratory (MBL), MAandBoston University Medical School, MA

Martin ChalfieColumbia University, N.Y.

Roger Y. TsienUCSD, La Jolla, CAJapanese citizen, born 1928 in Kyoto,

Japan. Ph.D. in organic chemistry1960, from Nagoya University, Japan.Professor emeritus at Marine BiologicalLaboratory (MBL), Woods Hole,MA, USA a nd Boston UniversityMedical School, MA, USA.

US citizen, born 1947, grew up inChicago, IL, USA. Ph.D. 1977 in neurobiologyfrom Harvard University.William R. Kenan, Jr. Professor ofBiological Sciences at ColumbiaUniversity, New York, NY, USA,since 1982.

US citizen, born 1952 in New York, NY,USA. Ph.D. in physiology 1977 fromCambridge University, UK. Professorat University of California, San Diego,La Jolla, CA, USA since, 1989.

2008 Nobel Prize in Chemistryfor the discovery and development of the green fluorescent protein, GFP

Fluorescence labeling in biology

Baby mice fathered by mice receiving a donation of spermatogonial stem cells from mice expressing green fluorescent protein. Only half the baby mice show the green color. This is because each spermatogonial stem cell has only one copy of the gene for green fluorescent protein.

http://www.nichd.nih.gov/news/releases/green_brown_mice.cfm

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Fluorescence Proteins in all colours – Roger Y. Tsien

Agar Plate of Fluorescent Bacteria Colonies

Using DNA technology, various amino acids in different parts of GFP were exchanged

Extended red proteins

Shcherbo D, et al. Nat Methods. 2007 Sep;4(9):741-6, Merzlyak EM, et al. Nat Methods. 2007 Jul;4(7):555-7

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Nanoparticles as fluorescence markers

• Liposoms• can be used as vehicles for any fluorophore and

thus modify its pharmacokinetic properties

• Quantum dots• strong fluorescence• long Stoke shift

• can avoid tissue autofluorescence• must excite at shorter wavelengths

• narrow and tunable emission • toxic substances

• Upconverting nanoparticles• autofluorescence free detection• non-toxic

Courtecy: Dr. Niels Bendsoe, Lund University Medical Laser Centre

► Ansamlas itumörvävnad (pgablodförsörjning, cellernasmembran, enzymer, pH etc)

► Karaktäristiskfluorescens

► Fotodynamisktumörterapi (PDT)

Tumörmarkörer

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2014-02-19


Thanks for the attention, Questions?

biomedicinsk optik ljusutbredning i vävnad · biomedicinsk optik ljusutbredning i vÄvnad...

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