Fluorescence Resonance Energy Transfer (FRET)

FRET

Resonance energy transfer can occur when the donor and acceptor molecules are less than 100 A of one another

Energy transfer is non-radiative which means the donor is not emitting a photon which is absorbed by the acceptor

Fluorescence RET (FRET) can be used to spectrally shift the fluorescence emission of a molecular combination.

Resonance Energy Transfer

FRET The mechanism of FRET involves a donor

fluorophore in an excited electronic state, which may transfer its excitation energy to a nearby

acceptor chromophore

non-radiative fashion through long-range dipole-dipole interactions

FRET The absorption spectrum of the acceptor must

overlap fluorescence emission spectrum of the donor

Donorfluorescnece

Flu

ores

cnec

e In

tens

ity

Wavelength

Acceptorabsorption

J(λ)

FRET

Energy Donor excitation state

Emission Acceptor excitation state

학교 제도 : 교육 제도 중 학교에 관한 제도사회적으로 가장 먼저 공인된 제도 , 형식적 교육 제도1)서구 사회의 학교 제도 - schola : 한가 , 여가를 뜻함 , 오늘날의 학교

school

- 고대 그리스 사회에서 지배계급의 지위와 신분을 유

지하기 위해 소수의 귀족계급을 위해 조직되어 교육 실시

- 중세 유럽사회의 학교는 소수의 성직자나 지도자 양

성 을 위한 교회부속의 사원 학교가 대부분

FRET

488nm light

excitation

excitation

630nm

light

FITC FITC

520nm

light

TRITC TRITC

FRET

Distance dependent interaction between the electronic excited states of two molecules

*not sensitive to the surrounding solvent shell of a fluorophore

*Donor-Acceptor 의 Energy transfer 는 거리에 의해 효율이 결정

(~10nm)

Spectral properties of involved chromophore

FRET

Calculation

Efficiency of Energy Transfer = E = kT/(kT + kf + k’) kT = rate of transfer of excitation energy kf = rate of fluorescence k’ = sum of the rates of all other deexcitation

processes

E = R60/ R60+ R6

FRET Förster Equation Ro= Forster radius

= Distance at which energy transfer is 50% efficient

= 9.78 x 103(n-4*fd*k2*J)1/6 Å

fd- fluorescence quantum yield of the donor in the absence of acceptor n- the refractive index of the solution k2- the dipole angular orientation of each molecule

j- the spectral overlap integral of the donor and acceptor

Typical values of R0

Donor Acceptor Ro(Ǻ)Fluorescein Tetramethlrho

damine55

IAEDANS Fluorescein 46

EDANS Dabcyl 33

Fluorescein Fluoresscein 44

BODIPY FL BODIPY FL 57

Fluorescein Qsy7&Qsy9 dyes

61

FRET

Critical Distance for Common RET Donor-Acceptor Pairs

FRET

Förster Equation

46

2

417

~

~~~108.8

dF

Rn

kW DA

Dr

DAFörster

Equation

FRET

Schematic diagram of FRET phenomena

FRET SUMMARY

Emission of the donor must overlap absorbance of the acceptor

Detect proximity of two fluorophores upon binding

Energy transfer detected at 10-80Ǻ

FRET

FRET

Inter-molecular FRET Intra-molecular FRET

Biological application using FRET (ex: cameleon)

FRET

Biological application using FRET

OutlineOutline

1. What is fluorescence??

2. Fluorescent molecules

3. Equipment for single-molecule fluorescence experiments

4. Some applications & examples

fluorescence from moleculesfluorescence from moleculesphysical fundamentsphysical fundaments

photon

photon

molecule in ground state

molecule in excited state

light can induce transitions between electronic states in a molecule

S0

S1

T0

transition involving emission/absorption of photon

radiationless transition

abso

rptio

n

+hν

fluor

esce

nce

-hν

inte

rnal

co

nver

sion

inte

rsys

tem

cr

ossi

ngin

tern

al

conv

ersi

on

fluorescencefluorescencethe Jablonski diagramthe Jablonski diagram

fluorescencefluorescenceproperties that can be measuredproperties that can be measured

• spectra (environmental effects)

• fluorescence life times

• polarization (orientation and dynamics)

• excitation transfer (distances ->

dynamics)

• location of fluorescence

fluorescencefluorescencerequirements for a good fluorophorerequirements for a good fluorophore

• good spectral properties

• strong absorber of light (large extinction coefficient)

• high fluorescence quantum yield

• low quantum yield for loss processes (triplets)

• low quantum yield of photodestruction

• small molecule / easily attachable to biomolecule to be studied

1.7 Fluorescence quantum yield

knrkr

nrrfluo kk 1

1nrr

rfluo kk

k

S0

S1

fluorescencefluorescencechromophores: intrinsic or synthetic??chromophores: intrinsic or synthetic??

• common intrinsic fluorophores like tryptophan, NAD(P)H are not good enough

• chlorophylls & flavins work

in most cases extrinsic fluorophores have to be added:

• genetically encoded (green fluorescence protein)

• chemical attachment of synthetic dyes

R

NH

O(H3C)2N N+(CH3)2

O

OCH3

R

NH

NN

N

O

O

CH3

CH3

R

fluorescencefluorescencea typical synthetic chromophore: a typical synthetic chromophore: tetramethylrhodaminetetramethylrhodamine

• extinction coefficient: ~100,000 Molar-1 cm-1 • fluorescence quantum yield: ~50%• triplet quantum yield <1%• available in reactive forms (to attach to amines,

thiols) and attached to many proteins and other compounds (lipids, ligands to proteins)

400 450 500 550 600 650 700

AbsorptionEmission

Abs

orp

tion

/ Em

issi

on (

a.u.

)

wavelength (nm)

550

550

580

580

extinction coefficient (): ~100 000 M-1 cm-1

the fluorescence of a single TMR can be measured easilythe fluorescence of a single TMR can be measured easily

absorption cross section () = · 2303 / N0: ~4·10-16 cm2

excitation power: ~100 W/cm2

excitation photon flux = power / photon energy: ~2.5 · 1020 photons·s -1·cm-2

photon energy = h·c/

#excitations·molecule-1·s-1 #exc = flux· ~105 photons·s -1·cm-2

= area of an opaque object with the same that blocks thelight as good as the molecule

dI/I = (·C·NAv/1000)·dL

dI/I = ·2.303·dL

#emitted photons·molecule-1·s-1 #em = #exc·QY ~105 photons·s -1·cm-2

single-molecule fluorescence microscopysingle-molecule fluorescence microscopy

• excitation source: laser

Lasers cw (ion), pulsed (Nd-YAG, Ti-sapphire, diodes

• detector: - CCD camera, PMT- eyes; PMT, APD, CCD

PhotoMultiplier Tube, Avalanche PhotoDiode,

Charge Coupling Device (signal is usually weak) + electronics

• optics to separate fluorescence from excitation light: filters / dichroic mirrorsmonochromators, spectrographs; filters: colored glass, notch holographic, multidielectric

• optical system with high collection efficiency: high NA objective

rotation of F1-ATPaserotation of F1-ATPase

Adachi, K., R. Yasuda, H. Noji, H. Itoh, Y. Harada, M. Yoshida, and K. Kinosita, Jr. 2000. Proc. Natl. Acad. Sci. U.S.A. 97:7243-7247

folding / unfolding of RNAfolding / unfolding of RNA((TetrahymenaTetrahymena ribozymes) ribozymes)

X. Zhuang, L. Bartley, H. Babcock, R. Russell, T. Ha, D. Herschlag, and S. Chu Science 2000 June 16; 288: 2048-2051.

FLUORESCENCE MEASUREMENTS

• Information given by each property of fluorescence photons:

- spectrum

- delay after excitation (lifetime)

- polarization

Spectra

Laser exc fluo

Spectrograph

DetectorSample

exc fluo

Fluo. intensity

Excitation spectrumFluorescence spectrum

Solvent effects

Non-polar solvent

Polar solvent

Energy

Static molecular dipole moment

S0

S1

S1

S0

S1

Fluorescence Lifetime

Pulsed laser

Sample

Detector

Filter

time

Laser pulses

photons delay

delay, t

number

fluote

/

Polarization

polarized depolarized

Rigid Fluid

Polarization memory during the fluorescencelifetime : fluo. anisotropy

Fluorescence Resonance Energy Transfer (FRET)

DAAD RRR

V

ˆˆ314

13

0

Dipole-dipole interaction(near-field)

Donor Acceptor

Transfer Efficiency

• Fraction of excitations transferred to acceptor

• R0 = Förster radius, maximum 10 nm for large overlap

6

01

1

RRkk

kE

fDDA

DA

Förster Resonance Energy Transfer

R>10 nm

R<10 nm

FRET studies of interaction and dynamics(molecular ruler)

Association of two biomolecules

Dynamics ofa biomolecule

Other specific labeling and imaging

• Possibility to specifically label certain biomolecules, sequences, etc. with fluorophores

• Staining and imaging with various colors• Detection of minute amounts (DNA assays)• Fluorescence lifetime imaging (FLIM)• Fluorescence recovery after photobleaching

multicolor2-photonmicroscopy

specific labeling with various colors

Fluorescence Correlation Spectroscopy

t

I(t)

I(t+

)()()2( tItIg

Keeps track of the fluctuations of the fluorescence intensity.

log

g(2)

Single molecule spectroscopy

• Single molecule tracking• dynamics of single enzyme• sp-FRET• orientation fluctuations• lifetime measurement