Fluorescence Resonance Energy
Transfer (FRET)
FRET
Resonance Energy Transfer



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.
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 dipoledipole interactions
FRET
The absorption spectrum of the acceptor must
overlap fluorescence emission spectrum of the
donor
Fluorescnece Intensity

J(λ)
Donor
fluorescnece
Acceptor
absorption
Wavelength
FRET

Energy
Donor excitation state
Emission
Acceptor excitation state
학교 제도 : 교육 제도 중 학교에 관한 제도
사회적으로 가장 먼저 공인된 제도, 형식적 교육 제도
1) 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
FITC
FITC
TRITC
TRITC
520nm
light
630nm
light
FRET

Distance dependent interaction
between the electronic excited states
of two molecules
*not sensitive to the surrounding solvent shell of a
fluorophore
*Donor-Acceptor의 Energy transfer는 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
55
IAEDANS
Tetramethlrhoda
mine
Fluorescein
EDANS
Dabcyl
33
Fluorescein
Fluoresscein
44
BODIPY FL
BODIPY FL
57
Fluorescein
Qsy7&Qsy9
dyes
61
46
FRET
Critical Distance for Common RET Donor-Acceptor Pairs
FRET

Förster Equation
Förster
Equation
D
2
~ F ~ d~

k



WDA  8.8 1017  r4  6   A ~D4
n R

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
Biological application using FRET (ex: cameleon)
Inter-molecular FRET
Intra-molecular FRET
FRET

Biological application using FRET
Outline
1. What is fluorescence??
2. Fluorescent molecules
3. Equipment for single-molecule
fluorescence experiments
4. Some applications & examples
fluorescence from molecules
physical fundaments
photon
molecule in
ground state
photon
molecule in
excited state
light can induce transitions
between electronic states in a molecule
intersystem
crossing
S1
internal
conversion
fluorescence
absorption
fluorescence
the Jablonski diagram
T0
-hν
internal
conversion
+hν
S0
radiationless transition
transition involving
emission/absorption of
photon
fluorescence
properties that can be measured
• spectra (environmental effects)
• fluorescence life times
• polarization (orientation and dynamics)
• excitation transfer (distances -> dynamics)
• location of fluorescence
fluorescence
requirements 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
1
 fluo
kr knr
S1
kr
S0
knr
kr
 fluo
1
kr knr
fluorescence
chromophores: intrinsic or synthetic??
• common intrinsic fluorophores like tryptophan,
NAD(P)H
are not good enough
R
NH
• chlorophylls & flavins work
O
H3C
N
H3C
N
NH
N
O
R
in most cases extrinsic fluorophores have to be added:
• genetically encoded (green fluorescence protein)
(H3C)2N
N+(CH3)2
O
• chemical attachment of synthetic dyes
OCH3
O
R
fluorescence
580
a typical synthetic chromophore: tetramethylrhodamine
Absorption / Emission (a.u.)
Absorption
Emission
400
•
•
•
•
450
500
550
600
wavelength (nm)
650
700
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)
the fluorescence of a single TMR can be measured easily
extinction coefficient ():
~100 000 M-1 cm-1
absorption cross section (s)
-16 cm2
s
=

·
2303
/
N
:
~4·10
0
s = area of an opaque
object with the same that blocks the
lightpower:
as good as the molecule
excitation
~100 W/cm2
excitation photon flux
=
power
/ photon
energy:
dI/I
= (s·C·N
Av/1000)·dL
photon energy = h·c/l
~2.5 · 1020 photons·s -1·cm-2
#excitations·molecule-1·s-1
#exc = flux·s
dI/I = ·2.303·dL
~105 photons·s -1·cm-2
#emitted photons·molecule-1·s-1
#em = #exc·QY
~105 photons·s -1·cm-2
single-molecule fluorescence microscopy
• excitation source:
laser
Lasers cw (ion), pulsed (Nd-YAG, Ti-sapphire, diodes
• optical system with high
collection efficiency:
high NA objective
• optics to separate fluorescence
from excitation light:
filters / dichroic mirrors
monochromators, spectrographs; filters: colored glass, notch holographic, multidielectric
• detector:
- CCD camera, PMT
- eyes; PMT, APD, CCD
PhotoMultiplier Tube, Avalanche PhotoDiode,
Charge Coupling Device (signal is usually weak) + electronics
rotation 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 RNA
(Tetrahymena 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
Sample
Laser lexc
Spectrograph
Fluo. intensity
Detector
lfluo
lexc
lfluo
Excitation spectrum
Fluorescence spectrum
Solvent effects
Energy
Non-polar solvent
Polar solvent
S1
S1
S1
S0
Static molecular dipole moment
S0
Fluorescence Lifetime
Sample
number
Pulsed laser
e
t /  fluo
Filter
delay, t
Detector
Laser pulses
photons
delay
time
Polarization
Rigid
polarized
Fluid
depolarized
Polarization memory during the fluorescence
lifetime : fluo. anisotropy
Fluorescence Resonance
Energy Transfer (FRET)
Dipole-dipole interaction
(near-field)
VAD 
1
40 R




ˆ
ˆ
 1  3RR  D
3 A
Donor
Acceptor
Transfer Efficiency
• Fraction of excitations transferred to
acceptor
E
k DA

k DA  k fD
1
 R
1   
 R0 
6
• R0 = Förster radius, maximum 10 nm for
large overlap
Förster Resonance Energy Transfer
R>10 nm
R<10 nm
FRET studies of interaction and dynamics
(molecular ruler)
Association of two
biomolecules
Dynamics of
a 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
multicolor
2-photon
microscopy
specific labeling with various
colors
Fluorescence Correlation Spectroscopy
I(t+ 
I(t)

g
(2)
  
I (t ) I (t   )
t
g(2)
log
Keeps track of the fluctuations of the fluorescence intensity.
Single molecule spectroscopy
•
•
•
•
•
Single molecule tracking
dynamics of single enzyme
sp-FRET
orientation fluctuations
lifetime measurement