Single Molecule FRET 01

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singlesingle--moleculemoleculefluorescence resonancefluorescence resonance

energy transfer energy transfer

(1) introduction

michael börsch

06/05/2004



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topics• light-matter interaction

• absorbance, luminescence, fluorescence

• fluorophores, quantum dots, GFP`s

• spectra, intensity, quantum yield,• lifetime

• anisotropy / polarization

• fluorescence resonance energy tranfer FRET

• single-molecule detection



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Born-Oppenheimer

approximation



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Franck-Condon Principle (1)



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Franck-Condon Principle (2)



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Fermi's Golden Rule

ħ

ħ



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Frank-Condon Factor



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absorption and emission (1)

Einstein coefficientsfor the

probability of transitions

ρ(ν) : radiation density



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VIS: all emission isspontaneous



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energy - frequency -

wavelength



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Jablonskidiagram



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fluorescence

(1) absorbance of one photon (fs)

(2) vibrational relaxation (ps)

(3) fluorescence parameters:

spectrumquantum yield

lifetime

anisotropy



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spectrum and linewidth

• Heisenberg uncertainty principle• Doppler effect

• solvent effects• ensemble



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solvent relaxation



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influencing fluorescence

• pressure, temperature• viscosity

• polarity

• hydrogen bonds, pH• ions

• quenching• electric potential



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quantum yield (for fluorescence)

• ratio of the number of emitted photons tothe number of absorbed photons

• ratio of the rate constant for radiative

deactivation (S1→ S0) to the sum of rate

constants for all deactivation processes

• ratio of the lifetime of S1

(fluorescence

lifetime) to the radiative lifetime (in

absence of any non-radiative deactivation)



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fluorescence lifetime



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anisotropy

î : exciting photon

p : polarized fluorescence photon



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transition dipole moment⇒ transition dipole

moments are

oriented in thegeometry of

the fluorophore

for excitationand fluorescence

from:

diphenyl-

hexatriene



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polarization [P] anisotropy [r]

definition by intensities

1. in isotropic solution: EX with linearly polarized light

⇒ selective excitation of absorption dipoles parallel tothe electric vector (photoselection).

2. rotational diffusion during fluorescence lifetime

⇒ depolarization



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example:dye

Cy3

© T. SchmidtUni Leiden



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fluorophores

fl h II



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fluorophores II

rhodamine 6 G

tetramethylrhodamine

cyanine 5

fl h III



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fluorophores III

green fluorescent protein GFP

(Uni Leiden; spectra: Clontech)

fl h IV



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fluorophores IV

QuantumDotCorp 565-585-605-streptavidin: kidney cells

Nanorods

T. Nann

Uni Freiburg

nanocrystals

semiconductor quantum dots, CdSe

with capping



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fluorescence observables

fl



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fluorescence spectrometer



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sample chamber SLM 8100



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fluorescence of R6G in H2O

• excitation volume: 5mm x 2mm x 2mm = 20 µl• R6G 200 pM ⇒ 2.4•109 Molecules

• H2O 55 M ⇒ 6.6•1020 Molecules

• ratio 1 : 2.8•1011

550 600 650 700 750

0

50

100

150

200

250

300

350

400

450

500

dark counts

R6G 200 pM, 11 µW 532 nm

R6G 200 pM, 45 µW 532 nm

p h o t o n c o u n t s /

5 0 0 m s

wavelength / nm

550 600 650 700 750

0

100

dark counts

H2O, 11 µW 532 nm

H2O, 45 µW 532 nm

p h o t o n c o u n t s /

5 0 0 m s

wavelength / nm

i i l f



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principle of

single-molecule fluorescence detection

S

S

T

0

1

1

photon burst

diffraction-

limited spot

1 fl = 10-15 l

1 nM conc.:1 molecule@1fl

laser induced fluorescence



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relaxation modes• internal conversion (IC)

• intersystem crossing (ISC)• fluorescence

• phosphorescence

• delayed fluorescence (after reverse ISC)• triplet-triplet annihilation

• triplet-triplet transition

• photoinduced intramolecular charge transfer (TICT)• intermolecular de-excitation (quenching or FRET)

• photochemical transformation

• excimer or exciplex formation



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fluorescence resonance

energy transfer : FRET (1)non-radiative



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efficiency of transfer E

D + A + h νex → D* + A→ D + A*→ A + D + h νA

kT

E =kT

kT + kD + k Di

kDi

D + A D + A

kAi

kD

D + A + h νD



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energy transfer : FRET (2)

spectral overlap (depends on λ):



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energy transfer : FRET (3)distance dependence and

Förster radius R0

5



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Förster radius R0 and

Donor quantum yield



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Förster radius R0 and

index of refraction n

fl



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orientational factor κ2

freely rotating: κ2 = 2/3

22 2

fl



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energy transfer : FRET (5)transfer efficiency and distance measurement

fl



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fluorophore interaction: energy, symmetry, distance



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single-molecule FRET imagingTIRFM with CCD



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reverible docking / dissociation of

P1 site @Tetrahymena ribozyme

Zhuang, X. et al. Science288, 2048–2051 (2000)




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distance dependence on nanometer scale: 1-10

intensity traces



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literature• http://www.biophys.leidenuniv.nl/Teaching/

Spectroscopy/Fluorescence.pdf

(T.Schmidt)

• Bernard Valeur, Molecular Fluorescence, Wiley-VCH

• Joseph Lakowicz, Principles of Fluorescence

Spectroscopy, Plenum Press



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• www.m-boersch.org slides + literature

• email: [email protected]

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Single Molecule FRET 01