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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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absorption and emission (1)
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absorption and emission (2)
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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absorption and emission (3)
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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fluorescence resonance
energy transfer : FRET (2)
spectral overlap (depends on λ):
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fluorescence resonance
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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fluorescence resonance
energy transfer : FRET (4)
orientational factor κ2
freely rotating: κ2 = 2/3
22 2
fl
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fluorescence resonance
energy transfer : FRET (5)transfer efficiency and distance measurement
fl
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fluorescence resonance
energy transfer : FRET (6)
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)
fluorescence resonance
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fluorescence resonance
energy transfer : FRET (7)
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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next 26/05/2003
• www.m-boersch.org slides + literature
• email: [email protected]