1. Biophysical Tools '04 - Fluorescence
9/18/2018

2. Biophysical Tools '04 - Fluorescence
Energy diagram
Energy diagram
Quantum Yield - Q
Energy diagram
Red shift
Some of the energy absorbed is lost by dropping through the vibrational levels of the excited state thus the emitted light is always less energetic (longer wavelength, red shifted) with respect to absorbed light.
Emission always from the lowest vibrational level of the excited state (absorption always from the lowest level of the ground state) to any of the vibrational levels of the ground state  spectral width of the emission line.
Quantum yield
Probability of emitting light =(# emitted photons)/(# absorbed photons)
No fluorescence if the vibrational states of the excited state overlap with the ground state, true for most molecules. Exception: rigid rings containing double bonds, most fluorophores.
Vibrations, energy transfer compete with fluorescence.
The overall rate of depopulation of the excited state:
quantum yield
is given then by:
( for unknown samples  is obtained by comparison to a standard:  of quinine sulfate in 0.5 M H2SO4 is 0.7)
Rate of depopulation
Red shift
Biophysical Tools '04 - Fluorescence
9/18/2018

3. Biophysical Tools '04 - Fluorescence
Fluorimeter
Time resolved experiment
Spectrofluorimeter
Frequency domain (phase/modulation)
Excitation/emmission spectrum:
Spectrofluorimeter
source (Xenon lamp)  excitation monochromator  excitation slits  sample  detection at 90o consisting of: emission monochromator  emission slits  photomultiplier.
Excitation spectrum:
scan of the excitation wavelength with detection at the single emission wavelength.
Emission spectrum:
scan of the emission wavelength with excitation at the single wavelength
Biophysical Tools '04 - Fluorescence
9/18/2018

4. Biophysical Tools '04 - Fluorescence
Fluorophores
Intrinsic fluorophores
Extrinsic probes
Solvent Effects
Intrinsic fluorophores
Tryptophan, tyrosine (quenched by Trp), phenylalanine
Nothing in nucleic acids
Extrinsic probes
protein labels consist of chromophore and in case of covalent labeling: functional group linking the chromophore to a specific aminoacid of a protein. Chromophores:
fluorescein (rhodamine) dansyl, anilino-naphtalene-sulhonates, pyrene,
Functional groups:
maleimide, iodoacetamide (for cysteines) ; activated esters and isothiocyanate (for lysines)
Probes must be: specific and must not perturb the structure. Probes are reporter molecules: good reporter reports news, does not make them.
Another group of probes are non-covalently binding cofactors and nucleotide analogs.
Solvent Effects
excited state more polarized than the ground state  rearrangement of water dipoles (or polar aminoacids) leads to loss of energy, red shift.
Non-polar environment: blue shift, increase of quantum yield
More polar: red shift, decrease of quantum yield
Biophysical Tools '04 - Fluorescence
9/18/2018

5. Biophysical Tools '04 - Fluorescence
Anisotropy
Time resolved anisotropy Iװ I┴
Anisotropy
Fluorescence depolarization
Anisotropy of the absorbance selects chromophores parallel to direction of polarization; emission will be also polarized (albeit in different direction) unless the molecule moves during the lifetime of the excited state.
Anisotropy
Degree of polarization;
Motional depolarization leads to reduction of intrinsic anisotropy:
multiexponential decays due to complex motions
residual anisotropy due to restricted motion
Phosphorescence
triplet probes: eosin, erythrosin
longer lifetimes, probing of slower motions
anisotropy decays like in fluorescence
Motional depolarization
Biophysical Tools '04 - Fluorescence
9/18/2018

6. Biophysical Tools '04 - Fluorescence
Phosphorescence
Phosphorescence
Øtriplet probes: eosin, erythrosin S T
Biophysical Tools '04 - Fluorescence
9/18/2018

7. Biophysical Tools '04 - Fluorescence
Anisotropic motion
Hindered rotation
Perrin Plot
A(t) A∞
Biophysical Tools '04 - Fluorescence
9/18/2018

8. Biophysical Tools '04 - Fluorescence
Quenching
the rate of quenching = k[Q]
Stern-Volmer plots 2
Fluor (no Q) / fluor (Q=x)
Quenching
dynamic
collisions of the quencher with the chromophore
the rate of quenching = k[Q]
Stern-Volmer plots
total rate of relaxation:
plot Io/Iq against [Q] to yield kt which indicates accessibility to quencher.
Quenchers: I- , acrylamide, nitroxides,
static
energy transfer before emission, deviation from linearity in the Stern-Volmer plots 1
Quencher conc.
Biophysical Tools '04 - Fluorescence
9/18/2018

9. E = kFET/(k + kFET ) = 1-tAD/tD = 1- FAD/FD
FRET
Efficiency of transfer:
E = rate of transfer /total rate of relaxation =
E = kFET/(k + kFET ) = 1-tAD/tD = 1- FAD/FD
dipole-dipole interactions
kFET  (R-3)2 ~ 1/R6
Ro E=0.5 i.e. when kFET = 1/t then:
E = R-6/(Ro-6 + R-6)
Orientation problem: k2
400
500
600
700 1 2 3 4 5
donor
donor/aceptor
acceptor
Wavelength (nm)
Intensity
acceptor (AF)
donor (AEDANS)
Forster Energy Transfer
fluorescence energy transfer when emission of one chromophore excites absorption of another
the emission of a donor must overlap with excitation of an acceptor
energy transfer measured as a stimulated emission of acceptor or loss of donor emission
Efficiency of transfer: E = rate of transfer /total rate of relaxation =
E = kFET/(k + kFET ) = 1-tAD/tD = 1- FAD/FD
dipole-dipole interactions between neighboring chromophores
 energy of dipole-dipole interaction ~ R-3, rate of energy transfer
Define Ro as a distance at which energy transfer is 50% efficient i.e. when kFET = 1/t then:
Thus from measurement of quantum yield (or better: lifetimes) in the presence and absence of acceptor the distance between the two might be calculated if Ro for the given donor-acceptor pair is known.
Orientation problem
The efficiency of transfer depends on the mutual orientation of the donor’s emission dipole and acceptor’s absorption dipole.
Usually, assume isotropic distribution leading to orientational factor = 2/3.
Biophysical Tools '04 - Fluorescence
9/18/2018