Biophysical Tools '04 - Fluorescence 9/18/2018

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

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

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

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

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

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

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

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 distance @ 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