Fluorescence Biophysical Tools 04 Fluorescence 9192021 1 Energy

Fluorescence Biophysical Tools '04 - Fluorescence 9/19/2021 1

Energy diagram Quantum Yield - Q Rate of depopulation Red shift Biophysical Tools '04 - Fluorescence 9/19/2021 2

Spectrofluorimeter Fluorimeter Time resolved experiment Frequency domain (phase/modulation) Excitation/emmission spectrum: Biophysical Tools '04 - Fluorescence 9/19/2021 3

Fluorophores Intrinsic fluorophores Extrinsic probes Solvent Effects Biophysical Tools '04 - Fluorescence 9/19/2021 4

Anisotropy Time resolved anisotropy I װ I┴ Anisotropy Motional depolarization Biophysical Tools '04 - Fluorescence 9/19/2021 5

Phosphorescence Øtriplet probes: eosin, erythrosin S T Biophysical Tools '04 - Fluorescence 9/19/2021 6

Anisotropic motion Perrin Plot A(t) Hindered rotation A∞ Biophysical Tools '04 - Fluorescence 9/19/2021 7

Quenching Fluor (no Q) / fluor (Q=x) the rate of quenching = k[Q] Stern-Volmer plots 2 1 Quencher conc. Biophysical Tools '04 - Fluorescence 9/19/2021 8

FRET Efficiency of transfer: E = rate of transfer /total rate of relaxation = E = k. FET/(k + k. FET ) = 1 -t. AD/t. D = 1 - FAD/FD dipole-dipole interactions k. FET (R-3)2 ~ 1/R 6 Ro distance @ E=0. 5 i. e. when k. FET = 1/t then: Orientation problem: k 2 5 Intensity 4 E = R-6/(Ro-6 + R-6) donor/aceptor 3 donor (AEDANS) 2 acceptor 1 acceptor (AF) 0 400 500 600 700 Wavelength (nm) Biophysical Tools '04 - Fluorescence 9/19/2021 9