Fluorescence recovery anisotropy

One of the most popular applications of molecular rotors is the quantitative determination of solvent viscosity (for some examples, see references [18, 23-27] and Sect. 5). Viscosity refers to a bulk property, but molecular rotors change their behavior under the influence of the solvent on the molecular scale. Most commonly, the diffusivity of a fluorophore is related to bulk viscosity through the Debye-Stokes-Einstein relationship where the diffusion constant D is inversely proportional to bulk viscosity rj. Established techniques such as fluorescent recovery after photobleaching (FRAP) and fluorescence anisotropy build on the diffusivity of a fluorophore. However, the relationship between diffusivity on a molecular scale and bulk viscosity is always an approximation, because it does not consider molecular-scale effects such as size differences between fluorophore and solvent, electrostatic interactions, hydrogen bond formation, or a possible anisotropy of the environment. Nonetheless, approaches exist to resolve this conflict between bulk viscosity and apparent microviscosity at the molecular scale. Forster and Hoffmann examined some triphenylamine dyes with TICT characteristics. These dyes are characterized by radiationless relaxation from the TICT state. Forster and Hoffmann found a power-law relationship between quantum yield and solvent viscosity both analytically and experimentally [28]. For a quantitative derivation of the power-law relationship, Forster and Hoffmann define the solvent s microfriction k by applying the Debye-Stokes-Einstein diffusion model (2)... 

A number of macromolecular diffusion and conformational properties can be studied using fluorescence anisotropy, fluorescence correlation spectroscopy (ECS), and fluorescence recovery after photobleaching (FRAP). These techniques most commonly are applied to proteins labelled with highly fluorescent probes, but can exploit intrinsic fluorescence in some instances. In fluorescence anisotropy studies, polarized light is used to selectively excite molecules whose transition dipole moments are aligned with the electric field vector. Steady-state... 

Figure 8. Fluorescence recovery anisotropy (FRA) of ConA labelled with fluorescein-5 -isothiocyanate. Conditions ...

Lee, J., Wang, Y., and Gibson, B. G. (1990). Recovery of components of fluorescence spectra of mixtures by intensity- and anisotropy decay-associated analysis the bacterial luciferase intermediates. Anal. Biochem. 185 220-229. 

Reticulum ATPase [105,106], Owing to the long-lived nature of the triplet state, Eosin derivatives are suitable to study protein dynamics in the microsecond-millisecond range. Rotational correlation times are obtained by monitoring the time-dependent anisotropy of the probe s phosphorescence [107-112] and/or the recovery of the ground state absorption [113— 118] or fluorescence [119-122], The decay of the anisotropy allows determination of the mobility of the protein chain that cover the binding site and the rotational diffusion of the protein, the latter being a function of the size and shape of the protein, the viscosity of the medium, and the temperature. 

---