Fluorescence isotropic rotational diffusion

The timescale of fluorescence emission is comparable to that of rotational diffusion of proteins and the timescale of segmental motions of protein domains or individual amino acid residues. The polarization or anisotropy of the emission provides a measure of these processes. Suppose a sample is excited with vertically polarized light (Fig. 11), and that the sample is viscous so that the fluorophores do not rotate during the lifetime of the excited state. Then the emission is polarized, usually also in the vertical direction. This polarization occurs because the polarized excitation selectively excites those fluorophores in the isotropic solution whose absorption... 

This relationship is modified by two constants the molecular shape factor/ (a function of the molecular dimensions) and the boundary coefficient C, which takes into account the interaction between the solvent and the solute. In principle, two-photon fluorescence anisotropy decays in isotropic media should yield the same diffusion times as for single photon excitation, but with significantly increased initial fluorescence anisotropy this can be seen in Figure 11.17, which compares single- and two-photon anisotropy decays for the fluorescent probe rhodamine 6G in ethylene glycol. Rotational drflusion times for small molecular probes vary from nanoseconds to hundreds of picoseconds for isotropic rotational drflusion in low viscosity solvents. 

Third, the interpretation of polarization measurements normally requires assumptions about the nature of the motion leading to emission depolarization. Isotropic motion is easy to interpret. In some instances one can get a very detailed description of anisotropic rotational diffusion by combining nmr, light scattering and fluorescence depolarization measurements. 

The kinetic interactions between solvent and solute molecules in free solution determine their rotational and translational diffusion characteristics. Fluorescence polarization is a spectroscopic technique that allows the determination of motional preferences of reporter molecules in fluids with respect to both the rate of motion and the orientational restriction of that motion [1,2], For spherical molecules in isotropic fluids at low concentrations, these motions can be described by the Stokes-Einstein and Perrin relationships, and if these motions have an equal probability of occurring in any dimension they are referred to as isotropic. However, when a fluid displays structure, or anisotropy, the motion of diffusing molecules may be restricted, generally to different extents in different dimensions, and these motions are said to be anisotropic. New approaches must then be taken in order to describe the probe s hydrodynamic behavior. By measuring the hydrodynamic properties of a fluorescent probe in solution, it is possible to extract valuable information on the physical structure and properties of a fluid. Knowledge of the physical structure and properties of food fluids and matrices is essential for solving practical problems in food research. 

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