Abstract
This chapter describes the kinetic models and data analysis methods for fluorescence anisotropy decay. Time-resolved fluorescence anisotropy is a powerful technique, for investigating macromolecular dynamics. In a time-resolved anisotropy measurement, a sample is excited, by linearly polarized light, and the anisotropy (or polarization) decay of the resulting emission is evaluated, by observing the fluorescence decay at polarizations parallel and perpendicular to the excitation. The emission can be depolarized, by a variety of dynamic and photophysical processes. Photophysical mechanisms of depolarization include the vibrational relaxation of the excited-state fluorophore as well as more unusual events, such as resonance energy transfer. Each of these depolarizing processes occurs with a characteristic correlation time and the influence of a given process on the anisotropy decay depends on the relative values of that correlation time and the lifetime of the excited state, as determined by a fluorescence intensity decay experiment. Time-resolved fluorescence anisotropy data may be analyzed, by several algorithms, including the method of moments, the Laplace transform, and nonlinear least squares (NLLS).
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