The precise measurement of the fluorescence polarisation anisotropy by single-photon counting

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Abstract
The rotational diffusion coefficients of a fluorescent molecule can be determined by measuring the time-dependent anisotropy r(t). The errors introduced in such measurements by misalignments in the setup are discussed. Since the single-photon sampling technique normally used is nonlinear, the necessary normalisation of the intensities I/sub /// and Iperpendicular to is considerably complicated. A correct normalisation procedure is described and the sensitivity of the method is shown in a measurement of micellar rotation.