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Review of fluorescence anisotropy decay analysis by frequency-domain fluorescence spectroscopy

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Abstract

This didactic paper summarizes the mathematical expressions needed for analysis of fluorescence anisotropy decays from polarized frequency-domain fluorescence data. The observed values are the phase angle difference between the polarized components of the emission and the modulated anisotropy, which is the ratio of the polarized and amplitude-modulated components of the emission. This procedure requires a separate measurement of the intensity decay of the total emission. The expressions are suitable for any number of exponential components in both the intensity decay and the anisotropy decay. The formalism is generalized for global analysis of anisotropy decays measured at different excitation wavelengths and for different intensity decay times as the result of quenching. Additionally, we describe the expressions required for associated anisotropy decays, that is, anisotropy decays where each correlation time is associated with a decay time present in the anisotropy decay. And finally, we present expressions appropriate for distributions of correlation times. This article should serve as a reference for researchers using frequency-domain fluorometry.

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Authors and Affiliations

  1. Center for Fluorescence Spectroscopy and Department of Biological Chemistry, University of Maryland School of Medicine, 108 North Greene Street, 21201, Baltimore, Maryland

    Joseph R. Lakowicz, Henryk Cherek, Józef Kuśba & Ignacy Gryczynski

  2. Department of Pharmacology, University of Virginia, Box 448, 22908, Charlottesville, Virginia

    Michael L. Johnson

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  1. Joseph R. Lakowicz
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Lakowicz, J.R., Cherek, H., Kuśba, J. et al. Review of fluorescence anisotropy decay analysis by frequency-domain fluorescence spectroscopy. J Fluoresc 3, 103–116 (1993). https://doi.org/10.1007/BF00865324

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  • DOI: https://doi.org/10.1007/BF00865324

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