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

Joseph R. Lakowicz, Henryk Cherek, Józef Kuśba, Ignacy Gryczynski, Michael L. Johnson

Research output: Contribution to journalReview articlepeer-review

50 Scopus citations


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.

Original languageEnglish
Pages (from-to)103-116
Number of pages14
JournalJournal of Fluorescence
Issue number2
StatePublished - Jun 1993


  • Fluorescence anisotropy decays
  • frequency-domain fluorescence spectroscopy
  • review


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