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Pure Appl. Chem., 2013, Vol. 85, No. 3, pp. 589-608

http://dx.doi.org/10.1351/PAC-REP-11-11-12

Published online 2013-01-11

ANALYTICAL CHEMISTRY DIVISION

ORGANIC AND BIOMOLECULAR CHEMISTRY DIVISION

PHYSICAL AND BIOPHYSICAL CHEMISTRY DIVISION

Fluorescence anisotropy measurements in solution: Methods and reference materials (IUPAC Technical Report)

Marcel Ameloot1*, Martin vandeVen1, A. Ulises Acuña2 and Bernard Valeur3

1 Universiteit Hasselt, BIOMED, Agoralaan, Building C, 3590-Diepenbeek, Belgium
2 Instituto de Química-Física Rocasolano, CSIC, 119 Serrano, 28006-Madrid, Spain
3 Département CASER, CNAM, 292 rue Saint-Martin, F-75141 Paris Cedex 03, and Laboratoire PPSM, ENS-Cachan, 61 Avenue du Président Wilson, F‑94235 Cachan Cedex, France

Abstract: After recalling the basic relations relevant to both steady-state and time-resolved fluorescence polarization, it is shown how the values of steady-state polarized intensities recorded experimentally usually need to be corrected for systematic effects and errors, caused by instrumentation and sample properties. A list of selected reference values of steady-state fluorescence anisotropy and polarization is given. Attention is also paid to analysis of time-resolved fluorescence anisotropy data obtained by pulse fluorometry or phase and modulation fluorometry techniques. Recommendations for checking the accuracy of measurements are provided together with a list of selected time-resolved fluorescence anisotropy data as reported in the literature.