Theory and Application of Differential Polarized Phase Fluorometry
As discussed in the preceding contributions, polarized pulse fluorometry is widely used to investigate the rotational motions of fluorophores. Differential polarized phase fluorometry (DPF) provides comparable information. For differential phase measurements, a sinusoidally modulated exciting light is employed, and the difference in phase angle between the parallel and perpendicular components of the fluorescence emissions measured. These phase angles depend on the rates of fluorophore rotation and the isotropy and freedom of these rotations. Although polarized phase measurements date back to 1935, the theory and practice of DPF have only recently become available and been applied in biological research. These advances are primarily a result of the efforts of Weber.1,2 In the following sections, the theory of differential phase fluorometry, and the measurement of phase angles will be described. In addition, examples from the literature will be reviewed, and DPF compared with pulse methods.
KeywordsPhase Angle Propylene Glycol Rotational Diffusion Pulse Method Exciting Light
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- 2.Weber, G., Limited rotational motion: Recognition by differential phase fluorometry, Acta Phys.Polon. A54 859–965 (1978)Google Scholar
- 5.Galanin, M.D., Dokl.Akad.Nauk SSSR. 57 883–886 (1947)Google Scholar
- 6.Tumerman, L.A., Soviet Physics (Uspekhi), 33 218–276 (1947)Google Scholar
- 7.Galanin, M.D. Trudy Fiz.Inst.Lebedev, Akad.Nauk SSSR, 5 339–386 (1950)Google Scholar
- 13.Lakowicz, J.R. and Prendergast, F.G., Detection of Hindered Rotations of 1, 6-Diphenyl-1, 3, 5-hexatriene in Lipid Bilayers by Differential Polarized Phase Fluorometry, Biophys.J. 24 213–231 (1978)Google Scholar
- 14.Lakowicz, J.R. and Prendergast, F.G., Quantitation of hindered rotations of diphenylhexatriene in lipid bilayers by differential polarized phase fluorometry, Science 200 1399–1401 (1978)Google Scholar
- 15.Zinsli, P.E., Anisotropic rotation and libration of perylene in paraffin, Chem.Phys. 20 299–309 (1977)Google Scholar
- 17.Weber, G. and Mitchell, G.W., Demonstration of Anisotropic Molecular Rotations by Differential Polarized Phase Fluorometry, in, Excited States of Biological Molecules, J.B. Birks, ed., John Wiley and Sons, New York, 1976, pp. 72–76.Google Scholar
- 18.Valeur, B. and Weber, G., Anisotropic rotations in lnaphthylamine. Existence of a red-edge transition normal to the ring plane, Chem.Phys.Lett. 45 140–144 (1977)Google Scholar
- 19.Haar, H.P., Klein, U.K.A., Hafner, F.W. and Hauser, M., Determination of the rotational diffusion by a picosecond phase fluorometer, Chem.Phys.Lett. 49 563–567 (1977)Google Scholar
- 20.Heyn, M.P., Determination of lipid order parameters and rotational correlation times from fluorescence depolarization experiments, FEBS Lett. 108 359–364 (1979)Google Scholar