A direct method for the correction of pressure-induced scrambling of polarized fluorescence intensities

A simple and direct method for the simultaneous correction of steady-state polarized fluorescence intensities, depolarized (or scrambled) by the effec ts of applied hydrostatic pressure, is described. In the method discussed h ere, it is not necessary to first determine the scrambling factors from a s eparate experiment with a dye immobilized in a rigid medium. Rather correct ion for depolarizing effects of the high-pressure spectroscopy cell windows is achieved by direct recalculation of the measured polarized data obtaine d for the sample of interest at the time of data collection. This method of correction is tested for common fluorescent dyes 1,6-diphenyl-1,3,5-hexatr iene (DPH) and 9,10-diphenylanthracene in glycerol where their rotational b ehavior is well understood. In addition, the pressure-induced "melt" profil e for the more complicated biologically relevant system of DPH imbedded wit hin dipalmitoylphosphatidylcholine small unilamellar vesicles has been reex amined. While the method discussed here is used for the correction of stead y-state polarized data, it may be easily adapted for use in time-resolved p olarized fluorescence measurements. Advantages and limitations of the new c orrection method are discussed. (C) 1999 Academic Press.