PubMed · 7765356
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R Bolger, D Thompson. 1994. A quantitative RNase assay using fluorescence polarization.. https://pubmed.ncbi.nlm.nih.gov/7765356/
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A simple and direct method for the simultaneous correction of steady-state polarized fluorescence intensities, depolarized (or scrambled) by the effects of applied hydrostatic pressure, is described. In the method discussed here, it is not necessary to first determine the scrambling factors from a separate experiment with a dye immobilized in a rigid medium. Rather correction for depolarizing effects of the high-pressure spectroscopy cell windows is achieved by direct recalculation of the measured polarized data obtained 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-hexatriene (DPH) and 9,10-diphenylanthracene in glycerol where their rotational behavior is well understood. In addition, the pressure-induced "melt" profile for the more complicated biologically relevant system of DPH imbedded within dipalmitoylphosphatidylcholine small unilamellar vesicles has been reexamined. While the method discussed here is used for the correction of steady-state polarized data, it may be easily adapted for use in time-resolved polarized fluorescence measurements. Advantages and limitations of the new correction method are discussed.
We observed emission from the tyrosine derivative N-acetyl-L-tyrosinamide (NATyrA) when excited with the fundamental output of a femtosecond Ti:Sapphire laser from 780 to 855 nm. The dependence on incident laser power indicates a three-photon process. The emission spectra and intensity decay in glycerol-water (30:70) at 5 degrees C were found to be identical for one- and three-photon excitation. Also the excitation spectrum of three-photon-induced fluorescence of NATyrA corresponds to the one-photon excitation spectrum. The time-zero or fundamental anisotropy spectrum was reconstructed from the frequency-domain anisotropy decays. The three-photon anisotropies are similar or larger than the one-photon anisotropies. These three-photon anisotropies are surprising given the near zero values known for tyrosine with two-photon excitation. The observations indicate that one- and three-photon excitation directly populates the same singlet excited states(s). However, the origin of the anisotropies with multi-photon excitation of tyrosine remain unclear and unpredictable.
A new approach to oxygen sensing based on the luminescence polarization observed from a novel type of sensor is described. The oxygen sensor consists of an oxygen-sensitive silicone film containing tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) chloride [Ru(dpp)3Cl2] and an oxygen-insensitive film of Styryl 7 in poly(vinyl alcohol). Polarizers are used to select orthogonally polarized emission components from Ru(dpp)3Cl2 and Styryl 7. The polarization of the combined emission was found to be highly sensitive to the partial pressure of oxygen. This method of polarization sensing is generic and can be used with any fluorophore which displays an analyte-dependent change in intensity.