Time-resolved fluorescence polarization measurements for entire emission spectra with a resistive-anode, single-photon-counting detector: The Fluorescence Omnilyzer

Abstract

We report a fluorescence analyzer that records simultaneously the temporal profiles for both orthogonal linear polarizations for all wavelengths in a fluorescence emission spectrum. The Analyzer combines a resistive-anode single-photon-counting photomultiplier, imaging spectrograph, Wollaston polarizer, multiparameter analyzer with histograming memory, and standard timing electronics. The spectrograph disperses the fluorescence spectrum across the photocathode of the photomultiplier, and the Wollaston polarizer separates the spectra of the two polarizations in opposite directions from the center of the photocathode perpendicular to the direction of spectral dispersion. The locations at which each photon reaches the photocathode is determined by the ratios of the charges read from the four corners of the resistive anode. One of the two address coordinates that determine where in histogramming memory each photon is recorded is obtained by measuring the time of arrival of the photon at the detector relative to the pulse of light that excites the fluorescence. The second address coordinate is obtained by combining the most-significant bit of the location of the event along the direction on the resistive anode corresponding to the polarization of the photon with the multibit digital value indicating photon wavelength. Storing the data directly into histogramming memory permits display of the data set as it is recorded. Both the spectral and temporal calibrations of the fluorescence analyzer are independent of the polarization of the fluorescence. The ≈100 ps temporal resolution of the resistive-anode detector is well matched to the ≈1 ns full width at half-maximum pulses of light produced by the synchrotron storage ring that we use as the excitation source, but laser excitation could also be used with this detector. Recording simultaneously all of the data required for the global analysis of the time evolution of both linear polarization components of fluorescence, and thus, time-resolved anisotropy, reduces the duration of exposure of the sample to the excitation beam, hence, facilitating studies of fragile or photosensitive biological specimens.