Double beam fluorescence lifetime spectrometer with subnanosecond resolution: Application to aqueous tryptophan

Abstract

An instrument capable of accurately measuring fluorescence lifetimes with subnanosecond resolution is described. The equipment consists of a mode-locked frequency doubled dye laser and a channel plate photomultiplier. The time dependence of the fluorescence is measured using the single photon counting technique. The apparatus response function which was found to be count rate dependent (FWHM = 150 ps at 10 kHz) is monitored simultaneously with the sample fluorescence with the same detection equipment by using a double beam and optical delay technique and a standard reference (p-terphenyl). With this technique, typical problems such as count rate and wavelength dependence of the time response of the channel plate photomultiplier are eliminated. The data are analyzed using a nonlinear least-squares procedure where the fluorescence of the reference is directly used to account for the time response of the instrument. The system is applied to the fluorescence decay of aqueous solutions of tryptophan excited at 295 nm and observed between 320 and 380 nm. The fluorescence decay can be described by a biexponential with 3.15 and 0.54 ns components, which are found to be independent of emission wavelength.