Photon-Counting Phase-Modulation Fluorometer

Abstract

We propose a phase-modulation fluorometer that is applicable to a very weak fluorescence intensity level. In order to counter the single-photon event situation, we have introduced a combination of a time-to-amplitude converter and a pulse height analyzer (PHA) to the fluorometer, the combination of which is usually used in the single-photon correlation method to measure fluorescence decay waveforms with pulsed excitation. In the proposed fluorometer, a sinusoidal response waveform that is shifted in phase over the reference one is obtained statistically as a histogram in the PHA memory, and then the fluorescence lifetime can be calculated by the same procedure as the conventional analog phase-modulation method. The excitation light source used was a current-modulated ultraviolet light-emitting diode, whose center wavelength was 370 nm and whose spectral bandwidth was 10 nm. Fluorescence lifetimes of 17.5 ns and 5.7 ns obtained for 10 ppb quinine sulfate in 0.1 N H2SO4 and for 10 ppb rhodamine 6G in ethanol, respectively, agreed well with those reported in the literature. In passing, we report that the fluorescence lifetime of benzo[a]pyrene in dichloromethane is 14.6 ns, which is known as one of the carcinogenic, environmental-pollution materials.