EVALUATION OF A TIME-RESOLVED FLUORESCENCE MICROSCOPE USING A PHOSPHORESCENT PT-PORPHINE MODEL SYSTEM

A time-resolved fluorescence microscope is presented that allows the s ensitive detection of delayed luminescent labels with decay times from one microsecond to several milliseconds. The microscope utilizes an a rgon ion laser chopped with an acousto-optical modulator as excitation light source in combination with a gated multichannel plate image int ensifier in the image plane. A theoretical model for the detection eff iciency of practically any time-resolved fluorescence microscope is ve rified using phosphorescent Pt-porphine-stained Sephadex beads. The de tection efficiency of the presented setup was shown to be 42%, which i s near the theoretical limit of 50% for non-saturated luminescent dyes . The suppression of prompt fluorescence signals was found to be 1:5,5 00. The Pt-porphine beads proved to be an excellent model system for t ime-resolved fluorescence microscopy, showing a high extinction coeffi cient and high phosphorescence quantum yield in aqueous environment un der room temperature conditions. Furthermore, for the microscope descr ibed the decay time of the Pt-porphine beads of 47 mu s is long enough to enable efficient suppression of the prompt fluorescence while main taining a high excitation and emission duty cycle. This is considered to be of vital importance in order not to saturate the luminescence wi th the excitation intensities commonly used in fluorescence microscopy . (C) 1996 Wiley-Liss, Inc.