Development of a laser-based fluorescence microscope with subnanosecond time resolution

This report describes the development of a fluorescence microscope based on a standard inverted optical microscope which incorporates a pulsed picosecond dye laser excitation source and a detector consisting of a gated image intensifier coupled to a CCD camera. Fluorescence images have been obtained using gate durations of 0.5 ns from this apparatus, representing a reduction in gate duration of an order of magnitude compared with similar instruments reported by others recently. Subnanosecond gated fluorescence images of V79-4 Chinese hamster lung fibroblasts stained with a phthalocyanine photosensitizer used in photodynamic therapy are presented. The results of these measurements are discussed in terms of the intracellular distribution of the sensitizer. Other potential applications and limitations of this technique are also outlined.