Short pulsed diode lasers as an excitation source for time-resolved fluorescence applications and confocal laser scanning microscopy in PDT

This article describes a setup for subcellular time-resolved fluorescence spectroscopy and fluorescence lifetime measurements using a confocal laser scanning microscope in combination with a short pulsed diodes laser for fluorescence excitation and specimen illumination. The diode laser emits pulses at 398 run wavelength with 70 ps full width at half maximum (FWHM) duration. The diode laser can be run at a pulse repetition rate of 40 MHz down to single shot mode. For time resolved spectroscopy a spectrometer setup consisting of an Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Subcellular fluorescence lifetime measurements were achieved using a time-correlated single photon counting (TCSPC) module instead of the spectrometer setup. The capability of the short pulsed diode laser for fluorescence imaging, fluorescence lifetime measurements and time-resolved spectroscopy in combination with laser scanning microscopy is demonstrated by fluorescence analysis of several photosensitizers on a single cell level.