Intracellular Fluorescence Lifetime Imaging of Ala-ppix Using Ps Diode Lasers and Tcspc Techniques in Laser Scanning Microscopes

A setup consisting on a laser scanning microscope (LSM 410, Zeiss, Germany) equipped with appropriate detection units was developed for time-resolved intracellular fluorescence spectroscopy and fluorescence lifetime imaging (FLIM) for on-line detection of structural changes of various biomolecules. Short-pulsed excitation was performed with a diode laser which emits pulses at 398 nm with 70 ps duration (LDH-C 400, PicoQuant, Germany). The laser was coupled to the laser scanning microscope. For time resolved spectroscopy a setup consisting on a Czerny Turner spectrometer and a MCP-gated and -intensified CCD camera was used. Time-gated spectra within the cells were acquired by placing the laser beam in “spot scan” mode. In addition, a time-correlated single photon counting module (SPC-730, Becker & Hickl, Germany) was used to determine the fluorescence lifetime of different photosensitizers from single spots and to record lifetime images (τ-mapping) [1].