Since their broad introduction in the early 90s confocal and two-photon laser scanning microscopes have initiated a breakthrough in biomedical imaging. The applicability of multi-photon excitation, the optical sectioning capability and the superior contrast of these instruments make them an ideal choice for fluorescence imaging of biological samples.

However, the fluorescence of organic molecules is not only characterised by the emission spectrum, it has also a characteristic lifetime. Any energy transfer between an excited molecule and its environment in a predictable way changes the fluorescence lifetime. Since the lifetime does not depend on the concentration of the chromophore fluorescence lifetime imaging is a direct approach to all effects that involve energy transfer. Typical examples are the mapping of cell parameters such as pH, ion concentrations or oxygen saturation by fluorescence quenching, or fluorescence resonance energy transfer (FRET) between different chromophores in the cell. Furthermore, combined intensity / lifetime imaging is a powerful tool to distinguish between different fluorescence markers in multi-stained samples and between different natural fluorophores of the cells themselves. These components often have ill-defined fluorescence spectra but are clearly distinguished by their fluorescence lifetime.

Recording time-resolved fluorescence images can be achieved by combining a Laser Scanning Microscope with pulsed laser excitation and a new Time-Correlated Single Photon Counting (TCSPC) Imaging technique introduced by Becker & Hickl. This note was written to assist upgrading of laser scanning microscopes for lifetime imaging

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