Lifetime imaging with the Zeiss LSM-510

The Zeiss LSM-510 NLO laser scanning microscope can be combined with a new TCSPC (time-correlated single photon counting) lifetime imaging technique developed by Becker & Hickl, Berlin. This technique is based on a three-dimensional histogramming process that records the photon density over the time within the fluorescence decay function and the x-y coordinates of the scanning area. The histogramming process avoids any time gating and therefore yields a counting efficiency close to one. Upgrading The LSM-510 for TCSPC imaging does not require changes in the microscope hardware or software. A fast detector is attached to the fibre output of the scanning head, and synchronisation of the TCSPC module with scanning is achieved via the user I/O of the scan controller. With an MCP-PMT as a detector, fluorescence decay components down to 30 ps can be resolved. The capability of the instrument is shown for the separation of chromphores by their fluorescence lifetime and for CFP/YFP FRET.