How fast can TCSPC FLIM be made?

The acquisition time of TCSPC FLIM depends on the number of pixels of the image, on the required lifetime accuracy, and on the count rate available from the sample. For samples with high fluorophore concentrations, such as stained tissue or plant cells the available count rates may come close to the maximum counting capability of the currently used TCSPC FLIM techniques. We describe the behaviour of TCSPC at high count rates and estimate the size of counting loss and pile-up effects. It turns out that systematic lifetime errors are smaller than previously believed. TCSPC FLIM can therefore be used to record fast sequences of fluorescence lifetime images. Fast sequential FLIM will be demonstrated for the measurement of chlorophyll transients in living plant tissue.