Fluorescence-lifetime can be used as a contrast mechanism for determining different fluorophores. The lifetime of fluorophores is in the nanosecond range and requires the use of a pulsed laser with Time Correlated Single Photon Counting (TCSPC) in order to measure the fluorescence decay. Our two-photon microscope is equipped with a TCSPC detector which, along with the 80 MHz repetition rate of the laser, allows lifetime imaging.
Below is an example of lifetime being used to distinguish a cyan fluorescent protein from autofluorescence with strongly overlapping spectra. Small regions of the image data are used to fit multi-exponential curves. An image of the fitted time-constants can then be plotted - giving an image of the lifetime constants.
