High-speed fluorescence lifetime measurement for investigation of dynamic phenomena

We present a novel method for high-speed measurements of fluorescence lifetime, in which fluorescence signal for precise lifetime determination is acquired in a short time on the order of microseconds. Our method is based on analog signal that contains a number of fluorescence photons in a pulse, on the contrary to the conventional time-correlated single-photon counting in which only a single photon is permitted for a fluorescence pulse. Because this method does not have any problem of photon counting pile-up, the measurement speed is not limited by the single-photon constraint and can increase up to the excitation repetition rate. In order to extract the accurate lifetime information from the analog signal contaminated by the slow instrumental response function (IRF), we have developed a new signal processing method, in which the lifetime is determined by difference between mean arrival time of the analog photo-electronic pulse of fluorescence signal and one of IRF signal. By both experimental and theoretical studies, we have verified that the measurement accuracy and precision are nearly independent of the width of the IRF so that inexpensive narrowbandwidth photo-detectors and low-speed electronics can be used for this method. Excellent accuracy and precision have been obtained experimentally for high-speed measurements completed in a few microseconds. These results suggest that our method can be well applied to measurement of fast dynamic phenomena and real time fluorescence lifetime imaging microscope with low cost.