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We demonstrate a high-speed confocal fluorescence lifetime imaging microscopy (FLIM) whose accuracy and photon economy are as good as that of a time-correlated single photon counting (TCSPC). It is based on a new lifetime determination scheme, the analog mean delay (AMD) method. Due to the technical advantages of multiple fluorescence photon detection capability, accurate lifetime determination scheme and high photon detection efficiency, the AMD method can be the most effective method for high-speed confocal FLIM. The feasibility of real-time confocal FLIM with the AMD method has been demonstrated by observing the dynamic reaction of calcium channels in a RBL-2H3 cell with respect to 4αPDD stimulus. We have achieved the photon detection rate of 125 times faster than a conventional TCSPC based system in this experiment.

High-speed confocal fluorescence lifetime imaging microscopy (FLIM) with the analog mean delay (AMD) method