Confocal fluorescence lifetime imaging microscopy based on a real-time sampling method

Abstract

We present a fluorescence lifetime imaging microscope (FLIM) based on a real-time waveform acquisition method. The fluorophores were excited by a 635-nm gain-switched laser diode, which produced short pulses with duration ~50 ps in a 20-MHz repetition rate. The fluorescence signals were detected by a silicon avalanche photo-diode (APD) in addition to a wide-band electric amplifier. The converted electric pulses were sampled by a high-speed digitizer of which sampling rate was 2 GS/s. In order to reduce the sampling interval for analyzing sub-nanosecond lifetimes, an interleaved data acquisition technique was used. The effective sampling rate was increased to 10 GS/s. In addition, the impulse response was measured simultaneously with the lifetime signals by an interleaving manner and was used in calibration of the system. By using these methods, accurate lifetime information was acquired in a short time less than 8 μs.