Parallelized fluorescence lifetime imaging microscopy (FLIM) based on photon reassignment

Abstract

We propose a novel parallelized fluorescence lifetime imaging microscopy (FLIM) method based on photon reassignment with a detector array and time-correlated single photon counting (TCSPC) array. The detector array composed of seven avalanche photon diodes (APDs) substitutes for original pinhole and detector to constitute an imaging scanning microscopy (ISM) system. The introduction of parallel detection can solve the pile-up effect caused by the dead-time in TCSPC measurement, which can enhance the effective photon detection efficiency and notably increase the fluorescence lifetime imaging speed. More importantly, the resolution and signal–noise ratio (SNR) can be enhanced based on photon reassignment. The simulation results embody the superiority of our FLIM system, and the experiments with different samples validate the improvement of the resolution and speed of fluorescence lifetime imaging. The experiment results show that the resolution of our method is enhanced by a factor of 1.5 and the imaging speed can be at least increased by a factor of 2.5 compared with conventional confocal TCSPC FLIM system. We envision that the proposed FLIM method based on parallel detection will allow for super-resolution fluorescence lifetime real-time imaging of live cell.