Abstract
SIGNIFICANCE: Fluorescence lifetime imaging microscopy (FLIM) has been gaining increasing attention due to its capability to provide robust, accurate, and quantitative measurements compared with conventional intensity-based fluorescence microscopy. However, the slow imaging speed of FLIM has long been a major limitation for expanding its applications in biology and medicine. AIM: We aim to discuss recent advancements in FLIM to enhance imaging speed and explore its biomedical applications and future prospects. APPROACH: We discuss high-speed FLIM techniques by categorizing them into time-domain and frequency-domain approaches, as well as wide-field and beam-scanning imaging schemes, with a focus on their combinations. RESULTS: Recent advances in high-speed FLIM have been primarily driven by innovations in fluorescence detection schemes for wide-field imaging, as well as fluorescence excitation strategies for beam-scanning imaging. By enhancing FLIM imaging speed to levels comparable with intensity-based fluorescence microscopy, the observation of fast cellular dynamics, such as neural spiking, and large-scale image-based analysis of heterogeneous cells have become feasible. In addition, promising directions for the future development of high-speed FLIM include further utilization of fit-free analysis, implementation of video-rate 3D FLIM, and realization of FLIM-based cell sorting. CONCLUSIONS: The development of high-speed FLIM promises new opportunities in cell biology, biophysics, and neurology.