High-speed fluorescence lifetime imaging microscopy: techniques, applications, and prospects

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.

We aim to discuss recent advancements in FLIM to enhance imaging speed and explore its biomedical applications and future prospects.

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.

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.

The development of high-speed FLIM promises new opportunities in cell biology, biophysics, and neurology.