2025/06/05 by Hiroshi Kanno, Fan Li, Jongchan Park +4 · 1 voice
Biochemistry, Genetics and Molecular Biology · Engineering · Physics and Astronomy · #Advanced Fluorescence Microscopy Techniques #Digital Holography and Microscopy #Optical Coherence Tomography Applications
paper · doi:10.1117/1.bios.2.3.030901
openalex publication_date 2025/06/05 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/29
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.