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Mini Review | Open Access

Emerging trends of fluorescence lifetime imaging microscopy (FLIM): advances, challenges, and prospects

Baoyi Wang1Meilin Li1Xiaoshuai Huang2Bei Liu1,3( )
National Biomedical Imaging Center, College of Future Technology, Peking University, Beijing 100871, China
Key laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Peking University Cancer Hospital & Institute, Beijing 100142, China
Beijing Laboratory of Biomedical Imaging, Beijing 100871, China
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Abstract

This mini-review provides a concise overview of the latest advances in fluorescence lifetime imaging microscopy (FLIM). It discusses both time-domain and frequency-domain techniques, analysis methods − including phasor approaches and deep learning, and highlights applications in multiplexed imaging and quantitative biosensing. Furthermore, FLIM-empowered multimodal imaging approaches aimed at enhancing spatial and temporal resolution are discussed. Persistent challenges, including photon efficiency, probe sensitivity, and achieving high-speed imaging in live-cell environments, are critically assessed, outlining pathways toward future innovations.

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Biophysics Reports
Pages 225-239

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Cite this article:
Wang B, Li M, Huang X, et al. Emerging trends of fluorescence lifetime imaging microscopy (FLIM): advances, challenges, and prospects. Biophysics Reports, 2026, 12(4): 225-239. https://doi.org/10.52601/bpr.2025.250024

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Received: 31 March 2025
Accepted: 03 June 2025
Published: 31 August 2026
© The Author(s) 2026

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