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

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.