image: FLIM principles. Schematic overview of fluorescence lifetime data acquisition and analysis in time−domain (td) and frequency−domain (fd) modes. In tdFLIM, photon arrival times are recorded after each pulsed excitation to build a decay histogram, which can be analyzed by multi-exponential curve fitting or transformed into the phasor space for model-free lifetime extraction. In fdFLIM, the emission is measured under high-frequency intensity modulation, yielding phase shift and demodulation factors that can likewise be processed by fitting or phasor analysis. Both approaches ultimately render lifetime maps as pseudocolor images for biological interpretation.
Credit: HIGHER EDUCATON PRESS
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.
Method of Research
Experimental study
Subject of Research
Not applicable
Article Title
Emerging trends of fluorescence lifetime imaging microscopy (FLIM): advances, challenges, and prospects
Article Publication Date
1-Jan-2026