Advancements in phasor-based FLIM: multi-component analysis and lifetime probes in biological imaging

Fluorescence lifetime imaging microscopy (FLIM) is a reliable method that achieves imaging by detecting fluorescence lifetimes within samples. Owing to its unique temporal characteristic, it can complement fluorescence intensity measurement. Technological and methodological advancements in FLIM have broadened its applications across various domains. The processing of fluorescence lifetime data is crucial for enhancing the speed and accuracy of imaging. Thus, various lifetime fitting algorithms have been developed to improve the imaging speed. The phasor analysis (PA) method is an approach for processing fluorescence lifetime data, capable of directly converting lifetime signals into visual graphics without fitting, which outperforms traditional approaches in speed. Furthermore, lifetime probes with distinct lifetimes are readily implemented for visualization and cluster analysis combined with PA, facilitating the prediction of specific biological states or functions. This review examines various lifetime probes employed in phasor-based FLIM and discusses their roles in the PA method. The methods for multi-component PA within complex biological environments were also described. Additionally, we focused on the advantages of the phasor vector rule and the unmixing of multi-component analysis based on PA. The integration of lifetime probes with phasor-based FLIM facilitates rapid and intuitive detection methods for analyzing complex biological environments.