Abstract
Fluorescence lifetime imaging has emerged as a promising modality to extract molecular information from biological systems, providing detailed and semi-quantitative characterisation of subcellular microenvironments. Fluorescence lifetime measurements offer robust insights into biological processes as they are less dependent on concentration and excitation power than intensity-based measurements. However, fluorescence lifetime imaging suffers from a paucity of fluorophores with wide dynamic ranges of lifetimes and responsiveness to biostimuli. This shortcoming has prompted the design of new chemical strategies to tailor the optical properties of organic fluorophores and their application in multiplexed live-cell imaging for the visualisation of molecular and cellular interactions. This Review article covers advances - primarily from the last 5 years - in the chemical design of fluorescence lifetime probes that combine optical reporters and targeting groups (e.g., ligands, peptides, proteins), their applications in bioimaging and related computational-based innovations for data acquisition and analysis. The perspectives and challenges in the design and applications of fluorescence lifetime probes are discussed, bridging chemistry and bioimaging as well as providing strategic insights for advancing fluorescence lifetime imaging.