Perspective: An outlook on fluorescence tracking

Abstract

Tracking single fluorescent molecules has provided new insights into dynamic molecular processes at the single-molecule level. This Perspective traces the evolution of single-molecule tracking, highlighting key methodological developments across fluorescence microscopy. In particular, we start by exploring what can be gleaned from each type of data and its resulting tracks, ranging from conventional widefield offline tracking to real-time confocal tracking. We subsequently consider emerging efforts to advance physics-inspired tracking techniques, the potential for parallelization and artificial intelligence, and both challenges and opportunities they present toward achieving higher spatiotemporal resolution as well as greater computational and data efficiency in next-generation single-molecule studies.