Perspective: An outlook on fluorescence tracking

Abstract: Tracking single fluorescent molecules has offered resolution into dynamic molecular processes at the single-molecule level. This perspective traces the evolution of single-molecule tracking, highlighting key developments across various methodological branches within fluorescence microscopy. We compare the strengths and limitations of each approach, ranging from conventional widefield offline tracking to real-time confocal tracking. In the final section, we explore emerging efforts to advance physics-inspired tracking techniques, a possibility for parallelization and artificial intelligence, and discuss challenges and opportunities they present toward achieving higher spatiotemporal resolution and greater computational and data efficiency in next-generation single-molecule studies.