Reversibly photoswitchable fluorescent proteins: integrating photophysics, photochemistry, bioimaging, and protein engineering

Reversibly photoswitchable fluorescent proteins (rsFPs) represent a unique class of genetically encoded probes that undergo light-driven transitions between non-fluorescent OFF and emissive ON states. Their distinctive switching properties enable repeated, non-destructive control of fluorescence and have become central to advanced bioimaging approaches. In this review, we provide a critical overview of the molecular mechanisms underlying rsFP function, focusing on GFP-like proteins and fluorogen-activating systems that employ external chromophores. We describe switching kinetics, ON/OFF contrast, and fatigue as fundamental performance parameters, and highlight mechanistic insights from spectroscopy, crystallography, and computational studies. The three subclasses of GFP-like rsFPs-negative, positive, and decoupled types-are discussed in detail, alongside external-chromophore systems such as FAST, UnaG, FbFPs, and biliverdin-binding near-infrared proteins. We further survey a wide range of applications, including super-resolution microscopy, functional biosensing, multiplex discrimination, anisotropy-based analyses, diffusion and transport studies, optical data storage, and optogenetic control. Finally, we outline emerging strategies for improving brightness, photostability, spectral diversity, and switching robustness, emphasizing opportunities for rational protein engineering guided by structural and computational approaches. Together, these developments establish rsFPs as versatile, chemically tunable tools that expand the frontiers of fluorescence imaging and quantitative biology.