Reversible photoswitching in fluorescent proteins: a mechanistic view.

Phototransformable fluorescent proteins (FPs) have received considerable attention in recent years, because they enable many new exciting modalities in fluorescence microscopy and biotechnology. On illumination with proper actinic light, phototransformable FPs are amenable to long-lived transitions between various fluorescent or nonfluorescent states, resulting in processes known as photoactivation, photoconversion, or photoswitching. Here, we review the subclass of photoswitchable FPs with a mechanistic perspective. These proteins offer the widest range of practical applications, including reversible high-density data bio-storage, photochromic FRET, and super-resolution microscopy by either point-scanning, structured illumination, or single molecule-based wide-field approaches. Photoswitching can be engineered to occur with high contrast in both Hydrozoan and Anthozoan FPs and typically results from a combination of chromophore cis-trans isomerization and protonation change. However, other switching schemes based on, for example, chromophore hydration/dehydration have been discovered, and it seems clear that ever more performant variants will be developed in the future.