Fast reversibly photoswitching red fluorescent proteins for live-cell RESOLFT nanoscopy

Reversibly photoswitchable fluorescent proteins (rsFPs) are gaining popularity as tags for optical nanoscopy because they make it possible to image with lower light doses. However, green rsFPs need violet-blue light for photoswitching, which is potentially phototoxic and highly scattering. We developed new rsFPs based on FusionRed that are reversibly photoswitchable with green-orange light. The rsFusionReds are bright and exhibit rapid photoswitching, thereby enabling nanoscale imaging of living cells. Bright reversibly switching red fluorescent proteins (rsFusionReds) with fast switching kinetics and low fatigue enable RESOLFT and MoNaLISA nanoscopy of live cells with green-orange illumination, which further reduces the risk of phototoxicity.