Lighting Up Live Cells with Smart Genetically Encoded Fluorescence Probes from GMars Family

As a special kind of delicate light-controllable genetically encoded optical device, reversibly photoswitchable fluorescent proteins (RSFPs) have been widely applied in many fields, especially various kinds of advanced nanoscopy approaches in recent years. However, there are still necessities for exploring novel RSFPs with specific biochemical or photophysical properties not only for bioimaging or biosensing applications but also for fluorescent protein (FP) mechanisms study and further knowledge-based molecular sensors or optical actuators' rational design and evolution. Besides previously reported GMars-Q and GMars-T variants, herein, we reported the development and applications of other RSFPs from GMars family, especially some featured RSFPs with desired optical properties. In the current work, in vitro FP purification, spectra measurements, and live-cell RESOLFT nanoscopy approaches were applied to characterize the basic properties and test the imaging performances of the selected RSFPs. As demonstrated, GMars variants such as GMars-A, GMars-G, or remarkable photofatigue-resistant GMars-L were found with beneficial properties to be capable of parallelized RESOLFT nanoscopy in living cells, while other featured GMars variants such as dark GMars-P may be a good candidate for further biosensor or actuator design and applications.