A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications.

Reversibly switchable fluorescent proteins (RSFPs) have attracted widespread interest for emerging techniques including repeated tracking of protein behavior and superresolution microscopy. Among the limited number of RSFPs available, only Dronpa is widely employed for most cell biology applications due to its monomeric and other favorable photochemical properties. Here we developed a series of monomeric green RSFPs with beneficial optical characteristics such as high photon output per switch, high photostability, a broad range of switching rate, and pH-dependence, which make them potentially useful for various applications. One member of this series, mGeos-M, exhibits the highest photon budget and localization precision potential among all green RSFPs. We propose mGeos-M as a candidate to replace Dronpa for applications such as dynamic tracking, dual-color superresolution imaging, and optical lock-in detection.