Fluorescent molecules that can be switched on and off or between two different colors are key to the advancement of super-resolution microscopy techniques. Reversibly switchable fluorescent proteins (RSFPs) have attracted widespread interest for emerging techniques including repeated tracking of protein behavior and super-resolution 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.

Recently, Professor Xu Tao and Professor Xu Pingyong at the Institute of Biophysics, Chinese Academy of Sciences discovered a unique strategy for generating RSFPs of different optical characteristics by mutating the first position of chromophore residues, but not the sites in the vicinity of the chromophore. They developed a series of 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. They propose mGeos-M as a candidate to replace Dronpa for applications such as dynamic tracking, dual-color superresolution imaging, and optical lock-in detection. They identified six reversibly photoswitchable monomeric green fluorescent proteins with distinguished photophysical properties. These mGeosexpanded the current tool kit of PAFPs and allow for broad applications in dynamic live cell imaging and superresolution imaging.

This work was published in the latest issue of PNAS as an original article titled “A unique series of reversibly switchable fluorescent proteins with beneficial properties for various applications”. It was supported by the Ministry of Science and Technology of China, the National Science Foundation of China and the Chinese Academy of Sciences. The latest issue of Nature Methods also published an article highlighted this work.