Green-to-red photoconvertible Dronpa mutant for multimodal super-resolution fluorescence microscopy.

Advanced imaging techniques crucially depend on the labels used. In this work, we present the structure-guided design of a fluorescent protein that displays both reversibly photochromic and green-to-red photoconversion behavior. We first designed ffDronpa, a mutant of the photochromic fluorescent protein Dronpa that matures up to three times faster while retaining its interesting photochromic features. Using a combined evolutionary and structure-driven rational design strategy, we developed a green-to-red photoconvertible ffDronpa mutant, called pcDronpa, and explored different optimization strategies that resulted in its improved version, pcDronpa2. This fluorescent probe combines a high brightness with low photobleaching and photoblinking. We herein show that, despite its tetrameric nature, pcDronpa2 allows for multimodal subdiffraction imaging by sequentially imaging a given sample using both super-resolution fluctuation imaging and localization microscopy.