Reversible photobleaching of photoconvertible SAASoti-FP

SAASoti is a green fluorescent protein suitable for use in super-resolution microscopy as it can be photoconverted to red fluorescence under 405 nm illumination. The green fluorescence of V127T SAASoti variant is reversibly photobleached under exposure to 470 nm light without photoconversion to the red form. The phenomenon can be explained by chromophore protonation that was confirmed by an increase in absorption at 400 nm (chromophore protonated form) and a decrease at 509 nm (anionic form). This light-induced photoswitching can be repeated with the same sample several times without loss of the initial fluorescence intensity. Subsequent sample exposures result in the same fluorescence recovery process. By changing the content of the H-form one can control photoswitching as only the protonated SAASoti may be converted to the red form. This property is extremely important for sub-diffraction microscopy.