Identification of the Product of Photoswitching of an Oxazine Fluorophore Using Fourier Transform Infrared Difference Spectroscopy

The oxazine fluorophore ATTO655 is routinely applied in super-resolution fluorescence microscopy due to its reversible photoswitching between a fluorescent and a nonfluorescent state by light in vitro and in living cells. The molecular basis of the oxazine switching has been unclear. Here the photoreaction of ATTO655 in the presence of a 1000-fold excess of the reductant 2-mercaptoethanol was studied using Fourier transform infrared (FT-IR) difference spectroscopy and quantum chemical calculations. The high sensitivity and selectivity of this approach allowed us to identify the chemical structure of the produce of photo-switching as the fully reduced and protonated form of the oxazine. This finding opens the door for both future synthetic as well as analytical work.