Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores.

The key events in the light-induced switching mechanism of the photochromic green fluorescent protein Padron have been investigated by employing femtosecond fluorescence up-conversion, femtosecond transient absorption, and time-correlated single photon counting techniques. In contrast to Dronpa, excitation of protein’s neutral state at 395 nm triggers an efficient and complex photoswitching to a dark state whereas irradiation with 495 nm light reverses the protein to its initial state restoring the bright fluorescence. On the basis of the kinetics observed upon irradiation of the chromophore in the protonated state, we suggest that the switching mechanism consists of a light-initiated excited state process (presumably ESPT) with a time constant of 1 ps producing an unstable intermediate state, tentatively assigned to the excited state of the cis-anionic form, that is followed by a cis- to trans- isomerization (14.5 ps) forming the trans-anionic state in which the dark chromophore resides. In the trans-state, the protonation equilibrium strongly favors the anionic form. Consequently, upon excitation of the formed anionic species a trans–cis isomerization of the chromophore was found to occur with a time constant as fast as 5.2 ps switching the chromophore quantitatively to the bright (anionic) state.