Electrochemical Control of Fluorescence Emission: From Intensity Modulation to Single Molecule Switching for Applications in Light Microscopy

ConspectusThe application of fluorescent probes in light microscopy has made microscopy a vital tool in biological research because of the high resolution, contrast, and sensitivity. The advances in spatiotemporal control of fluorescence emission continue to push fluorescence microscopy to higher levels. One example is the single molecule localization microscopy (SMLM) that allows imaging of entities well below the diffraction limit. In the SMLM approach, the fluorophores are photoswitched between an emitting state and a nonemitting state. In each frame, a small subset of the fluorophores emit, and their positions are determined by fitting the point spread functions (PSFs). Accumulating these localization events yields a map of the positions of fluorophores with nanometer scale resolution. These developments show that precise control over when and where fluorophores emit is the key driver of progress in fluorescence microscopy, and this control has been achieved almost entirely using light.Here we present our recent advances to control fluorescence emission using electrochemistry instead of conventional photochemical pathways. The progress of the work initiated by our group commenced with the observation that the organic dye Alexa 647 was sensitive to external electrochemical potentials. In the presence of the triplet scavenger Trolox, which is also electrochemically redox active, the fluorophore became brighter at positive potential and dimmer under negative potential. This finding motivated us to extend electrochemical fluorescence modulation to multiple organic dyes and fluorescent proteins. We found that, in a low oxygen buffer containing redox mediators, all tested fluorophores respond to applied potentials. More interestingly, different fluorophores respond in distinct ways. We refer to this potential dependent fluorescence curve as the electrochemical spectrum. Using differences in these electrochemical spectra, we imaged four fluorophores with similar excitations and emissions and then separated them by linear unmixing. This strategy expands the multicolor capability in fluorescence microscopy using a single excitation laser and enables simultaneous visualization of more structures.We also explored using electrochemistry for fluorescence switching in SMLM super resolution microscopy, specifically stochastic optical reconstruction microscopy (STORM). In STORM, organic dyes undergo photochemical switching. Under strong excitation, a substantial fraction populates the triplet state and, in the presence of primary thiols, enters the dark state via reversible thiol adduction. The emissive state can be recovered by photolysis or oxidation. Building on the same reversible chemistry, we developed an electrochemical switching strategy that drives this reaction by applying an external potential. The applied potential shifts the steady state of the dye thiol equilibrium by modulating local reactive radical concentrations through surface electrochemical redox processes, thereby tuning both the thermodynamics and the kinetics. As a result, the ON and OFF populations and switching rates can be set reproducibly by the potential. By varying the potential, the ON and OFF ratio can be adjusted so emitter density matches labeling density, suppressing PSF overlap and improving effective resolution in crowded structures. High frequency oscillatory potentials further accelerate switching and generate rapid, uniform blinking suited to super-resolution optical fluctuation imaging (SOFI), shortening acquisition times, and improving resolution through higher-order cumulants.