Stochastic optical reconstruction microscopy (STORM) allows widefield imaging with single-molecule resolution by calculating the coordinates of individual fluorophores from the separation of the fluorophore emission in both time and space. Such separation is achieved by photoswitching the fluorophores between a long-lived OFF state and an emissive ON state. While STORM can image single molecules, molecular counting remains challenging due to undercounting errors from photobleached or overlapping dyes and overcounting artifacts from the repetitive random blinking of the dyes. Here, we show that fluorophores can be switched electrochemically for STORM imaging (EC-STORM), with excellent control over the switching kinetics, duty cycle, and recovery yield. Using EC-STORM, we demonstrate molecular counting by using electrochemical potential to control the photophysics of dyes. The random blinking of dyes is suppressed by a negative potential but the switching ON event can be activated by a short pulsed positive potential, such that the frequency of ON events scales linearly with the number of underlying dyes. We also demonstrate the EC-STORM of tubulins in fixed cells with a spatial resolution as low as ~28 nm and counting of single Alexa 647 fluorophores on various DNA nanoruler structures. This control over fluorophore switching will enable EC-STORM to be broadly applicable in super-resolution imaging and molecular counting.

The TIRF images were collected on Zeiss Elyra SP2 Super-resolution PALM microscope. The collimated and linearly p-polarized 642 nm laser was reflected from the 642 nm long pass dichroic mirror and focused at the back focal plane of the 100 X 1.46 NA Oil objective. The focus is laterally shifted alone in the back focal plane to provide either EPI (0o) or TIRF (66.7o) illumination. The TIRF angle was identical between the glass and ITO surface. The fluorescence was collected by the same objective and guided to a cooled electron-multiplying charge-coupled Device EMCCD camera (iXon DU-897). The localization analysis was performed in Zen 2.3 software (black version) and exported as PALM.txt files.

Cyclic voltammetry, chronoamperometry, and different pulsed voltammetry were performed by SP-200 Potentiostat (Bio-Logic, France). All the raw data were exported directly from the software EC-Lab14.2 as txt files.