Source data for: Electrochemically controlled blinking of fluorophores for quantitative STORM imaging

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.