Electrochemically controlled blinking of fluorophores for quantitative STORM imaging

Abstract

AbstractStochastic optical reconstruction microscopy (STORM) allows wide-field imaging with single-molecule resolution by calculating the coordinates of individual fluorophores from the separation of 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. Although STORM can image single molecules, molecular counting remains challenging due to undercounting errors from photobleached or overlapping dyes and overcounting artefacts from the repetitive random blinking of dyes. Here we show that fluorophores can be electrochemically switched 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 positive-potential pulse, such that the frequency of ON events scales linearly with the number of underlying dyes. We also demonstrate EC-STORM of tubulin 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.