Electrochemically controlled blinking of fluorophores to enable quantitative stochastic optical reconstruction microscopy (STORM) imaging

Abstract

Stochastic optical reconstruction microscopy (STORM) allows widefield imaging with single molecule resolution through 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. Despite STORM having revolutionized cellular imaging it remains challenging for quantitative imaging of single molecules due to a number of limitations, such as photobleaching caused under counting, overlapping emitters related fitting error, and repetitive but random blinking induced over counting. To overcome these limitations, we develop an electrochemical approach to switch the fluorophores between ON and OFF states for STORM (EC-STORM). The approach provides greater control over the fluorophore recovery yield, emitter density, and random blinking than photochemically switching. The result is EC-STORM has superior imaging capability than conventional photochemical STORM and can perform molecular counting; a significant advance.