Toward Accurate Molecular Counting in EC-STORM via Reversible Stochastic Dye Switching.

Molecular counting using stochastic optical reconstruction microscopy (STORM), is often limited by random fluorophore reblinking, which makes repeated localization events hard to assign to the same or different molecules. Here, we turn reblinking from a source of uncertainty into a counting strategy by modulating fluorophore ON-events with electrochemical STORM (EC-STORM). Potential cycling converts long ON-events into repeated fluorescence bursts. This enables counting to be based on the total modulated ON-events accumulated over repeated cycles. Using DNA origami with spatially resolvable dyes, we validate EC-STORM counting and show that ON-event frequency scales linearly with dye number. We next applied the method to DNA origami with up to 13 labeling sites, where individual dyes could not be spatially resolved. A weighted Poisson model accounting for incomplete labeling can successfully describe the resulting ON-event distributions. Together, these results establish EC-STORM as an actively controlled approach for quantitative molecular counting in super-resolution microscopy.