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.