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 sh..., The TIRF images were collected on Zeiss Elyra SP2 Super-resolution PALM microscope. The collimated and linearly p-polarized 642 nm laser was reflected from the 642 nm long pass dichroic mirror and focused at the back focal plane of the 100 X 1.46 NA Oil objective. The focus is laterally shifted alone in the back focal plane to provide either EPI (0o) or TIRF (66.7o) illumination. The TIRF angle was identical between the glass and ITO surface. The fluorescence was collected by the same objective and guided to a cooled electron-multiplying charge-coupled Device EMCCD camera (iXon DU-897). The localization analysis was performed in Zen 2.3 software (black version) and exported as PALM.txt files.
Cyclic voltammetry, chronoamperometry, and different pulsed voltammetry were performed by SP-200 Potentiostat (Bio-Logic, France). All the raw data were exported directly from the software EC-Lab14.2 as txt files., , # **Source data for: Electrochemically controlled blinking of fluorophores for quantitative STORM imaging**

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DOI: 10.5061/dryad.7pvmcvdx9

The provided data consists of the raw imaging data file and Matlab scripts utilized in the publication titled \"Electrochemically Controlled Blinking of Fluorophores for Quantitative STORM Imaging\" by Yang et al. (2024), published in *Nature Photonics*. A description of the data collection and analysis methodologies is outlined in the Methods section, with additional specifics available in the manuscript's Methods section. For further inquiries or information, please feel free to contact the corresponding author.

## Description of the data and file structure

Zipped folders in the dataset: Fig. 1, Fig.2, Fig. 3, Fig. 4, SI Table 1, Sup Fig.2, Sup Fig.3, Sup Fig.4, Sup Fig.5, Sup Fig.6, Sup Fig.7, Sup Fig.8, and Sup Fig.9.

The relationship between the files is structured according to their association with the figure numbers in the manuscript. E...