DOI: 10.5061/dryad.7pvmcvdx9
title: Source data for: Electrochemically controlled blinking of fluorophores for quantitative STORM imaging

description: 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.
description: 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.
description: # **Source data for: Electrochemically controlled blinking of fluorophores
 for quantitative STORM imaging** --- 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.
 Each file contains the raw data employed in generating the corresponding
 graph in the figure, along with the Matlab script utilized for analysis,
 where applicable. Further elaboration on the analysis procedures can be
 found within the Methods section of the manuscript, as well as in the
 comments section of the Matlab script.