DOI: 10.5061/dryad.z34tmpgq9
title: Electrochemically controlled switching of dyes for enhanced super-resolution optical fluctuation imaging (EC-SOFI)

description: In super-resolution optical fluctuation imaging (SOFI), molecules spaced
 closer than the diffraction limit can be identified through spatial and
 temporal correlation analysis of the fluorescence intensity fluctuations.
 The resolution and speed of SOFI imaging greatly depend on the
 characteristics of these fluorescence fluctuations. Fluorophores with
 stochastic and rapid fluorescence fluctuations are favourable for
 improving SOFI imaging resolution and speed, especially in high order
 cumulant analysis. Stochastic blinking dyes in specific buffers are
 well-suited for SOFI due to their high brightness and strong fluorescence
 fluctuations. However, their overall blinking rate can sometimes be too
 slow, presenting practical limitations for high-resolution and fast SOFI
 imaging. Furthermore, dye blinking can be non-uniform, creating
 nonlinearity in SOFI image. To address these challenges, we introduce
 electrochemically controlled dye switching for SOFI (EC-SOFI). By applying
 an oscillating electrochemical potential to a transparent electrode
 surface, we increase the overall dye switching rate and uniformity. Using
 Alexa 647 dye as an example, EC-SOFI reduces the average ON time by over
 3-fold and switching variance by more than 2-fold compared to conventional
 photochemical switching, achieving ~60 nm spatial resolution in 6th order
 EC-SOFI image. We further demonstrate that EC-SOFI achieves ~130 nm and
 ~80 nm resolution with 100 and 300 frames, respectively, enabling fast,
 large-area tile-scan super-resolution imaging. This advancement in EC-SOFI
 significantly enhances the practical potential of SOFI technique.
description: <strong>Electrochemistry:</strong><br>Electrochemical measurements, including chronoamperometry and large-amplitude sinusoidal voltammetry, were performed using an SP-200 potentiostat (Bio-Logic, France) in a custom EC imaging chamber (Chamlide EC 22, Live Cell Instrument, Korea). The setup featured a three-electrode system: an ITO-coated coverslip (SPI Supplies, USA) as the working electrode, an Ag|AgCl|3M KCl reference electrode (LF-1-45, Innovative Instruments, USA), and a Pt mesh counter electrode. The chamber was mounted on the microscope stage for simultaneous EC control and fluorescence imaging. <strong>Image acquisition:</strong><br>SOFI and STORM images were acquired on a Zeiss Elyra 7 Lattice SIM TIRF microscope with a 642 nm laser and sCMOS camera (edge 4.2, PCO). Illumination (EPI or TIRF) was controlled by shifting focus laterally in the objective's back focal plane. A 1.6× OptoVar lens reduced pixel size to 100 nm. Fluorescence was collected by the same 100× 1.46 NA oil objective and detected by the cooled sCMOS camera.
description: # Data for: Electrochemically controlled switching of dyes for enhanced
 super-resolution optical fluctuation imaging (EC-SOFI) **Dataset DOI:**
 10.5061/dryad.z34tmpgq9\ **Corresponding Author:** To whom correspondence
 may be addressed. Email:
 [yuanqing.ma@unsw.edu.au](mailto:yuanqing.ma@unsw.edu.au) or
 [justin.gooding@unsw.edu.au](mailto:justin.gooding@unsw.edu.au). **1.
 Included Files** This dataset contains the raw imaging data used to
 generate Figures 1-4 in the associated publication. All image data are
 provided wither as .csv or`.tif` files inside `.zip` archives, directly
 exported from Zeiss Zen Blue.
 **Fig_1_single_molecule_photophysics_using_origami.zip**\ This folder
 includes the sinple molecule localization data in .csv format used for
 generating Figure 1, which shows single-molecule fluorescence trajectories
 using DNA origami labelled with Alexa 647. Four distinct regions of
 interest (ROI 1–4) are included for two experimental conditions: o   
 **Electrochemical (EC)**: 0.5 kW cm−2 642 nm laser were on, large
 amplitude square wave (LASV) potential was sinusoidally oscillated between
 −0.8 V to 0.1 V with a frequency of 10 Hz o    **UV Illumination**: 0.5 W
 cm−2 of UV laser and 0.5 kW cm−2 642 nm laser were on. These files of
 single-molecule localization results were directly exported from Zeiss Zen
 Blue, with each row corresponds to a detected fluorescent molecule. Below
 are the definitions of each column: * **Index**: Unique identifier for
 each localization event. * **First Frame**: Frame number when the molecule
 first appeared. * **Number Frames**: Total number of frames the molecule
 was detected. * **Frames Missing**: Number of skipped frames during a
 blinking event. * **X Position (nm)** / **Y Position (nm)**: Lateral
 coordinates of the molecule in nanometers. * **Precision (nm)**: Estimated
 localization uncertainty in X/Y, based on fitting. * **Photons**: Total
 photon count collected from the molecule. Reflects signal brightness. *
 **Background Variance**: Variability of background noise in the fitting
 region. * **Chi Square**: Goodness-of-fit indicator for the Gaussian
 model. * **PSF width (nm)**: Width of the fitted point spread function in
 nanometers. * **Channel**: Color or channel label used during imaging. *
 **Z-Slice**: Z-stack slice index (for 3D acquisitions). * **Z Position
 (nm)**: Axial (depth) position of the molecule in nanometers. * **Z
 Precision (nm)**: Estimated localization uncertainty in Z direction.
 **Fig_2_UV_vs_EC_SOFI.zip**\ Contains raw SOFI image sequences used to
 generate Figure 2, comparing UV- and EC-SOFI in fixed COS-7 cells. The
 data highlight differences in switching uniformity and kinetics.
 **Fig_3_High_order_SOFI.zip**\ Includes raw data used in high-order SOFI
 reconstruction shown in Figure 3. These sequences were used to calculate
 up to 6th-order SOFI images, demonstrating resolution enhancement using
 electrochemically modulated dye switching. **Fig_4_Tile_Scan.zip**\
 Contains one example for raw tiled image stacks used in Figure 4, more
 similar tiles were collected to achieve wide-area EC-SOFI imaging. ## 2.
 Recommended Software The .tif files provided in this dataset can be opened
 using freely available software: **Fiji (ImageJ distribution)** –
 Recommended for most users; supports stack viewing, analysis, and basic
 SOFI processing.\ Website:
 [https://imagej.net/software/fiji](https://imagej.net/software/fiji)
 **ImageJ** – A lightweight alternative to Fiji, with broad compatibility
 for viewing and simple operations.\ Website:
 [https://imagej.nih.gov/ij/](https://imagej.nih.gov/ij/) No proprietary
 software or plugins are needed to access the data. ## 3. Imaging settings:
 ·       **Excitation wavelength**: For most experiments, a 640 nm laser
 was used; UV conditions refer to 400 nm laser illumination. ·      
 **Applied potential**: In EC conditions, potentials were alternating
 between –0.8 V and +0.15 V versus Ag/AgCl/3M KCl reference. Additional
 experimental parameters such as sample preparation, buffer composition,
 and detailed EC and imaging settings are available in the
 manuscript's Methods section. ## 4. Citation If you use this dataset,
 please cite: Y. Yang,Y. Ma,R.D. Tilley,& J.J. Gooding, 
 Electrochemically controlled switching of dyes for enhanced
 superresolution optical fluctuation imaging (EC-SOFI), Proc. Natl. Acad.
 Sci. U.S.A. 122 (28) e2425390122,
 [https://doi.org/10.1073/pnas.2425390122](https://doi.org/10.1073/pnas.2425390122) (2025).