Electrochemically controlled switching of dyes for enhanced super-resolution optical fluctuation imaging (EC-SOFI)
Data files
Jul 18, 2025 version files 5.74 GB
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Fig_1_single_molecule_photophysics_using_origami.zip
379.69 MB
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Fig_2_UV_vs_EC_SOFI.zip
2 GB
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Fig_3_High_order_SOFI.zip
3.21 GB
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Fig_4_Tile_Scan.zip
144.61 MB
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README.md
4.54 KB
Abstract
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.
Dataset DOI: 10.5061/dryad.z34tmpgq9
Corresponding Author: To whom correspondence may be addressed. Email: yuanqing.ma@unsw.edu.au or 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
ImageJ – A lightweight alternative to Fiji, with broad compatibility for viewing and simple operations.
Website: 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 (2025).
Electrochemistry:
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.
Image acquisition:
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.