Electrochemically controlled switching of dyes for enhanced super-resolution optical fluctuation imaging (EC-SOFI)

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.

Methods
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.