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

In super-resolution optical fluctuation imaging (SOFI), the locations of molecules spaced closer than the diffraction limit of light can be identified through spatial and temporal correlation analysis of the fluorescence intensity fluctuation. Using organic dyes as fluorophore probes, the fast but stochastic switching of the individual dyes is favourable for improving SOFI imaging resolution and speed, especially in the case of high-order cumulant analysis. While in practice, fluorophore switching can be non-uniform, with some fluorophores remaining in ON or OFF state for extended periods. Furthermore, in some cases the overall rate of switching of the fluorophores can be too slow, presenting practical limitations for high-resolution and fast SOFI imaging. In this work, we demonstrate how to overcome these challenges using electrochemical controlled fluorophore switching. The oscillating electrochemical potential setting with high frequency increases the switching rate and reduces the switching heterogeneity of fluorophores. The dye Alexa Fluor 647, applied here as an example, exhibits over 3-fold decrease in average ON time and over 2-fold decrease in switching variance, resulting in significantly improved SOFI image resolution with fewer frames. We demonstrated that this new electrochemically controlled SOFI imaging modality can achieve a SOFI image with [~]130 nm resolution in 2 seconds of acquisition time, and 80 nm resolution in 6 seconds. This advancement enables fast, large area tile-scan super-resolution imaging, which opens the full potential of SOFI imaging.