Yang Ying, Ma Yuanqing, Tilley Richard D, Gooding J Justin
School of Chemistry and Australian Centre for NanoMedicine, University of New South Wales, Sydney, NSW 2052, Australia.
European Molecular Biology Laboratory Australia Node in Single Molecule Science, School of Medical Sciences, University of New South Wales, Sydney, NSW 2052, Australia.
Proc Natl Acad Sci U S A. 2025 Jul 15;122(28):e2425390122. doi: 10.1073/pnas.2425390122. Epub 2025 Jul 10.
In superresolution optical fluctuation imaging (SOFI), molecules spaced closer than the diffraction limit can be separated 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 favorable for improving SOFI imaging resolution and speed, especially in high-order cumulant analysis. Stochastic blinking of organic dyes in oxygen scavenging and thiol reducing buffers are well suited for SOFI due to their high brightness. However, the blinking rates can be too slow and nonuniform, which introduces 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 threefold and switching variance by more than twofold 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 superresolution imaging. This advancement in EC-SOFI significantly enhances the practical potential of the SOFI technique.
在超分辨率光学涨落成像（SOFI）中，间距小于衍射极限的分子可通过对荧光强度涨落进行空间和时间相关分析来分离。SOFI成像的分辨率和速度很大程度上取决于这些荧光涨落的特性。具有随机且快速荧光涨落的荧光团有利于提高SOFI成像的分辨率和速度，尤其是在高阶累积量分析中。由于其高亮度，有机染料在除氧和硫醇还原缓冲液中的随机闪烁非常适合用于SOFI。然而，闪烁速率可能过慢且不均匀，这会在SOFI图像中引入非线性。为应对这些挑战，我们引入了用于SOFI的电化学控制染料切换（EC - SOFI）。通过向透明电极表面施加振荡电化学势，我们提高了整体染料切换速率和均匀性。以Alexa 647染料为例，与传统光化学切换相比，EC - SOFI将平均开启时间缩短了三倍多，切换方差降低了两倍多，在六阶EC - SOFI图像中实现了约60纳米的空间分辨率。我们进一步证明，EC - SOFI分别通过100帧和300帧实现了约130纳米和约80纳米的分辨率，从而实现了快速、大面积的平铺扫描超分辨率成像。EC - SOFI的这一进展显著增强了SOFI技术的实际应用潜力。
