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.

在超分辨光学波动成像（super-resolution optical fluctuation imaging，简称SOFI）中，对于间距小于衍射极限的分子，可通过对荧光强度波动进行时空相关分析实现识别。SOFI成像的分辨率与成像速度极大程度上依赖于上述荧光波动的特性。具备随机且快速荧光波动特性的荧光团，有助于提升SOFI成像的分辨率与速度，在高阶累积量分析中尤为如此。特定缓冲液中的随机闪烁染料，因亮度高且荧光波动强烈，非常适配SOFI技术。但这类染料的整体闪烁速率有时过慢，对高分辨率、高速SOFI成像造成了实际限制。此外，染料闪烁的非均匀性还会在SOFI图像中引入非线性效应。为解决上述挑战，本研究提出了电化学调控染料闪烁技术（electrochemically controlled dye switching for SOFI，简称EC-SOFI）。通过向透明电极表面施加振荡电化学电势，可提升染料整体闪烁速率与闪烁均匀性。以Alexa 647染料为例，相较于传统光化学闪烁方式，EC-SOFI可将染料的平均亮态时长缩短3倍以上，将闪烁方差降低2倍以上，在6阶EC-SOFI图像中实现了约60 nm的空间分辨率。本研究进一步证实，EC-SOFI在100帧与300帧成像条件下，分别可实现约130 nm与约80 nm的分辨率，支持高速、大区域拼接扫描式超分辨成像。EC-SOFI的这项技术进步，极大拓展了SOFI技术的实际应用潜力。