近日，澳大利亚新南威尔士大学的J. Justin Gooding及其研究团队取得一项新进展。经过不懈努力，他们实现用于定量STORM成像荧光团的电化学受控闪烁。相关研究成果已于2024年4月19日在国际知名学术期刊《自然—光子学》上发表。
该研究团队成功证明了荧光团可用于STORM成像（EC-STORM）的电化学开关，实现了对开关动力学、占空比和恢复率的精准控制。通过EC-STORM技术，研究人员展示了利用电化学电位调控染料光物理性质的分子计数方法。在实验中，染料的随机闪烁被负电位有效抑制，而开关-ON事件则可以通过短暂的正电位脉冲激活，确保开关-ON事件的频率与潜在染料的数量呈线性关系。
此外，该团队还在固定细胞中成功展示了微管蛋白的EC-STORM成像，其空间分辨率高达约28纳米，并成功计数了各种DNA纳米尺结构上的单个Alexa 647荧光团。这种对荧光团开关的精准控制，将使EC-STORM在超分辨率成像和分子计数领域得到广泛应用。
据悉，随机光学重构显微术(STORM)通过计算单个荧光团在时间和空间上发射分离的坐标，允许单分子分辨率的宽视场成像。这种分离是通过在长寿命的OFF状态和发射的ON状态之间进行荧光团的光开关来实现的。虽然STORM可以对单个分子进行成像，但由于光漂白或重叠染料造成的计数错误和重复随机闪烁染料造成的人工制品计数过高，分子计数仍然具有挑战性。
附：英文原文
Title: Electrochemically controlled blinking of fluorophores for quantitative STORM imaging
Author: Yang, Ying, Ma, Yuanqing, Berengut, Jonathan F., Lee, Lawrence K., Tilley, Richard D., Gaus, Katharina, Gooding, J. Justin
Issue&Volume: 2024-04-19
Abstract: Stochastic optical reconstruction microscopy (STORM) allows wide-field imaging with single-molecule resolution by calculating the coordinates of individual fluorophores from the separation of fluorophore emission in both time and space. Such separation is achieved by photoswitching the fluorophores between a long-lived OFF state and an emissive ON state. Although STORM can image single molecules, molecular counting remains challenging due to undercounting errors from photobleached or overlapping dyes and overcounting artefacts from the repetitive random blinking of dyes. Here we show that fluorophores can be electrochemically switched for STORM imaging (EC-STORM), with excellent control over the switching kinetics, duty cycle and recovery yield. Using EC-STORM, we demonstrate molecular counting by using electrochemical potential to control the photophysics of dyes. The random blinking of dyes is suppressed by a negative potential but the switching-ON event can be activated by a short positive-potential pulse, such that the frequency of ON events scales linearly with the number of underlying dyes. We also demonstrate EC-STORM of tubulin in fixed cells with a spatial resolution as low as ~28nm and counting of single Alexa 647 fluorophores on various DNA nanoruler structures. This control over fluorophore switching will enable EC-STORM to be broadly applicable in super-resolution imaging and molecular counting.
DOI: 10.1038/s41566-024-01431-0
Source: https://www.nature.com/articles/s41566-024-01431-0