Benchmarking Thiolate-Driven Photoswitching of Cyanine Dyes.
Benchmarking Thiolate-Driven Photoswitching of Cyanine Dyes.
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DOI：
10.1021/acs.jpcb.2c06872
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发表时间：
2023-01-26
影响因子：
3.3
通讯作者：
van de Linde, Sebastian
中科院分区：
文献类型：
--
作者：
Herdly, Lucas;Tinning, Peter W.;Geiser, Angeline;Taylor, Holly;Gould, Gwyn W.;van de Linde, Sebastian
碳菁是单分子定位显微镜（SMLM）中性能最好的染料之一，但它们的性能严重依赖于优化的光开关缓冲液。在这里，我们研究了多功能的作用，硫醇在花青的光开关在不同的强度产生在一个单一的采集由微机电系统（MEMS）的镜子放置在激发path. The关键指标，我们已经分析了作为一个函数的硫醇浓度的光子预算，开状态和关状态的寿命和相应的影响图像分辨率。我们表明，硫醇盐作为一个浓度的带通滤波器的最大可实现的分辨率和确定的最小值为1001 mM是必要的，以促进SMLM测量。我们还确定了1-16 mM的浓度带宽，其中光开关性能可以在高分子亮度和高关断时间与接通时间比之间平衡。此外，我们监测了流行的氧清除剂系统的葡萄糖和葡萄糖氧化酶随着时间的推移的性能，并显示简单的措施，以避免在长时间的测量酸化。最后，定量测试缓冲液设置对脂肪细胞质膜内葡萄糖转运蛋白4分布的影响。我们的工作提供了一个通用的策略，实现最佳分辨率的SMLM与相关的新的缓冲液和染料的发展。
Carbocyanines are among the best performing dyes in single-molecule localization microscopy (SMLM), but their performance critically relies on optimized photoswitching buffers. Here, we study the versatile role of thiols in cyanine photoswitching at varying intensities generated in a single acquisition by a microelectromechanical systems (MEMS) mirror placed in the excitation path. The key metrics we have analyzed as a function of the thiolate concentration are photon budget, on-state and off-state lifetimes and the corresponding impact on image resolution. We show that thiolate acts as a concentration bandpass filter for the maximum achievable resolution and determine a minimum of ∼1 mM is necessary to facilitate SMLM measurements. We also identify a concentration bandwidth of 1–16 mM in which the photoswitching performance can be balanced between high molecular brightness and high off-time to on-time ratios. Furthermore, we monitor the performance of the popular oxygen scavenger system based on glucose and glucose oxidase over time and show simple measures to avoid acidification during prolonged measurements. Finally, the impact of buffer settings is quantitatively tested on the distribution of the glucose transporter protein 4 within the plasma membrane of adipocytes. Our work provides a general strategy for achieving optimal resolution in SMLM with relevance for the development of novel buffers and dyes.
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影响因子：
15
作者：
Gidi Y;Payne L;Glembockyte V;Michie MS;Schnermann MJ;Cosa G
通讯作者：
Cosa G
影响因子：
48
作者：
Dempsey, Graham T.;Vaughan, Joshua C.;Chen, Kok Hao;Bates, Mark;Zhuang, Xiaowei
通讯作者：
Zhuang, Xiaowei
DOI：
10.1073/pnas.0907866106
发表时间：
2009-12-29
影响因子：
11.1
作者：
Dertinger, T.;Colyer, R.;Enderlein, J.
通讯作者：
Enderlein, J.
影响因子：
14.7
作者：
Ha T;Tinnefeld P
通讯作者：
Tinnefeld P
优化单分子定位显微镜的成像速度和激发强度。
影响因子：
48
作者：
Diekmann R;Kahnwald M;Schoenit A;Deschamps J;Matti U;Ries J
通讯作者：
Ries J