Experimental Guidelines to Image Transient Single-Molecule Events Using Graphene Liquid Cell Electron Microscopy,ACS Nano

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Experimental Guidelines to Image Transient Single-Molecule Events Using Graphene Liquid Cell Electron Microscopy
ACS Nano ( IF 15.8 ) Pub Date : 2022-10-18 , DOI: 10.1021/acsnano.2c06766
Huan Wang _{1,

2} , Zhun Xu ₁ , Sheng Mao ₃ , Steve Granick _{4,

5}

Affiliation

In quest of the holy grail to “see” how individual molecules interact in liquid environments, single-molecule imaging methods now include liquid-phase electron microscopy, whose resolution can be nanometers in space and several frames per second in time using an ordinary electron microscope that is routinely available to many researchers. However, with the current state of the art, protocols that sound similar to those described in the literature lead to outcomes that can differ. The key challenge is to achieve sample contrast under a safe electron dose within a frame rate adequate to capture the molecular process. Here, we present such examples from different systems─synthetic polymer, lipid assembly, DNA–enzyme─in which we have done this using graphene liquid cells. We describe detailed experimental procedures and share empirical experience for conducting successful experiments, starting from fabrication of a graphene liquid cell, to identification of high-quality liquid pockets from desirable shapes and sizes, to effective searching for target sample pockets under electron microscopy, and to discrimination of sample molecules and molecular processes of interest. These experimental tips can assist others who wish to make use of this method.

中文翻译：

使用石墨烯液体细胞电子显微镜成像瞬态单分子事件的实验指南

为了寻求“观察”单个分子如何在液体环境中相互作用的圣杯，单分子成像方法现在包括液相电子显微镜，使用普通电子显微镜，其空间分辨率可以达到纳米级，时间分辨率可以达到每秒几帧许多研究人员通常都可以使用它。然而，就目前的技术水平而言，听起来与文献中描述的协议相似的协议会导致可能不同的结果。关键挑战是在足以捕获分子过程的帧速率内，在安全电子剂量下实现样本对比度。在这里，我们展示了来自不同系统的示例——合成聚合物、脂质组装、DNA-酶——我们在这些系统中使用石墨烯液体细胞完成了这项工作。我们描述了详细的实验程序并分享了进行成功实验的经验，从石墨烯液体电池的制造开始，到从理想的形状和尺寸中识别高质量的液体袋，到在电子显微镜下有效搜索目标样品袋，以及到区分样品分子和感兴趣的分子过程。这些实验技巧可以帮助其他希望使用此方法的人。

更新日期：2022-10-18

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