冷冻电子显微镜技术进展及环境研究应用★

doi:10.6023/A23040166

化学学报 ›› 2023, Vol. 81 ›› Issue (8): 990-1001.DOI: 10.6023/A23040166 上一篇下一篇

所属专题：庆祝《化学学报》创刊90周年合辑

综述

冷冻电子显微镜技术进展及环境研究应用^★

杨宇洁^a, 巩宇锈^a, 顾天航^a^,^*(), 张伟贤^a^,^b^,^*()

a 同济大学环境科学与工程学院污染控制与资源化研究国家重点实验室上海 200092
b 岭南现代农业科学与技术广东省实验室广州 510642

投稿日期:2023-04-26 发布日期:2023-09-14

作者简介:

杨宇洁, 同济大学环境科学与工程学院2021级硕士生, 研究方向为纳米零价铁环境应用.

巩宇锈, 同济大学环境科学与工程学院2017级博士, 研究方向为纳米零价铁修复重金属污染土壤.

顾天航, 同济大学环境科学与工程学院2018级博士生, 研究方向为纳米零价铁富集水中稀有元素, 已发表SCI论文10余篇.

张伟贤, 教授、博士生导师, 国家特聘专家, 2011年起任污染控制与资源化研究国家重点实验室主任. 1984年毕业于同济大学, 1996年获美国约翰•霍普金斯大学(The Johns Hopkins University)环境工程博士学位, 曾任美国里海大学(Lehigh University)教授. 主持过国家自然科学基金海外及港澳学者合作研究基金及多项国家自然科学基金项目. 长期致力于环境中重金属及持久性有机污染物的基础与应用研究, 是环境纳米技术的先驱之一, 纳米零价铁技术的创始研究者. 在纳米零价铁合成、表征、污染物反应机理、应用于地下水修复及废水处理方面发表了系列经典论文.

^★庆祝《化学学报》创刊90周年.

基金资助:
项目受广东省重点领域研发计划(2020B0202080001); 国家自然科学基金(51978488)

Progress and Environmental Research Applications of Cryo-Electron Microscopy^★

Yujie Yang^a, Yuxiu Gong^a, Tianhang Gu^a(), Wei-xian Zhang^a^,^b()

a State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 200092
b Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou 510642

Received:2023-04-26 Published:2023-09-14
Contact: *E-mail: tianhanggu@tongji.edu.cn; zhangwx@tongji.edu.cn
About author:
^★Dedicated to the 90th anniversary of Acta Chimica Sinica.
Supported by:
Key-Area Research and Development Program of Guangdong Province(2020B0202080001); National Natural Science Foundation of China(51978488)

文章分享

摘要/Abstract

电子显微镜是表征微观粒子结构的必要手段, 但预处理过程会对含水样品结构造成破坏, 导致结果失真. 冷冻电子显微镜(Cryo-EM)技术通过将样品在低温下快速冷冻, 使水合样品处于无定形玻璃态冰层内, 在保障高真空度要求的同时减少电子辐射对样品的破坏, 目前已成为高分辨地观察含水样品原始状态下结构最先进的手段, 在生物、材料等领域应用广泛. 本综述总结了冷冻电镜在电子元件、成像技术与分辨率等方面的进展, 综述了样品制备方法及三维重构技术. 鉴于绝大多数环境样品为含水样品, 冷冻电镜技术在大气、水体、土壤等环境介质中的应用扩宽和深化了对环境中微纳米粒子本身形态结构和粒子间相互作用关系的理解. 我们期待冷冻电镜为环境科学研究带来突破性贡献.

关键词: 冷冻电镜, 快速冷冻, 含水样品, 玻璃态冰, 三维重构, 环境化学

Electron microscopy (EM) is one of the most important techniques to characterize the morphology and structure of micro- and nano-particles. However, conventional procedures for sample preparation often alter the structure and morphology of samples with high water contents, causing distortions and misinterpretations for EM characterizations. The invention of Cryo-electron microscopy (Cryo-EM) has largely solved this problem. Via rapidly freezing the hydrated samples at low temperatures, cryogenic techniques instantaneously transform liquid water into amorphous ice to ensure high vacuum and reduce electron radiation damage, allowing researchers to observe hydrated samples in their native state with high resolution. For example, applications of Cryo-EM during the COVID-19 pandemic have demonstrated the amazing ability of Cryo-EM to visualize the detailed structure of SARS-CoV-2 viruses, which provides vital knowledge for rapid and reliable detection and diagnosis of the disease, transmission mitigation, and vaccine development. So far, Cryo-EM technology has been widely used in materials, biology, pharmaceuticals, and other fields of research, successfully broadening and deepening the understanding of the interactions between micro- and nano-particles. This review summarizes the recent development of Cryo-EM from aspects of electronic components, imaging technology, and resolution and introduces the sample preparation methods. Furthermore, the three-dimensional reconstruction method is highlighted to advance the EM method from 2D to 3D. As most environmental samples are highly hydrated, Cryo-EM will likely become an essential tool to investigate microscopic particles in the environmental field. This review gives examples of the applications of Cryo-EM in the formation of aerosol particles in the atmosphere, observing biofilm morphology in the water treatment process, the pore structure of activated sludge flocs, and the potential mechanism of soil microorganisms on heavy metal remediation. Finally, the prospects of Cryo-EM are summarized and discussed. We expect that with software, hardware, and artificial intelligence development, Cryo-EM technology can achieve faster data acquisition and higher resolution and make breakthrough contributions to environmental chemistry research.

Key words: Cryo-electron microscopy (Cryo-EM), rapid freezing, hydrated samples, glassy ice, three-dimensional reconstruction (3D reconstruction), environmental chemistry

引用此文

杨宇洁, 巩宇锈, 顾天航, 张伟贤. 冷冻电子显微镜技术进展及环境研究应用^★[J]. 化学学报, 2023, 81(8): 990-1001.

Yujie Yang, Yuxiu Gong, Tianhang Gu, Wei-xian Zhang. Progress and Environmental Research Applications of Cryo-Electron Microscopy^★[J]. Acta Chimica Sinica, 2023, 81(8): 990-1001.

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图/表 11

冷冻电镜种类	原理	代表性研究	参考文献
冷冻透射电子显微镜	将平行电子束投射到样品上, 电子与样品中的原子碰撞散射, 从而形成投影图像	金、银纳米颗粒	[19-20]
		噬菌体的蛋白质外壳	[21]
		蒙脱石-二氧化硅悬浊液	[22]
		气溶胶颗粒	[23]
		MS2噬菌体	[24]
冷冻扫描电子显微镜	利用高能电子束扫描样品, 在其表面形成二次电子、背散电子等, 从而转换产生图像	石油工业中的乳液和悬浮液	[25]
		A. Borkumensis细菌生物膜	[26]
		乳胶/陶瓷纳米颗粒涂层	[27]
		海藻酸盐水凝胶	[28]
		活性污泥	[29-30]
冷冻聚焦离子束扫描电子显微镜	利用聚焦离子束扫描样品得到图像	枯草芽孢杆菌孢子	[31]
		新型冠状病毒	[5]
		黏土的孔隙结构	[32]
冷冻扫描透射电子显微镜	利用聚焦后的电子束斑逐点扫描样品, 在样品下方收集散射或透射电子, 从而形成明、暗场像	铁蛋白	[33]
冷冻扫描透射电子显微镜	利用聚焦后的电子束斑逐点扫描样品, 在样品下方收集散射或透射电子, 从而形成明、暗场像	锂金属电池中固液界面和枝晶的结构	[34]

表1. 冷冻电镜技术应用实例

Table 1. Examples of Cryo-EM application

图1. 冷冻电镜发展史

Figure 1. Development history of Cryo-EM

图2. Cryo-FIB-SEM工作原理[53]. (a)聚焦镓离子束铣削样品. (b)镓离子与样品碰撞中的动能转移促使原子克服其表面结合能, 作为溅射物质被喷射出来

Figure 2. Operating principle of Cryo-FIB-SEM[53]. (a) A focused gallium ion beam removes material from the sample. (b) Kinetic energy transfer during gallium ion and sample collisions causes surface atoms to overcome their surface binding energy and to become ejected as a sputtered species

图3. 冷冻透射电镜样品制备方法示意图

Figure 3. Schematic illustration for Cryo-TEM sample preparation method

图4. 冷冻聚焦离子束扫描电镜样品制作方法的分步示意图[56]. (a)预冷样品上的水冷凝. (b)在液氮雪泥中快速冷冻. (c)真空转移和金属镀膜. (d)冷冻聚焦离子束扫描电镜铣削和成像

Figure 4. Step-by-step schematic of the Cryo-FIB-SEM method[56]. (a) Water condensation on precooled sample. (b) Rapid plunge freezing in liquid nitrogen slush. (c) Vacuum transfer and metal coating. (d) Cryo- FIB-SEM milling and imaging

图5. 新冠病毒S糖蛋白的冷冻电镜图[93]. (a) ACE2诱导SARS-CoV-2 S三聚体构象转变的机制. 从闭合的预融合状态到瞬时打开状态(步骤1)的构象过渡, 具有与S1向下运动(红色箭头)相关的解扭曲运动(深灰色箭头突出显示), 从打开状态到动态ACE2接合状态(步骤2), 然后一直到重新折叠的融合后状态(步骤3). ACE2-RBD在S三聚体内的连续摆动运动用红色箭头表示. (b) SARS-CoV-2 S-ACE2复合体的冷冻电镜图. (c)来自S-ACE2结构的ACE2(紫红色)结合S1(浅绿色)的整体视图, 以及ACE2和RBD之间相互作用界面的放大视图. (d) RBD向上/向外旋转71.0°, 与原S1中SD1的向下移动有关

Figure 5. Cryo-EM map of SARS-CoV-2 S[93]. (a) The proposed mechanism of ACE2-induced conformational transitions of SARS-CoV-2 S trimer. Conformational transitions from the closed ground prefusion state (with packed FP, in red) to the transiently open state (step 1) with an untwisting motion (highlighted in dark gray arrow) associated with a downward movement of S1 (red arrow), from the open state to the dynamic ACE2 engaged state (step 2), and then all the way to the refolded postfusion state (step 3). The continuous swing motions of ACE2-RBD within S trimer are indicated by red arrows. (b) Cryo-EM map of SARS-CoV-2 S-ACE2 complex. (c) The overall view of ACE2 (violet red) bound S1 (light green) from S-ACE2 structure, and zoom-in view of the interaction interface between ACE2 and RBD. (d) There is a 71.0° upward/outward rotation of RBD associated with a downward shift of SD1 in S1

图6. 颗粒甲烷单加氧酶的冷冻电镜图[100]. (a) pMMO原型的原子模型, 其中PmoA, PmoB和PmoC分别用金色, 红色和绿色表示. 突出显示了A、B和D点以及E群集的位置. (b) D点的冷冻电镜图谱特写视图: 该位点由PmoA His38, Met42, Asp47, Asp49, Glu100(在此视图中未显示)和PmoC Glu154组成, 表示为Glu154(C). 这些残基主要是亲水的, 形成了催化中心. (c)为D点提出的甲烷氧化化学方案

Figure 6. Cryo-EM map of pMMO[100]. (a) Atomic model of the pMMO protomer, where PmoA, PmoB, and PmoC are depicted by ribbons in gold, red, and green, respectively. The A, B, and D sites and the location of the E clusters are highlighted. (b) Close-up view of the Cryo-EM map at the D site in stereopair: This site is composed of PmoA His38, Met42, Asp47, Asp49, Glu100 (not shown in this view), and of PmoC Glu154, denoted as Glu154(C). These residues, largely hydrophilic, form the proposed catalytic center. (c) Chemistry scheme of methane oxidation proposed for the D site

图7. 纳米零价铁与含蓝藻的污泥相互作用的扫描电镜图片. (a)常规扫描电镜照片. (b~c)冷冻扫描电镜照片(Gong和Zhang未发表的图片)

Figure 7. The SEM of nZVI-treated cyanobacteria-containing sludge. (a) Traditional SEM micrograph. (b~c) Cryo-SEM micrograph (Gong and Zhang, unpublished results)

图8. 细菌细胞与金纳米棒之间的相互作用[103]. (a~c)表皮链球菌与金纳米棒混合的冷冻电镜图: (a) 670 nm, 紫色. (b) 717 nm, 绿色. (c) 860 nm, 红色. (d~f)大肠杆菌与金纳米棒混合的冷冻电镜图: (d) 670 nm, 紫色. (e) 717 nm, 绿色. (f) 860 nm, 红色. 与大肠杆菌细胞表面相比, 纳米颗粒与表皮链球菌细胞表面紧密结合, 因为前者具有更高的负表面电荷. 比例尺表示500 nm

Figure 8. Interaction between bacterial cells and gold nanorods[103]. (a~c) Cryo-EM images of S. epidermidis mixed with NRs: (a) 670 nm, purple. (b) 717 nm, green. (c) 860 nm, red. (d~f) Cryo-EM images of E. coli mixed with NRs: (d) 670 nm, purple. (e) 717 nm, green. (f) 860 nm, red, respectively. The nanoparticles show tight binding to S. epidermidis cell surface compared to E. coli cell surfaces, as the former has a higher negative surface charge. Scale bar indicates 500 nm

图9. 银纳米颗粒吸附和合成的冷冻扫描透射电子显微照片[110]. (a, a’) 表面吸附银的成熟细菌细胞的暗场和明场扫描图像. (b, b’)银纳米颗粒的吸附和合成. 蓝色箭头表示细胞内的银纳米颗粒, 黄色箭头表示细胞外的银纳米颗粒

Figure 9. Cryo-scanning transmission electron micrographs showing the adsorption and synthesis of AgNPs[110]. (a, a’) dark field and bright field scanning images of mature bacterial cells with silver adsorbed on the surface. (b, b’) Adsorption and synthesis of AgNPs. Intracellular AgNPs and extracellular AgNPs are highlighten with blue arrows and yellow arrows

图10. 污泥样品内部的冷冻扫描电镜图像[30]. (a, b)絮状污泥样品内部. (c, d)颗粒状污泥样品内部

Figure 10. Cryo-SEM images of sludge[30]. (a, b) The interior of a floccular sludge sample. (c, d) The interior of a granular sludge sample

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