An introduction to cryo-FIB-SEM cross-sectioning of frozen, hydrated Life Science samples.
An introduction to cryo-FIB-SEM cross-sectioning of frozen, hydrated Life Science samples.
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DOI：
10.1111/jmi.12951
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发表时间：
2021-03
影响因子：
2
通讯作者：
DE Winter DAM
中科院分区：
文献类型：
--
作者：
Hayles MF;DE Winter DAM
将冷冻技术引入聚焦离子束扫描电子显微镜（FIB‐SEM）为研究生命科学领域的冷冻、水合样品带来了新的机会。冷冻技术长期以来一直用于电子显微镜。通过冷冻超薄切片术制备薄的电子透明切片，用于在冷冻透射电子显微镜（TEM）中观察。Cryo-TEM目前正在实现大分子结构的成像。同时，通过cryo‐SEM研究了散装材料的低温断裂表面。cryo-TEM和cryo-SEM都提供了丰富的信息，尽管是2D技术。Cryo-TEM断层扫描确实提供了3D信息，但体积的厚度最大为200-300 nm，这限制了特定结构背景下的3D信息。FIB铣削通过创建垂直于低温断裂表面的横截面（例如横截面或特定部位X截面），使其他平面成像，从而为低温SEM增加了第三个成像维度。本文讨论了如何从冷冻、水合的生命科学样品中产生合适的cryo‐FIB‐SEM横截面结果，重点是“常识”和重复观察。生命科学研究生命到最小的细节。可视化最小的细节需要使用高真空室的电子显微镜。在真空条件下保持生命科学样品完整性的一种方法是冷冻。冷冻样品可以保持在悬浮状态。因此，可以在不改变样品的化学或内部物理结构的情况下进行研究。两种类型的电子显微镜配备了冷冻样品处理设施，用于研究样品：扫描电子显微镜（SEM），用于研究表面;透射电子显微镜（TEM），用于研究薄的电子透明切片（称为laminate）。第三种研究方法将SEM与聚焦离子束（FIB）结合起来，形成cryo-FIB-SEM，这是本文的基础。电子束对低温样品表面进行成像，而离子束米尔斯则对表面进行研磨，以暴露样品的内部。后者被称为横截面，其结果提供了一种研究样品三维的方法。本文着眼于以这种方式制作横截面，这源于多年来使用这种技术获得的知识和经验。此信息适用于新人和有经验的冷冻显微镜研究人员。
The introduction of cryo‐techniques to the focused ion‐beam scanning electron microscope (FIB‐SEM) has brought new opportunities to study frozen, hydrated samples from the field of Life Sciences. Cryo‐techniques have long been employed in electron microscopy. Thin electron transparent sections are produced by cryo‐ultramicrotomy for observation in a cryo‐transmission electron microscope (TEM). Cryo‐TEM is presently reaching the imaging of macromolecular structures. In parallel, cryo‐fractured surfaces from bulk materials have been investigated by cryo‐SEM. Both cryo‐TEM and cryo‐SEM have provided a wealth of information, despite being 2D techniques. Cryo‐TEM tomography does provide 3D information, but the thickness of the volume has a maximum of 200–300 nm, which limits the 3D information within the context of specific structures. FIB‐milling enables imaging additional planes by creating cross‐sections (e.g. cross‐sectioning or site‐specific X‐sectioning) perpendicular to the cryo‐fracture surface, thus adding a third imaging dimension to the cryo‐SEM. This paper discusses how to produce suitable cryo‐FIB‐SEM cross‐section results from frozen, hydrated Life Science samples with emphasis on ‘common knowledge’ and reoccurring observations. Life Sciences studies life down to the smallest details. Visualising the smallest details requires electron microscopy, which utilises high‐vacuum chambers. One method to maintain the integrity of Life Sciences samples under vacuum conditions is freezing. Frozen samples can remain in a suspended state. As a result, research can be carried out without having to change the chemistry or internal physical structure of the samples. Two types of electron microscopes equipped with cryo‐sample handling facilities are used to investigate samples: The scanning electron microscope (SEM) which investigates surfaces and the transmission electron microscope (TEM) which investigates thin electron transparent sections (called lamellae). A third method of investigation combines a SEM with a focused ion beam (FIB) to form a cryo‐FIB‐SEM, which is the basis of this paper. The electron beam images the cryo‐sample surface while the ion beam mills into the surface to expose the interior of the sample. The latter is called cross‐sectioning and the result provides a way of investigating the 3rd dimension of the sample. This paper looks at the making of cross‐sections in this manner originating from knowledge and experience gained with this technique over many years. This information is meant for newcomers, and experienced researchers in cryo‐microscopy alike.
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影响因子：
3
作者：
Hsieh, Chyongere;Schmelzer, Thomas;Kishchenko, Gregory;Wagenknecht, Terence;Marko, Michael
通讯作者：
Marko, Michael
影响因子：
44.1
作者：
Bartels-Rausch, Thorsten;Bergeron, Vance;Uras-Aytemiz, Nevin
通讯作者：
Uras-Aytemiz, Nevin
DOI：
10.1126/science.aan7904
发表时间：
2017-08-18
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Böck D;Medeiros JM;Tsao HF;Penz T;Weiss GL;Aistleitner K;Horn M;Pilhofer M
通讯作者：
Pilhofer M
影响因子：
3
作者：
de Winter, D. A. Matthijs;Mesman, Rob J.;Post, Jan A.
通讯作者：
Post, Jan A.
影响因子：
2
作者：
Hayles, M. F.;Stokes, D. J.;Findlay, K. C.
通讯作者：
Findlay, K. C.