Cryo-FIB preparation of whole cells and tissue for cryo-TEM: use of high-pressure frozen specimens in tubes and planchets.
Cryo-FIB preparation of whole cells and tissue for cryo-TEM: use of high-pressure frozen specimens in tubes and planchets.
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DOI：
10.1111/jmi.12943
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发表时间：
2021-03
影响因子：
2
通讯作者：
Hayles MF
中科院分区：
文献类型：
--
作者：
DE Winter DAM;Hsieh C;Marko M;Hayles MF
在细胞环境中研究大分子复合物的需求，要求具备制备适合低温透射电子显微镜（cryo - TEM）研究的薄样品的能力。在本文中，我们讨论两种相似的方法，它们分别在荷兰乌得勒支和美国奥尔巴尼独立研发。这些方法尤其适用于高压冷冻仪（HPF）制备的组织样本和细胞悬浮液。在阐述工作流程时特别关注了潜在的陷阱，同时强调了基本原理（“为什么要这样做”）。尽管两种工作流程成功率都很高，但完整操作需要丰富的经验且要求很高。此外，通量较低。我们希望鼓励世界各地的其他研究小组接受改进高压冷冻 - 低温聚焦离子束扫描电镜 - 低温透射电子显微镜工作流程的挑战。为此我们讨论了一些建议。
生命最终由我们身体内分子的相互作用所决定。人们正在使用并进一步研发高度复杂的设备来研究这些相互作用。本文描述了在低温透射电子显微镜（cryo - TEM）中研究细胞时制备小而极薄的薄片（面积为5×5 µm²，厚度为50 - 300 nm）的方法。必须特别注意在自然环境中保持分子的自然状态。对于低温透射电子显微镜而言，样品必须冷冻并保持冷冻状态，以适应显微镜中的真空条件。冷冻条件带来了一些技术挑战，文中对此进行了探讨。文中描述了获得薄片的两种方法。两种方法都使用聚焦离子束（FIB）显微镜。聚焦离子束通过将一束电离原子（镓离子）聚焦到样品上，能够以纳米精度去除材料。对聚焦离子束的精细控制可以切割出所需的薄片。在这两种策略中，薄片都仍然附着在原始样品上，并且带有切片的样品和样品架的整体在保持冷冻的状态下从聚焦离子束显微镜转移到透射电子显微镜。
The desire to study macromolecular complexes within their cellular context requires the ability to produce thin samples suitable for cryo‐TEM (cryo‐transmission electron microscope) investigations. In this paper, we discuss two similar approaches, which were developed independently in Utrecht (the Netherlands) and Albany (USA). The methods are particularly suitable for both tissue samples and cell suspensions prepared by a high‐pressure freezer (HPF). The workflows are explained with particular attention to potential pitfalls, while underlying principles are highlighted (‘why to do so’). Although both workflows function with a high success rate, full execution requires considerable experience and remains demanding. In addition, throughput is low. We hope to encourage other research groups worldwide to take on the challenge of improving the HPF– cryo‐FIB‐SEM – cryo‐TEM workflow. We discuss a number of suggestions to this end. Life is ultimately dictated by the interaction of molecules in our bodies. Highly complex equipment is being used and further developed to study these interactions. The present paper describes methods to prepare small, very thin lamellae (area of 5×5 µm2, thickness 50–300 nm) of a cell to be studied in a cryo‐transmission electron microscope (cryo‐TEM). Special care must be taken to preserve the natural state of molecules in their natural environment. In the case of cryo‐TEM, the samples must be frozen and kept frozen to be compatible with the vacuum conditions in the microscope. The frozen condition imposes technical challenges which are addressed. Two approaches to obtain the thin lamellae are described. Both make use of a focused ion beam (FIB) microscope. The FIB allows removal of material with nanometre precision by focusing a beam of ionised atoms (gallium ions) onto the sample. Careful control of the FIB allows cutting out of the required thin lamellae. In both strategies, the thin lamellae remain attached to the original sample, and the ensemble of sample with section and sample holder is transported from the FIB microscope to the TEM while being kept frozen.
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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：
3
作者：
Hsieh, CE;Leith, A;Marko, M
通讯作者：
Marko, M
影响因子：
3
作者：
Agronskaia, Alexandra V.;Valentijn, Jack A.;Gerritsen, Hans C.
通讯作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
2
作者：
Hayles MF;DE Winter DAM
通讯作者：
DE Winter DAM