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Cryo‐FIB preparation of whole cells and tissue for cryo‐TEM: Use of high‐pressure frozen specimens in tubes and planchets
Journal of Microscopy ( IF 2 ) Pub Date : 2020-07-28 , DOI: 10.1111/jmi.12943
D A M DE Winter 1 , C Hsieh 2 , M Marko 2, 3 , M F Hayles 4
Affiliation  

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.

中文翻译:

用于冷冻 TEM 的全细胞和组织的冷冻 FIB 制备:在试管和平板中使用高压冷冻标本

在细胞环境中研究大分子复合物的愿望需要能够生产适用于低温透射电子显微镜(低温透射电子显微镜)研究的薄样品。在本文中,我们讨论了两种类似的方法,它们是在乌得勒支(荷兰)和奥尔巴尼(美国)独立开发的。这些方法特别适用于通过高压冷冻 (HPF) 制备的组织样本和细胞悬液。解释工作流程时特别注意潜在的陷阱,同时强调基本原则(“为什么这样做”)。尽管这两种工作流程的成功率都很高,但完全执行需要相当多的经验并且仍然要求很高。此外,吞吐量低。我们希望鼓励世界各地的其他研究小组接受改进 HPF-cryo-FIB-SEM-cryo-TEM 工作流程的挑战。为此,我们讨论了一些建议。
更新日期:2020-07-28
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