Central European Institute of Technology, Masaryk University.
Central European Institute of Technology, Masaryk University;
J Vis Exp. 2021 Nov 20(177). doi: 10.3791/62351.
Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in situ. Owing to the strong interaction of an electron with the matter, high-resolution cryo-ET studies are limited to specimens with a thickness of less than 200 nm, which restricts the applicability of cryo-ET only to the peripheral regions of a cell. A complex workflow that comprises the preparation of thin cellular cross-sections by cryo-focused ion beam micromachining (cryo-FIBM) was introduced during the last decade to enable the acquisition of cryo-ET data from the interior of larger cells. We present a protocol for the preparation of cellular lamellae from a sample vitrified by plunge freezing utilizing Saccharomyces cerevisiae as a prototypical example of a eukaryotic cell with wide utilization in cellular and molecular biology research. We describe protocols for vitrification of S. cerevisiae into isolated patches of a few cells or a continuous monolayer of the cells on a TEM grid and provide a protocol for lamella preparation by cryo-FIB for these two samples.
如今，冷冻电子断层扫描（cryo-ET）是唯一能够提供原位的大分子复合物的近原子分辨率结构数据的技术。由于电子与物质的强烈相互作用，高分辨率的 cryo-ET 研究仅限于厚度小于 200nm 的样本，这限制了 cryo-ET 仅适用于细胞的外围区域。在过去十年中，引入了一种由 cryo-FIBM（cryo-聚焦离子束微加工）制备薄细胞横截面的复杂工作流程，以能够从较大细胞的内部获取 cryo-ET 数据。我们提出了一种从通过跌落冷冻法进行玻璃化的样本中制备细胞薄片的方案，以酿酒酵母为例，作为在细胞和分子生物学研究中广泛应用的真核细胞的原型。我们描述了将酿酒酵母玻璃化到 TEM 网格上的几个细胞的孤立斑块或细胞的连续单层中的方案，并为这两种样品提供了 cryo-FIB 制备薄片的方案。
