Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box
Improving the technique of vitreous cryo-sectioning for cryo-electron tomography: Electrostatic charging for section attachment and implementation of an anti-contamination glove box
复制标题
DOI：
10.1016/j.jsb.2009.10.001
复制
发表时间：
2010-02-01
影响因子：
3
通讯作者：
Peters, Peter J.
中科院分区：
文献类型：
--
作者：
Pierson, Jason;Jesus Fernandez, Jose;Peters, Peter J.
玻璃体冷冻切片的冷冻电子断层扫描是探索大到不能完整成像的生物样品的三维组织的最合适的方法。然而，生产高质量的玻璃体冷冻切片是具有挑战性的。在这里，我们专注于成功的主要障碍：切片内和周围的污染，以及将切片带状附着到电子显微镜网格支撑膜上。传统的将部分连接到网格上的方法涉及到由粗糙的冲压或压制设备产生的机械力，但这会破坏玻璃体冷冻部分的完整性。此外，附着性差，并且部分带状部分经常远离支撑膜。这会导致标本在图像采集过程中不稳定，并随后难以对齐投影图像。在此，我们在冷冻超微切割机周围安装了一个保护手套箱，以减少切片过程中切片机周围和内部的湿度。我们还介绍了一种利用静电将玻璃体冷冻切片连接到EM栅极支撑膜上的新方法。玻璃体冰冻切片的带子在转移和储存过程中保持在适当的位置，没有冲压相关的文物。我们通过探索酿酒酵母玻璃体冰冻切片中50 nm内假定的细胞80S核糖体的结构来说明这些改进。(C)2009 Elsevier Inc.保留所有权利。
Cryo-electron tomography of vitreous cryo-sections is the most suitable method for exploring the 3D organization of biological samples that are too large to be imaged in an intact state. Producing good quality vitreous cryo-sections, however, is challenging. Here, we focused on the major obstacles to success: contamination in and around the microtome, and attachment of the ribbon of sections to an electron microscopic grid support film. The conventional method for attaching sections to the grid has involved mechanical force generated by a crude stamping or pressing device, but this disrupts the integrity of vitreous cryo-sections. Furthermore, attachment is poor, and parts of the ribbon of sections are often far from the support film. This results in specimen instability during image acquisition and subsequent difficulty with aligning projection images.Here, we have implemented a protective glove box surrounding the cryo-ultramicrotome that reduces the humidity around and within the microtome during sectioning. We also introduce a novel way to attach vitreous cryo-sections to an EM grid support film using electrostatic charging. The ribbon of vitreous cryo-sections remains in place during transfer and storage and is devoid of stamping related artefacts. We illustrate these improvements by exploring the structure of putative cellular 80S ribosomes within 50 nm, vitreous cryo-sections of Saccharomyces cerevisiae. (C) 2009 Elsevier Inc. All rights reserved.