Zhang Jianguo, Ji Gang, Huang Xiaojun, Xu Wei, Sun Fei
Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.
Center for Biological Imaging, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing 100101, China.
J Struct Biol. 2016 May;194(2):218-23. doi: 10.1016/j.jsb.2016.02.013. Epub 2016 Feb 12.
Cryo-electron tomography (cryo-ET) provides great insights into the ultrastructure of cells and tissues in their native state and provides a promising way to study the in situ 3D structures of macromolecular complexes. However, this technique has been limited on the very thin specimen, which is not applicable for most cells and tissues. Besides cryo-sectioning approach, cryo focused ion beam (cryo-FIB) appeared recently to achieve 'artifact-free' thin frozen hydrated lamella via fabrication. Considering that the current cryo-FIB methods need modified holders or cartridges, here, with a "D-shaped" molybdenum grid and a specific shutter system, we developed a simple cryo-FIB approach for thin frozen hydrated lamella fabrication, which fits both standard transmission cryo-electron microscopes with side-entry cryo-holders and state-of-the-art ones with AutoGrids. Our approach will expand the usage of cryo-FIB approach in many labs.
冷冻电子断层扫描（cryo-ET）能深入了解细胞和组织在其天然状态下的超微结构，并为研究大分子复合物的原位三维结构提供了一种很有前景的方法。然而，该技术一直局限于非常薄的样本，这不适用于大多数细胞和组织。除了冷冻切片方法外，冷冻聚焦离子束（cryo-FIB）最近出现，通过加工制备出“无伪像”的薄冷冻水合薄片。鉴于目前的冷冻聚焦离子束方法需要改进的样品架或样品盒，在此，我们利用一个“D形”钼网和一个特定的快门系统，开发了一种简单的冷冻聚焦离子束方法来制备薄冷冻水合薄片，该方法适用于配备侧入式冷冻样品架的标准透射冷冻电子显微镜以及配备自动样品网的先进显微镜。我们的方法将扩大冷冻聚焦离子束方法在许多实验室中的应用。
