Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried, Germany.
Proc Natl Acad Sci U S A. 2012 Mar 20;109(12):4449-54. doi: 10.1073/pnas.1201333109. Epub 2012 Mar 5.
Cryoelectron tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (< 0.5-1 μm) that is accessible with today's intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200-500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell's interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.
冷冻电子断层扫描技术为研究接近生理状态下细胞的大分子和超分子结构提供了前所未有的视角。然而，由于目前的中压电子显微镜的有限厚度范围（<0.5-1μm），只能直接检查小的原核细胞或真核细胞的周边区域。克服这一限制的关键是制备足够薄的样品的能力。冷冻切片可以用来制备足够薄的切片，但会产生严重的伪影，如大量压缩。在这里，我们描述了一种基于聚焦离子束（FIB）铣削的方法，用于从在电子显微镜（EM）网格上生长的玻璃化细胞中制备薄（200-500nm）的薄片。这些自支撑的薄片显然没有扭曲或其他伪影，并且为细胞内部打开了很大的窗口，可以对细胞的任何选定部分进行断层扫描研究。我们用从单个断层扫描图获得的核孔复合体结构来说明样品保存的质量。
