Electron tomography of vitreous sections from cultured mammalian cells
Electron tomography of vitreous sections from cultured mammalian cells
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DOI：
10.1016/j.jsb.2007.10.008
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发表时间：
2008-03-01
影响因子：
3
通讯作者：
Leis, Andrew
中科院分区：
文献类型：
--
作者：
Gruska, Manuela;Medalia, Ohad;Leis, Andrew
适当薄的冷冻水合生物标本的低温电子断层扫描对于研究真核细胞和组织的三维大分子结构具有极好的潜力。由于心肌细胞太厚，无法在完整状态下观察，我们将永生化细胞系HL-1培养至亚融合，并在高压冷冻、超微切片和断层扫描之前通过酶分离获得细胞。我们通过实现两个新功能来提高玻璃体冷冻切片层析成像的效率：(1)在低温下使用荧光显微镜来搜索感兴趣的特征，而不会在次要（非成像）任务上花费任何可耐受的电子剂量；(2)使用胶体金作为基准标记。玻璃体切片的荧光染色和随后的冷冻荧光显微镜用于定位线粒体位于适合获得倾斜序列的位置，同时考虑到切片的平直度、污染的存在和靠近网格条。为了提供一种简单而可靠的层图对齐方法，我们开发了一种普遍适用的将胶体金沉积到玻璃切片上的方法，类似于Masich等人描述的应用量子点的方法[Masich， S., Ostberg， T., Norlen， L., Shupliakov， O., Daneholt， B., 2006]。在玻璃体冷冻切片上沉积基准标记物用于细胞断层扫描的一种方法。j . Struct。生物医学工程学报，2004,26(2):481 - 481。心肌线粒体薄片（名义厚度65 ~ 85 nm）的断层扫描显示嵴间的互联性以及与线粒体内膜的连接。在某些情况下，ATP合酶可以毫无歧义地识别。这些发现证实了在6- 8nm分辨率下三维研究哺乳动物细胞结构生物学的可行性。(C) 2007爱思唯尔公司版权所有。
Cryo-electron tomography of appropriately thin, frozen-hydrated biological specimens has excellent potential for investigating the 3D macromolecular architecture of eukaryotic cells and tissues. Since cardiomyocytes are too thick to be visualised in an intact state, we grew immortalised cell line HL-1 to sub-confluency and harvested the cells by enzymatic detachment prior to hyperbaric freezing, ultramicrotomy, and tomography. We improved the efficiency of tomographic acquisition from vitreous cryosections by implementing two new features: (1) fluorescence microscopy at cryogenic temperatures to search for features of interest without expending any of the tolerable electron dose on secondary (non-imaging) tasks, and (2) the use of colloidal gold as fiducial markers. Vital fluorescent staining and subsequent cryo-fluorescence microscopy of vitreous sections were used to localise mitochondria lying in positions suitable for acquiring tilt series, taking into account section flatness, presence of contamination and proximity to grid bars. To provide a simple and robust means of aligning tomograms, we developed a universally applicable protocol for depositing colloidal gold onto vitreous sections, analogous to the method for applying quantum dots described by Masich et al. [Masich, S., Ostberg, T., Norlen, L., Shupliakov, O., Daneholt, B., 2006. A procedure to deposit fiducial markers on vitreous cryo-sections for cellular tomography. J. Struct. Biol. 156, 461-468]. Tomograms of thin sections (nominal thickness 65-85 nm) of cardiac mitochondria revealed the interconnectivity of cristae and junctions with the inner mitochondrial membrane. In some cases, ATP synthases could be identified without ambiguity. These findings confirm the feasibility of investigating the structural biology of mammalian cells in three dimensions and at a resolution of 6-8 nm. (C) 2007 Elsevier Inc. All rights reserved.