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Author:
Date:
2015-09-05
[Abstract] Cryo-electron tomography (CET) is a well-established technique for imaging cellular and molecular structures at sub-nanometer resolution. As the method is limited to samples that are thinner than 500 nm, suitable sample preparation is required to attain CET data from larger cell volumes. Recently, cryo-focused ion beam (cryo-FIB) milling of plunge-frozen biological material has been shown to reproducibly yield large, homogeneously thin, distortion-free vitreous cross-sections for state-of-the-art CET. All eukaryotic and prokaryotic cells that can be plunge-frozen can be thinned with the cryo-FIB technique. Together with advances in low-dose microscopy, this has shifted the frontiers of in situ structural biology. In this protocol we describe the typical steps of the cryo-FIB technique, ...
[摘要] 低电子层析成像（CET）是用于在亚纳米分辨率下成像细胞和分子结构的成熟技术。 由于该方法仅限于比500nm薄的样品，因此需要合适的样品制备以从较大的细胞体积获得CET数据。 最近，冷冻生物材料的低温聚焦离子束（cryo-FIB）研磨已被证明可再现地产生大的，均匀的，无失真的玻璃体横截面用于最先进的CET。 可以使用cryo-FIB技术稀释所有可以浸入冷冻的真核细胞和原核细胞。 连同低剂量显微镜的进展，这已经转移了原位结构生物学的前沿。 在本协议中，我们描述了冷冻FIB技术的典型步骤，从完全生长的细胞培养开始。 最近研究的三个生物样品作为例子。
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