Schaffer Miroslava, Engel Benjamin D, Laugks Tim, Mahamid Julia, Plitzko Jürgen M, Baumeister Wolfgang
Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany.
Bio Protoc. 2015 Sep 5;5(17). doi: 10.21769/bioprotoc.1575.
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, starting with fully grown cell cultures. Three recently investigated biological samples are given as examples.
冷冻电子断层扫描（CET）是一种成熟的技术，可在亚纳米分辨率下对细胞和分子结构进行成像。由于该方法仅限于厚度小于500 nm的样品，因此需要合适的样品制备才能从更大的细胞体积中获取CET数据。最近，对 plunge-frozen生物材料进行冷冻聚焦离子束（cryo-FIB）铣削已被证明能够为先进的CET可重复地产生大尺寸、均匀薄且无变形的玻璃态横截面。所有能够被plunge-frozen的真核细胞和原核细胞都可以用cryo-FIB技术进行减薄。再加上低剂量显微镜技术的进步，这已经改变了原位结构生物学的前沿。在本方案中，我们描述了cryo-FIB技术的典型步骤，从完全生长的细胞培养物开始。给出了三个最近研究的生物样品作为示例。
