Yang Wen, Briegel Ariane
Department of Biology, Leiden University, Leiden, The Netherlands.
Methods Mol Biol. 2018;1729:173-185. doi: 10.1007/978-1-4939-7577-8_16.
Cryo-electron microscopy (cryo-EM) allows the imaging of intact macromolecular complexes in the context of whole cells. The biological samples for cryo-EM are kept in a near-native state by flash freezing, without the need for any additional sample preparation or fixation steps. Since transmission electron microscopy only generates 2D projections of the samples, the specimen has to be tilted in order to recover its 3D structural information. This is done by collecting images of the sample with various tilt angles in respect to the electron beam. The acquired tilt series can then be computationally back-projected. This technique is called electron cryotomography (ECT), and has been instrumental in unraveling the architecture of chemoreceptor arrays. Here we describe the method of visualizing in vivo bacterial chemoreceptor arrays in three main steps: immobilization of bacterial cells on EM grids by plunge-freezing; 2D image acquisition in tilt series; and 3D tomogram reconstruction.
冷冻电子显微镜（cryo-EM）能够在全细胞环境下对完整的大分子复合物进行成像。用于cryo-EM的生物样品通过快速冷冻保持在接近天然的状态，无需任何额外的样品制备或固定步骤。由于透射电子显微镜仅生成样品的二维投影，因此必须倾斜样品以获取其三维结构信息。这是通过收集样品相对于电子束的不同倾斜角度的图像来实现的。然后可以对获取的倾斜系列进行计算反投影。这种技术称为电子冷冻断层扫描（ECT），在揭示化学感受器阵列的结构方面发挥了重要作用。在这里，我们分三个主要步骤描述在体内可视化细菌化学感受器阵列的方法：通过 plunge-freezing 将细菌细胞固定在电子显微镜网格上；在倾斜系列中进行二维图像采集；以及三维断层图像重建。
