Institut de Génétique et de Biologie Moléculaire et Cellulaire, Department of Integrated Structural Biology, Equipe labellisée Ligue Contre le Cancer, Illkirch, France; Centre National de la Recherche Scientifique, UMR7104, Illkirch, France; Institut National de la Santé et de la Recherche Médicale, U1258 Illkirch, France; Université de Strasbourg, Illkirch, France.
Electron Microscopy Core Unit, Department of Neurogenetics, Max Planck Institute of Experimental Medicine, Göttingen, Germany.
J Struct Biol. 2020 Jul 1;211(1):107528. doi: 10.1016/j.jsb.2020.107528. Epub 2020 May 6.
Focused Ion Beam-Scanning Electron Microscopy (FIB-SEM) is an invaluable tool to visualize the 3D architecture of cell constituents and map cell networks. Recently, amorphous ice embedding techniques have been associated with FIB-SEM to ensure that the biological material remains as close as possible to its native state. Here we have vitrified human HeLa cells and directly imaged them by cryo-FIB-SEM with the secondary electron InLens detector at cryogenic temperature and without any staining. Image stacks were aligned and processed by denoising, removal of ion beam milling artefacts and local charge imbalance. Images were assembled into a 3D volume and the major cell constituents were modelled. The data illustrate the power of the workflow to provide a detailed view of the internal architecture of the fully hydrated, close-to-native, entire HeLa cell. In addition, we have studied the feasibility of combining cryo-FIB-SEM imaging with live-cell protein detection. We demonstrate that internalized gold particles can be visualized by detecting back scattered primary electrons at low kV while simultaneously acquiring signals from the secondary electron detector to image major cell features. Furthermore, gold-conjugated antibodies directed against RNA polymerase II could be observed in the endo-lysosomal pathway while labelling of the enzyme in the nucleus was not detected, a shortcoming likely due to the inadequacy between the size of the gold particles and the voxel size. With further refinements, this method promises to have a variety of applications where the goal is to localize cellular antigens while visualizing the entire native cell in three dimensions.
聚焦离子束扫描电子显微镜（FIB-SEM）是一种非常有价值的工具，可以用于观察细胞成分的 3D 结构并绘制细胞网络。最近，无定形冰包埋技术与 FIB-SEM 结合使用，以确保生物材料尽可能保持其天然状态。在这里，我们将人宫颈癌细胞（HeLa）进行玻璃化固定，并通过 cryo-FIB-SEM 直接进行低温下的二次电子 InLens 检测成像，无需任何染色。对图像堆栈进行对齐和处理，包括去噪、去除离子束铣削伪影和局部电荷失衡。将图像组装成一个 3D 体积，并对主要的细胞成分进行建模。该数据说明了该工作流程的强大功能，可以提供完全水合、接近天然状态的整个 HeLa 细胞内部结构的详细视图。此外，我们还研究了将 cryo-FIB-SEM 成像与活细胞蛋白检测相结合的可行性。我们证明，内部化的金颗粒可以通过在低千伏下检测背散射的初级电子来可视化，同时从二次电子检测器获取信号来成像主要的细胞特征。此外，还可以观察到针对 RNA 聚合酶 II 的金结合抗体在内体溶酶体途径中，而在核中未检测到该酶的标记，这一缺点可能是由于金颗粒的大小与体素大小不匹配所致。通过进一步改进，这种方法有望在各种应用中具有多种应用，其目标是在可视化整个天然细胞的同时定位细胞抗原。
