Compared with conventional two-dimensional transmission electron microscopy (TEM), focused ion beam scanning electron microscopy (FIB-SEM) can provide more comprehensive 3D information on cell substructures at the nanometer scale. Biological samples prepared by cryofixation using high-pressure freezing demonstrate optimal preservation of the morphology of cellular structures, as these are arrested instantly in their near-native states. However, samples from cryofixation often show a weak back-scatter electron signal and bad image contrast in FIB-SEM imaging. In addition, it is impossible to do large amounts of heavy metal staining. This is commonly achieved via established osmium impregnation (OTO) en bloc staining protocols. Here, we compared the FIB-SEM image quality of brain tissues prepared using several common freeze-substitution media, and we developed an approach that overcomes these limitations through a combination of osmium tetroxide, uranyl acetate, tannic acid, and potassium permanganate at proper concentrations, respectively. Using this optimized sample preparation protocol for high-pressure freezing and freeze-substitution, perfect smooth membrane morphology, even of the lipid bilayers of the cell membrane, was readily obtained using FIB-SEM. In addition, our protocol is broadly applicable and we demonstrated successful application to brain tissues, plant tissues, Caenorhabditis elegans, Candida albicans, and chlorella. This approach combines the potential of cryofixation for 3D large volume analysis of subcellular structures with the high-resolution capabilities of FIB-SEM.

与传统的二维透射电子显微镜 (TEM) 相比，聚焦离子束扫描电子显微镜 (FIB-SEM) 可以提供更全面的纳米级细胞亚结构 3D 信息。使用高压冷冻通过冷冻固定制备的生物样品证明了细胞结构形态的最佳保存，因为它们在接近天然的状态下立即被捕获。然而，冷冻固定的样品在 FIB-SEM 成像中通常显示出微弱的背散射电子信号和较差的图像对比度。另外，不可能做大量的重金属染色。这通常是通过已建立的锇浸渍 (OTO) 整体染色方案来实现的。在这里，我们比较了使用几种常见冷冻替代培养基制备的脑组织的 FIB-SEM 图像质量，我们开发了一种方法，通过将四氧化锇、乙酸双氧铀、单宁酸和高锰酸钾分别以适当的浓度组合来克服这些限制。使用这种用于高压冷冻和冷冻替代的优化样品制备方案，使用 FIB-SEM 可以轻松获得完美光滑的膜形态，甚至细胞膜的脂质双层。此外，我们的协议具有广泛的适用性，我们证明了对脑组织、植物组织、使用 FIB-SEM 很容易获得完美光滑的膜形态，即使是细胞膜的脂质双层。此外，我们的协议具有广泛的适用性，我们证明成功应用于脑组织、植物组织、使用 FIB-SEM 很容易获得完美光滑的膜形态，即使是细胞膜的脂质双层。此外，我们的协议具有广泛的适用性，我们证明了对脑组织、植物组织、秀丽隐杆线虫、白色念珠菌和小球藻。这种方法结合了冷冻固定对亚细胞结构的 3D 大体积分析的潜力和 FIB-SEM 的高分辨率能力。