The two-dimensional observation of ultrathin sections from resin-embedded specimens provides insufficient understanding of the three-dimensional (3D) morphological information of membranous organelles. The osmium maceration method, developed by Professor Tanaka’s group over 40 years ago, is the only technique that allows direct observation of the 3D ultrastructure of membrane systems using scanning electron microscopy (SEM), without the need for any reconstruction process. With this method, the soluble cytoplasmic proteins are removed from the freeze-cracked surface of cells while preserving the integrity of membranous organelles, achieved by immersing tissues in a diluted osmium solution for several days. By employing the maceration method, researchers using SEM have revealed the 3D ultrastructure of organelles such as the Golgi apparatus, mitochondria, and endoplasmic reticulum in various cell types. Recently, we have developed new SEM techniques based on the maceration method to explore further possibilities for this method. These include: (1) a rapid osmium maceration method that reduces the reaction duration of the procedure, (2) a combination method that combines agarose embedding with osmium maceration to elucidate the 3D ultrastructure of organelles in free and cultured cells, and (3) a correlative immunofluorescence and SEM technique that combines cryosectioning with the osmium maceration method, enabling the correlation of the immunocytochemical localization of molecules with the 3D ultrastructure of organelles. In this paper, we review the novel osmium maceration methods described above and discuss their potential and future directions in the field of biology and biomedical research.

中文翻译：

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通过扫描电子显微镜对细胞内结构进行三维分析

对树脂包埋标本超薄切片的二维观察对膜细胞器的三维（3D）形态信息的理解不够。田中教授团队在 40 多年前开发的锇浸渍法是唯一可以使用扫描电子显微镜 (SEM) 直接观察膜系统 3D 超微结构的技术，无需任何重建过程。通过这种方法，可溶性细胞质蛋白从冷冻破裂的细胞表面去除，同时保留膜细胞器的完整性，这是通过将组织浸入稀释的锇溶液中几天来实现的。通过浸渍方法，研究人员使用 SEM 揭示了各种细胞类型中高尔基体、线粒体和内质网等细胞器的 3D 超微结构。最近，我们开发了基于浸渍方法的新扫描电镜技术，以探索该方法的进一步可能性。其中包括：(1) 一种快速锇浸渍方法，可缩短程序的反应时间，(2) 将琼脂糖包埋与锇浸渍相结合的组合方法，以阐明游离细胞和培养细胞中细胞器的 3D 超微结构，以及 (3)一种相关的免疫荧光和 SEM 技术，将冷冻切片与锇浸渍法相结合，使分子的免疫细胞化学定位与细胞器的 3D 超微结构相关联。在本文中，我们回顾了上述新型锇浸渍方法，并讨论了它们在生物学和生物医学研究领域的潜力和未来方向。