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Vol:.(1234567890)Anatomical Science International (2025) 100:598–613https://doi.org/10.1007/s12565-025-00888-5ORIGINAL ARTICLEDirect imaging of the three?dimensional ultrastructure of neuronal organellesDaisuke Koga 1  · Ryosuke Morinaga 1  · Satoshi Kusumi 2Received: 12 May 2025 / Accepted: 16 July 2025 / Published online: 5 August 2025 © The Author(s) 2025AbstractIn the context of cell morphological analyses observing organelles embedded within the cell matrix is dif f i cult. The osmium maceration method is a unique technique used to directly observe the three-dimensional structure of organelles through scanning electron microscopy, without requiring time-consuming and labor-intensive reconstruction. In this method, tissues are immersed in a diluted osmium solution for several days to remove cytosolic soluble proteins and fi lamentous structures, including microf i laments, intermediate fi laments, and microtubules, from the freeze-cracked surfaces of cells, leaving the subcellular structures, Golgi apparatus, mitochondria, and smooth and rough endoplasmic reticulum intact. Specimen preparation involves several key steps, specif i cally pre-f i xation with aldehyde fi xatives, tissue excision, trimming, post-f i xation with osmium tetroxide solution, dimethyl sulfoxide cracking (i.e., freeze-cracking), the thawing of cracked tissues, osmium maceration, osmium fi xation, conductive staining (tannin–osmium method), dehydration, drying, mounting, metal coating, and scanning electron microscopy observations. Here, we present a step-by-step protocol based on the maceration method using neural cells as an example, ensuring reproducibility and consistent results for neurons and various other cell types. Moreover, the results presented indicate that the osmium maceration method is ef f ective for elucidating the three-dimensional intracellular ultrastructure of neurons. Keywords Osmium maceration method · Scanning electron microscopy · Three-dimensional · Organelles; neuronsIntroductionIn contrast to the transmission electron microscopy (TEM) analysis of resin-embedded tissue sections, scanning elec-tron microscopy (SEM) uses secondary electron signals to enable the direct three-dimensional (3D) visualization of the surface topography of tissues and cells. Using conventional specimen preparations, SEM provides valuable morphologi-cal information regarding hollow organs, such as the intes-tines and trachea. For instance, for intestinal specimens, the 3D structure of villi and ultrastructural details of the apical surfaces of absorptive and goblet cells, such as microvilli and secretory granules, can be clearly observed using SEM. In addition, SEM has revealed the 3D ultrastructural fea-tures of various cell types, such as podocytes in the kid-ney glomeruli (Takahashi-Iwanaga 2002), hair cells of the inner ear cochlea (Grillet 2022), and cells in hollow organs. As glands, blood vessels, nerves, and muscles are covered by the basal membrane and wrapped by the surrounding connective tissue, observing the surface structures of cells located in the connective tissues is dif f i cult using conven-tional SEM preparations. To address this issue, connective tissue digestion methods have been established to remove the fi brous components of connective tissues and basal mem-brane covering the basal surfaces of cells, leaving only the cellular components (Takahashi-Iwanaga and Fujita 1986; Ushiki and Ide 1988). For SEM, the alkali-water macera-tion method, which removes cellular components while preserving the collagen fi brillar network, has contributed to elucidating the 3D architectures of reticular tissues, such Neuroanatomy Across Scales * Daisuke Koga daisukek@asahikawa-med.ac.jp1 Department of Microscopic Anatomy and Cell Biology, Asahikawa Medical University, 2-1-1-1 Midorigaoka-Higashi, Asahikawa, Hokkaido 078-8510, Japan2 Department of Morphological Sciences, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan