Direct imaging of the three-dimensional ultrastructure of neuronal organelles

In the context of cell morphological analyses observing organelles embedded within the cell matrix is difficult. 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 filamentous structures, including microfilaments, intermediate filaments, 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, specifically pre-fixation with aldehyde fixatives, tissue excision, trimming, post-fixation with osmium tetroxide solution, dimethyl sulfoxide cracking (i.e., freeze-cracking), the thawing of cracked tissues, osmium maceration, osmium fixation, 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 effective for elucidating the three-dimensional intracellular ultrastructure of neurons.