The osmium maceration method with scanning electron microscopy (SEM) enabled to demonstrate directly the three-dimensional (3D) structure of membranous cell organelles. However, the polarity of the Golgi apparatus (that is, the cis–trans axis) can hardly be determined by SEM alone, because there is no appropriate immunocytochemical method for specific labelling of its cis- or trans-faces. In the present study, we used the osmium impregnation method, which forms deposits of reduced osmium exclusively in the cis-Golgi elements, for preparation of specimens for SEM. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically visualised the cis-elements of the Golgi apparatus, with osmium deposits that were clearly detected by backscattered electron-mode SEM. Prolonged osmication by osmium impregnation (2% OsO4 solution at 40°C for 40 h) and osmium maceration (0.1% OsO4 solution at 20°C for 24 h) did not significantly impair the 3D ultrastructure of the membranous cell organelles, including the Golgi apparatus. This novel preparation method enabled us to determine the polarity of the Golgi apparatus with enough information about the surrounding 3D ultrastructure by SEM, and will contribute to our understanding of the global organisation of the entire Golgi apparatus in various differentiated cells.Lay Description
The Golgi apparatus consists of several layers of flattened cisternae with distinct polarity. Its entry and exit sides are termed the cis-faces (or forming) and trans-faces (or maturing), respectively. The 3D fine structure of the Golgi apparatus, as well as the rough endoplasmic reticulum and mitochondria, can be observed by scanning electron microscopy (SEM), after the soluble cytoplasmic proteins have been selectively removed from the cells by immersing the tissue samples in diluted OsO4 for several days (osmium maceration method). We have analysed the 3D organisation of the Golgi apparatus in various differentiated cells by high-resolution SEM of osmium-macerated tissues, and have demonstrated the diversity in the organisation of the Golgi apparatus. However, we have often faced difficulties in specifying the polarity of the Golgi apparatus, because there were no appropriate ways to label the cis- or trans-faces of the Golgi apparatus by SEM. To overcome this problem, in the present study, we used osmium impregnation for preparation of the specimens for SEM. Impregnation of OsO4 into tissue samples at a relatively high temperature (40°C) for several tens of hours resulted in accumulation of osmium deposits exclusively in the cis-Golgi elements. The newly developed procedure combining osmium impregnation with subsequent osmium maceration specifically labelled the cis-elements of the Golgi apparatus with osmium deposits. The intensity of backscattered electron (BSE)-mode signals depends on the atomic number of the specimen composition (osmium, atomic number 76). Osmium accumulated in the cis-Golgi elements could be clearly visualised owing to the intense signals of BSE-mode SEM, compared with the low background signals of the surrounding organic compounds. Prolonged exposure to OsO4 during the impregnation and maceration processes hardly damaged the 3D fine structure of the cell organelles, including the Golgi apparatus. This novel preparation method enabled us to analyse the polarity of the Golgi apparatus by SEM, with enough information about the surrounding 3D ultrastructure. The method will contribute to our understanding of the organisation of the entire Golgi apparatus in various differentiated cells that has rarely been achieved by transmission electron microscopy.