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Scanning Electron Microscopy

Abstract

With the recent developments of the specimen preparation techniques, intracellular organization has been observed three-dimensionally by scanning electron microscopy (SEM). A suitable preparation method is the most important factor for observing intracellular structures at high resolution. Since intracellular structures are usually hidden in the cytoplasmic matrix, SEM observation of them is impossible merely by cracking the fixed cell. To remove the fixed cytoplasm, an osmic maceration technique is the most effective method which is applicable to almost all kinds of specimen preparation. In our laboratory, we developed some methods for observing intracellular structures. Those are the O-D-O method, the A-O-D-O method, O-D-W method, freeze-polishing method, and so on. Since each specimen preparation method has merits and demerits, it is necessary to select the suitable method for each purpose. To observe the intra-cellular membranous structures such as endoplasmic reticulum and mitochondria, the A-O-D-O method is recommended. An exfoliating method by surface tension is effective to observe submembranous structures. The freeze-polishing method is applied for observing intracellular structures of thin materials such as mesothelial cells or cultured cells. By these methods, some new findings on the three-dimensional architecture of the intracellular organelles were obtained. On the surface of sarcoplasmic reticulum ribosomes were sometimes attached forming spiral polysomes. In Golgi apparatus, the cisternae were composed of compiled cisternae which showed a whorl-like appearance seen from above. Although the newly revealed findings must be investigated further in the near future, it is obvious that three-dimensional cytology by high resolution SEM is emerging.

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