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Biomedical subjects

A Mitsushima

Publications and source records attributed to A Mitsushima.

9 recordsLinked to original sources
PubMed1993

A simple method of chromium coating using a ferrotype plate for scanning electron microscopy.

A simple method of chromium coating was developed. A chromium ferrotype plate for drying photoprints was used as a target of a planar magnetron ion-sputter coater. As a result, a high quality chromium film was obtained on dried specimens at room temperature without cooling. The grain size, measuring 1-2 nm in diameter, was small enough for observations of ultrastructures such as F1 particles on the inner mitochondrial membrane. In routine studies under x 100,000, however, chromium coating was not recommended because of its low emission of secondary electrons.

Chromium↗
PubMed1991

Direct visualization of colloidal gold-bound molecules and a cell-surface receptor by ultrahigh-resolution scanning electron microscopy.

The structure of protein A-coated colloidal gold particles, and of macrophage cell-surface receptors conjugated with immunogold particles, was studied using an ultrahigh-resolution scanning electron microscope. Protein A, when conjugated with 15-nm gold, formed a coat completely surrounding the particle. Particles conjugated with both protein A and immunoglobulin G (IgG) were similar, but with additional protrusions formed by the IgG. IgG molecules directly bound to gold were resolved sometimes as complexes of three units, sometimes as more filamentous, V-shaped structures. On the cell surface of macrophage reacted with a monoclonal antibody to Mac-1 antigen (the murine C3bi receptor) followed by protein A-gold, gold particles were seen to be linked via the IgG to the receptor, visualized as a round granule.

Animals↗
PubMed1991

Application of high-resolution scanning electron microscopy to biological macromolecules.

The development of ultrahigh-resolution scanning electron microscopes (SEMs) has made the observation of biological macromolecules feasible, but adequate preparation methods have not yet been established. Although it has been possible to observe some molecules after they have been spread on a carbon substrate, this method has not proved suitable for other molecules which exhibit lower contrast, or are more susceptible to damage by the electron beam. In this study we have applied heavy-metal impregnation methods using phosphotungstic acid, uranyl acetate, or osmium tetroxide mordanted by tannic acid. In addition, contamination due to the electron beam was reduced by improving the vacuum in the specimen chamber, and by the use of a heated specimen stage. Using these measures, haemocyanin, ferritin, apoferritin, thyroglobulin and immunoglobulin M were successfully image. Ultrahigh-resolution SEM seems likely to become an important means for studying the morphology of biological macromolecules.

Animals↗
PubMed1990

The role of the vimentin intermediate filaments in rat 3Y1 cells elucidated by immunoelectron microscopy and computer-graphic reconstruction.

The three-dimensional arrangement of vimentin intermediate filaments (IF) was studied in 3Y1, rat fibroblastic cell line, to elucidate its biological role in the cell. While actin filaments were observed exclusively in the superficial part of the cell, vimentin IF were found to be abundantly present in the inside of the cell where microtubules were occasionally discovered. By whole-mount immunoelectron microscopy and computer-graphic reconstruction of serial thin sections, it was observed in more detail that vimentin IF are located very close to the nucleus, endoplasmic reticulum, and mitochondria. Vimentin IF were observed to be attached to these organelles laterally or terminally. Thus, we can reasonably assume that vimentin IF are major cytoskeletal structures deep inside the cell and that they play an important role in supporting the location of the organelles. This is the first report which has visualized the three-dimensional relationship between vimentin IF and the organelles of the cell.

Animals↗
PubMed1990

A preparation technique for observing cytoskeletons by high resolution scanning electron microscopy.

The three-dimensional architecture of the cytoskeleton in cultured fibroblasts was studied using a newly devised technique for revealing cell interiors and an ultrahigh resolution scanning electron microscope, the UHS-T1. Both the cytoskeleton and membranous structures such as the plasma membrane and endoplasmic reticulum were well preserved, and we could observe the relationship between both components. Actin filaments, intermediate filaments, and microtubules were identified by immunogold staining with 5-15 nm gold particles. Actin filaments, measuring 10 nm in diameter in material not metal coated, formed thick bundles (stress fibers), sheaths or meshworks. Just beneath the plasma membrane, actin filaments could be seen in a two-dimensional network, with fibers linked laterally to the membrane. Intermediate filaments, 12 nm in diameter in uncoated material, were observed mainly in the perikaryon. Microtubules (26 nm) and clathrin-coated vesicles were also clearly seen.

Actin Cytoskeleton↗
PubMed1989

Application of an ultrahigh-resolution scanning electron microscope (UHS-T1) to biological specimens.

In 1985 we developed an ultrahigh-resolution scanning electron microscope with a resolution of 0.5 nm. It is equipped with a field emission gun and an objective lens with a very short focal length. In this study we report a survey of some different preparation techniques and biological specimens using the new scanning electron microscope. Intracellular structures such as cell organelles were observed surprisingly sharper than those observed by ordinary scanning electron microscopes. However, at magnifications over 250,000 x, platinum particles could be discerned as scattered pebbles on the surface of all structures in coated materials. Using an uncoated but conductively stained specimen, we successfully observed ribosomes on a rough endoplasmic reticulum at a direct magnification of 1 million. In these images some protrusions were recognized on the ribosomes. Ferritin and immunoglobulin G were used as samples of biological macromolecules. These samples were observed without metal coating and conductive staining. The ferritin particles appeared as rounded bodies without any substructure on the surface and immunoglobulin G as complexes of three-unit bodies. In the latter the central body might correspond to the Fc fragment and two side ones to Fab fragments. We assume that ultrahigh-resolution scanning electron microscopy is an effective means for observation of the cell fine structures and biological macromolecules. It will open a new research field in biomedicine.

Animals↗
PubMed1986

Three-dimensional architecture of the Golgi complex observed by high resolution scanning electron microscopy.

In spite of many light and transmission electron microscopic studies of the Golgi complex, many important points remain unsettled. Recently, we have devised a method of observing the three-dimensional configurations of intracellular structures by scanning electron microscopy. We have used this method to study the three-dimensional structure of the Golgi complex in several tissues of the rat, paying special attention to three problems: the overall configuration; the spatial construction of each stack, and the relationship to neighbouring organelles. The interconnected stacks which form the Golgi complex are arranged in a manner characteristic of the particular cell type. In rat extraorbital lacrimal gland the stacks form an irregular network spread throughout the cell, while in anterior pituitary cells they form a hollow sphere. Continuities were seen between cisternae within a stack, and in one case a stack actually consisted of one helically wound cisterna. We also observed slender tubules connecting Golgi stacks to neighbouring rough endoplasmic reticulum. Vesicles, presumably transport vesicles, were frequently observed on the surface of the cisternae, most commonly at the cisternal rims. However the continuities among stacks and connections with cisternae and with the rough endoplasmic reticulum demonstrated here offer alternative routes for movement of substances through the cellular endomembrane system.

Animals↗
PubMed1984

A preparation method for observing intracellular structures by scanning electron microscopy.

In order to observe intracellular structures by scanning electron microscopy, excess cytoplasmic matrix must be removed from the fractured surface of cells. Previously we reported an Osmium-DMSO-Osmium method devised for this purpose. This method is very effective in revealing intracellular structures, but requires osmium tetroxide for initial fixation with some consequent disadvantages. In the present study, a revised Osmium-DMSO-Osmium method is reported, in which an aldehyde mixture is used as the initial fixative instead of osmium tetroxide. As fixation is carried out by perfusion in this revised method, better preservation of fine structures is achieved than by the original method, especially in the central nervous tissue which tends to suffer from post-mortem degeneration. Moreover this method can be applied to cytochemical studies of intracellular structures with a scanning electron microscope (SEM). In this study, acid phosphatase of lysosomes is demonstrated in a coloured SEM micrograph.

Animals↗