SUMMARY 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.

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.

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.

SUMMARY 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 a 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.

SUMMARY 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, thyro‐globulin and immunoglobulin M were successfully imaged. Ultrahigh‐resolution SEM seems likely to become an important means for studying the morphology of biological macromolecules.