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Scanning electron microscopy (SEM) using osmium-maceration methods has been used for analyzing the three-dimensional structure of cell organelles in tissue samples, but it has been quite difficult to observe free and cultured cells with this technique. The present study was performed to develop a method that can be applied to free and cultured cells for SEM studies of intracellular structures after osmium maceration. The method was also applied to light microscopy (LM) and to transmission electron microscopy (TEM). HeLa cells and human leukocytes were fixed with a mixture of 0.5% paraformaldehyde and 0.5% glutaraldehyde followed by an additional fixation with 1% osmium tetroxide. These cells were embedded in low-melting-point agarose. A temperature-responsive dish was also used for collection of cultured cells before embedding. For LM and TEM, the cell-embedded agarose was further embedded in epoxy resin, and semi- and ultrathin sections were examined conventionally. For SEM, the agarose was freeze-fractured in 50% dimethyl sulfoxide, processed for osmium maceration and observed in a high-resolution SEM. Low-melting-point agarose was useful as an embedding medium for SEM, because it was well preserved during prolonged osmication for SEM. Thus, the fine structure of cell organelles was clearly analyzed by SEM after osmium-maceration treatment. These SEM images could also be compared with those of LM and TEM of the agarose-embedded tissues.

Cathodoluminescence (CL) properties of various kinds of organic material were investigated for the purpose of staining biological specimens and obtaining CL images. Several kinds of organic light emitting device (OLED) material exhibited CL. The europium complex, Eu(dbm)3(phen), showed the strongest CL signal and was chemically modified for biological staining. However, the CL intensity from the stained biological specimen was too weak to build CL images. We discussed the CL properties of organic materials considering their chemical structure and charge distribution in the molecules.

To observe bone cells by scanning electron microscopy (SEM), the mouse parietal bones were processed by decalcification with EDTA and digestion of collagen fibers with KOH to remove the bone matrix, in addition to the conventional preparation for SEM. The critical-point-dried specimens were split into two membranous pieces along the gaps formed by removing the bone matrix. By this method, osteoclasts showing full three-dimensional images of ruffled borders, osteoblasts showing special structures on the surfaces facing the bone matrix, and osteocytes extending many slender processes were clearly demonstrated in SEM. This new method may provide new viewpoints in bone cell biology.

The present study examined procedures of specimen preparation for observing elastin components in the formic acid-digested tissue by scanning electron microscopy (SEM). The rat thoracic aorta fixed in 4% paraformaldehyde was treated with 90% formic acid for 85-96 hr at 45 degrees C and processed in two different ways: 1) the tissues were freeze-dried directly from water and 2) they were treated with tannic acid and osmium followed by critical point drying. The former procedure caused marked deformation of elastin components, while the latter preserved them well in shape and arrangement. Thus, the three-dimensional organization and ultrastructure of the elastin components were precisely demonstrated in this study.

The structures of the bony modiolus of the human cochlea are studied by scanning electron microscopy after macerating the organic material with a NaOCl solution. The modiolus on the scala vestibuli side had a canal for the spiral modiolar artery. Ducts for the radiating arterioles spread radially from the canal, and opened to furrows in the roof of the scala vestibuli. The surface of the modiolus facing the scala tympani was lace-like with numerous pores. Just beneath the bone surface were a modiolar canal for the spiral ganglion and spirally-arranged spaces for the anterior and posterior spiral veins. The spiral spaces for the veins had windows (fenestrations) leading to the scala tympani. The openings in the bone on the scala vestibuli and tympani sides of the modiolus are considered to be communication routes for the transport of fluids from the perineural and perivascular spaces in the modiolus to the perilymph in the scalae.

A predenervated sciatic nerve segment, which had been treated by repeated freezing and thawing to kill Schwann cells, was grafted to the original sciatic nerve in the rat. Three to five days later, the graft was chemically fixed and treated by KOH-collagenase digestion, a treatment which selectively removes almost all non-cellular elements including the collagen fibrils and basal laminae from the tissue, thus making it possible to observe regenerating axons by scanning electron microscopy. Debris of degraded Schwann cells and myelin sheaths remained in the form of "columns", and several thick (2-3 microns in diameter) and thin (less than 1 micron in diameter) axons ran singly or in bundles on such "cell debris columns." Thick axons have an almost straight contour, while there were various swellings at intervals along the thin axons. In most cases, the growing tips of regenerating axons were swollen as growth cones ranging from 2 microns to 5 microns in diameter. Growth cones exhibited fusiform to polygonal variations in structure and had only a few filopodial processes on the surface.