For comparison with chemically fixed osteoclasts, we prepared chick osteoclasts by quick freezing followed by freeze-substitution. In spite of technical difficulties this demonstrated that osteoclasts can be satisfactorily frozen in situ by the metal contact method. Ultrastructural differences were revealed between conventional fixation and quick freezing. Compared with conventional fixation, the quick freezing method appeared to improve preservation : (1) a discrete trilaminar plasma membrane and other intracellular membranes showed a smooth profile without undulation or rupture ; (2) cytoskeletal components appeared to be clearer, straighter, and more numerous ; (3) the interior of the ruffled finger contained interconnected lattice structures whereas highly organised microfilaments were seen in the clear zone ; (4) well developed tubulovesicular structures (TVSs) that branched or anastomosed with each other were revealed in the cytoplasm ; (5) the contents of intracellular membrane systems including the nuclear envelope, endoplasmic reticulum, and Golgi complex were stained to a various extent ; (6) vesicles and vacuoles were much smaller, round and well-defined with electron-dense contents ; (7) crystalline structures were seen at the extracellular channels of the ruffled border, in the lumen of TVSs, and in vesicles ; (8) in some instances mitochondrial granules were visible ; (9) within the resorptive lacuna, osteoclasts adhered to the degraded bone matrix without any intervening empty space.

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: In the present study, the initial dentine forming areas were stained with ruthenium hexammine trichloride (RHT) and examined under an electron microscope , consequently, the following conclusions were obtained. 1) Basal lamina had a marked affinity for RHT , in particular, and lamina lucida reacted intensely rather than lamina densa.2) The intercellular spaces between preodontoblasts were filled with electron opaque materials which were intensely stained with RHT and appeared almost homogeneous.On the other hand , RHT-positive granules, approximately 30 nm in diameter, from which faint filamentous structures extended radially, were scattered in the predentine matrix. Some of them were situated on the surface of collagen fibrils in the periodic array.3) Large amounts of RHT-positive materials were attached to the outer surface membrane of matrix vesicles.

Abstract:In the present study,the precipitate formed in situ by the antimonate -osmium mixed fixative was examined using both energy dispersive X-ray microanalysis(EDX)and electron energyloss spectroscopy(EELS)in order to elucidate its chemical composition .EDX spectrum from the antimonate reaction product suggested the presence of complex Ca and Sb .However,since the peaks of Ca and Sb overlap considerably,it was difficult to identify both simultaneously .On the other hand, EELS spectrum clearly indicated the presence of Ca(L2,3,350,346eV)and Sb(M4 ,5,537,528eV)in the electron dense precipitate within secretory granules of preodontoblasts and on the inner face of the plasma membrane of preameloblasts.Thus,EELS analysis is very useful to confirm the presence of Ca and Sb in the antimonate reaction product.