The simultaneous elemental analysis and observation of small surface areas and nuclei of urinary tract calculi was attempted with a conventional transmission electron microscope. This microscope was fitted with a scanning device, side-entry goniometer stage and an energy dispersive type X-ray microanalyzer unit coupled to a computer system. This method is advantageous in that in addition to quantitative analysis, a clear electron image may be observed. Apatite, whewellite and cystine calculi derived from five patients as determined by X-ray diffraction, were studied. In three samples of apatite calculus, calcium (Ca) and phosphorus (P) were detected, while magnesium (Mg) was observed in one case. In one sample of whewellite calculus, P as well as Ca was detected in the nucleus. In one sample of cystine calculus, Ca and P were detected in addition to sulfur (S). These results indicate that the crystal structure of calculi does not always consist of a single substance.
In order to improve immunohistochemical staining of standard paraffin sections, and to explore the possibility of a single procedure for both light and electron microscopic observations, the plastic embedding method was investigated. The rat pituitary glands fixed with Acrolein and embedded in Epon showed excellent light microscopic staining with the peroxidase-labeled antibody method on 1μm sections cut by a glass knife. Antigenicity of both peptide hormones (ACTH, prolactin, GH) and glycoprotein hormone (LH) was preserved even after osmification. Compared to the Zamboni fixed or Bouin fixed paraffin sections, this method resulted in much more sharply defined and better preserved positively stained cells. Furthermore, this method is especially useful in the neoplastic condition, i.e. pituitary adenomas, where fixation is extremely difficult. Ultrastructural observations of cells positively stained with this method revealed the presence of the immunoreactive hormones on the secretory granules and probably on the adjacent membranous structures. In considering these advantages, it seems probable that this method will replace paraffin sections in localizing pituitary hormones, and possibly other peptide or glycoprotein hormones with the peroxidase-labeled antibody method.
Epithelial cells from the weanling rat liver could proliferate easily if repeated colonial selection of the epithelial cells was performed in order to prevent overgrowth of the non-epithelial cells. The newly started two epithelial cell lines were studied ultrastructurally and cytochemically in order to examine whether or not these cells in vitro were hepatocytes. These cell lines possessed features common to other rat liver cell lines described in various studies. However, no definite evidence suggesting a hepatocytic origin was found, although some features were characteristic of the hepatocytes, especially those of the fetal rat. Cytochemically, these cells exhibited many types of enzyme activity, except for glucose 6-phosphatase. Cytochemical study of the cells in the primary culture suggested that the cell lines were not derived from the fully differentiated hepatocytes.
A simple procedure used to isolate epidermal cells for the cytofluorometric estimation of their nuclear DNA content is described. The principal part of this method involves the microdissection of fixed tissue sections as well as ultrasonic irradiation. The loss of Feulgen-stainable nuclear DNA during treatment with ultrasonics, using lymphocytes and spermatozoa in control experiments, was estimated at approx. 2% of the original content. In using this method for determining the nuclear DNA content of human epidermis, it was found that the presence of individual cells was maximal in the stratum basale and minimal in the uppermost layer of stratum granulosum.
The localization of lipid droplets within the liver lobules of normal mice was studied. In 40μm thick sections of liver lipid droplets were localized mainly in mid- and centrilobular cells, showing more deposition in animals killed in the afternoon than in the morning. The accumulation and distribution of droplets in animals killed in the afternoon appeared similar to those in animals fasted from 10:00 A. M. to 4:00 P. M. Therefore, the increased amount of lipid deposition in animals killed in the afternoon is probably due to the length of time from the last feeding, i.e., relatively poor nutritional state caused by low intake of food in the afternoon. In 10μm thick sections, however, almost all cells showed no staining for lipid in animals killed in the morning, although a few small lipid droplets were seen mainly in the mid- and centrilobular areas in animals killed in the afternoon.
Fine structural changes in the hepatic microbodies of male adult rats treated with a newly developed hypolipidemic agent, gemfibrozil, were compared with results of clofibrate treatment. Both drugs caused a hepatic microbody proliferation and elevation of the catalase activity in the liver, in spite of their different chemical structures. A significant difference was observed, however, in the ultrastructure of the microbodies of the two drugs. An appearance of tubular substructure was evident in the matrix of hepatic microbodies due to the effect of gemfibrozil, in contrast to clofibrate which caused an occurrence of fibrous substructures.
The dithionite reduction method was shown to be an excellent one for removing iron from ultrathin sections, because it inflicts the least harm upon tissues and exhibits the strongest effectiveness for desiderization among several methods for removing iron from tissues reported until today. It is expected that ferritin or hemosiderin, if necessary, can be identified by using this procedure.
Electron microscopic localization of acid phosphatase (ACPase) activity was investigated in cultured human fibroblasts from cystic fibrosis homozygotes and heterozygotes and from normal subjects using the Teflon-treated coverglass method (8). Our data suggest the following: 1) ACPase activity was found in lysosomes, in strikingly well-developed Golgi complexes and occasionally in rough endoplasmic reticulum (RER), irrespective of cell lines. 2) The ACPase activity of the Golgi complex was localized in the cisternal portions arranged in parallel and also in the thick reticular portions which may correspond to the GERL (20). 3) No clear-cut differences were observed in the localization or in the intensity of the ACPase activity among cells from cystic fibrosis homozygotes and heterozygotes and from normal subjects. 4) Cells in the confluent stage showed a tendency to have more lysosomes and less-developed Golgi complexes than those in the preconfluent stage. 5) The increase in the lysosome number in the confluent stage is mainly due to the change of the characteristic membrane-bounded bodies into secondary lysosomes. 6) RER may directly supply ACPase to secondary lysosomes during their formation.