Two types of nuage aggregates, the mitochondria-associating nuage and the freely-existing nuage, appeared in the cytoplasm of the growing Chinese hamster oocytes in a growing stage-specific manner. Enzyme digestion studies on the ultrathin sections obtained from the glycol methacrylateembedded materials showed that both types of nuage contain RNA and proteins. The ethanol-phosphotungstic acid staining and also the cobalt thiocyanate staining used to examine the localization of basic proteins at the ultrastructural level revealed that the mitochondria-associating nuage contains a considerable amount of basic proteins, whereas only a few may be present in the freely-existing nuage.
It was considered that the mitochondria-associating nuage may lose its basic proteins and transform into the freely-existing nuage, along with the oocyte's growth.

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³H-thymidine autoradiography and acetylcholinesterase (AChE) histochemistry were performed on the same plastic sections to examine the origin of myotome cells in the chick embryo.
The myotome first forms in the dorso-medial angle of the dermomyotome and elongates ventro-laterally along the inner surface of the dermomyotome with development. The myotome consists of numerous AChE-positive cells and some AChE-negative cells.
Two hr after injection of ³H-thymidine, a few AChE-negative cells but none of the AChE-positive myotome cells were labeled. Four hr after the injection, a few labeled AChE-positive and AChE-negative cells were observed throughout the myotome and thereafter, the labeling of the AChE-positive as well as AChE-negative cells increased with time. Twenty-four hr after the treatment, many labeled cells were distributed throughout the myotome, but unlabeled ACNE-positive cells were also evident in the myotome.
In the present study, the proliferating ability and origin of myotome cells in the chick embryo are discussed.

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A cytochemical procedure for p-nitrophenyl phosphatase (p-NPPase) activity was applied to epiphyseal growth-plate cartilage in order to study maturational changes in some of the phosphatases present in this tissue. In the plasma membrane and matrix vesicles, two types of p-NPPases were observed. One was insensitive to aldehyde fixation, was located on the outside of the membrane, and diminished in potency as maturation progressed. The other was sensitive to fixation, occurred on the inside of the membranes, and was most intense in the hypertrophic zone. Matrix vesicles rich in p-NPPase activity contained crystals of mineral while those which had no activity did not calcify. In fixed samples, p-NPPase was also detectable in mitochondria, Golgi complex and lysosomes, while in unfixed samples the only non-plasma membrane location observed was in the endoplasmic reticulum. A variety of inhibitors with known specificities (ouabain, vanadate, NaF, duramycin, levamisole) had no effect on activity. K-dependency was not demonstrable. Consequently, it is not possible to assign the p-NPPase activity to a specific phosphatase. However, the p-NPPase activity is distinct from Na⁺, K⁺-ATPase, Ca-ATPase, K⁺, H⁺-ATPase and alkaline phosphatase. The results of this study provide further evidence for the plasma-membrane origin of matrix vesicles. Further, the phosphatase which hydrolyzes p-NPPase observed is associated with matrix-vesicle calcification.

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To compensate uneven section thickness in the cytophotometric determination of dehydrogenase activity demonstrated with nitroblue tetrazolium (NBT), the protein staining with naphthol yellow S (NYS) was superimposed and the amount of formazan was estimated as the ratio of tissue protein content instead of the direct correction of section thickness. The absorbance of superimposed section was measured at 700nm, 570nm and 450nm for the background amount of formazan, formazan and tissue protein, respectively. Then, the enzyme activity was expressed as a relative activity (RA) as follows:
RA=A570/(A450-A700)
where, A570 and (A450-A570) represent the amounts of formazan and tissue protein, respectively. The quantitative reliability was tested with the polyacrylamide gel film containing mouse liver soluble fraction. For rapid and semi-automatic determination of RA, a special microphotometer, tridensity-automicrophotometer (TRIDENT), was devised. The method with TRIDENT was applied to show the quantitative distribution pattern of succinate dehy-drogenase activity in various tissues. A quantitative topography has been revealed in various structural units of the liver lobule, gastric gland and muscle fiber for succinate dehydrogenase activity.

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An autostage cytofluorometry system for multiparametric analysis is described. The system consists of a compact epi-illumination cytofluorometer equipped with an autostage device, a small data processing unit, an autostage controller and a microcomputer. In this system, repeated cytofluorometry and morphological observations for the same series of cells can be made at predetermined cell positions (X, Y-values) on a slideglass, permitting accurate multiparametric cytofluorometry in the same cells, even for a mixed cell population. Fluorochromes can be applied in any combination, if they can be utilized sequentially by staining and destaining cells. Application experiments demonstrated that the present system allows detailed cell cycle analysis.

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So far quantitative fluorescence histochemistry has been carried out separately by two different methods, cytofluorometry for densitometry and image analysis for morphometry. We described here a method of quantitative fluorescence image analysis to realize simultaneous densitometric and morphometric measurements. This method gives complementary informations of quantity of substances of interest together with data on shape and pattern of the biological materials.
DAPI staining for DNA is employed. The fluorescence images are successively taken from SIT (Silicon Intensifier Target) camera and sent to an intelligent color terminal. The data obtained from the image analyzer are compared with cytofluorometric data. Integration technique can remove random noise from the image.
Brightness values of all pixels in the frame buffer or brightness of the pixels on nuclear area were summed up. Both parameters responded linearly and proportionally to fluorescence values determined by cytofluorometry within an adequate range. Nuclear DNA measurements revealed that rat hepatocytes are composed of diploid, tetraploid and octaploid subpopulations with proportional DNA contents. All cerebellar neurons showed DNA values strictly in diploid class.
The conclusion is that both densitometric and morphometric informations can be obtained simultaneously by quantitative fluorescence image analysis within an adequate dynamic range. This will enable us to extend quantitative investigation on histochemical reaction and shape analysis in variety of biological systems.

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A new principle to obtain sectional images of a thick specimen by a conventional optical microscope without physically slicing is developed. An optical microscope with an off-axis pupil is used to project a three-dimensional (3-D) distribution of a specimen in various directions within the numerical aperture of the objective lens onto a fixed TV camera position. The obtained images of the thick sample are combined to reconstruct its 3-D density distribution. Since the system is strictly angularly-limited, a strong constraint is needed to form the 3-D structure with a moderate spatial resolution from the projections. The conjugate gradient method with the object boundary constraint which is a priori known solves this problem. Experimental results with a biological sample verify the capability of the proposed method as a promising instrumentation of optical tomography in microscopic size.

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Fluorescence measurement of intensity, anisotropy and lifetime with Hoechst 33258 (H33258) and ethidium bromide (EB) fluorochromes were applied for structural study of DNA. The fluorescence of complexes of H33258 with DNA, chromatin and nucleosome core with and without NaCl were compared. The complex with the nucleosome core showed the lowest intensity of the three complexes, but the highest anisotropy in the absence of NaCl. At the higher NaCl concentration than 1M, the complexes with the chromatin and nucleosome core showed the same fluorescence as that of DNA complex. The anisotropic fluorescence with H33258 was applied for the determination of cell number suspended in a solution. A background fluorescence due to unreacted free dye was optically removed, without removal of the dye, through the polarized component of the fluorescence. The fluorescence decay measurement was carried out to determine the molar ratio of DNA- phosphorus to dye (P/D ratio) in situ in cell nuclei stained with EB. The fluorescence lifetime of DNA-EB complex increased with the increase of the P/D ratio.

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It has been examined using a video-intensified microscope photon counting system (VIMPCS) whether preferential digestion of chloroplast gemone of male origin in young zygotes of Chlamydomonas reinhardtii affects the chloroplast genomes of female origins in number. To distinguish between the chloroplasts of male origin and the chloroplasts of female origin in young zygotes, a cross, that is, mt⁺ (gametes with normal chlorophyll, 137c wild type)×mt^- (gametes with diminished chlorophyll, ac29a mutant) was prepared. The timing of initiation of the digestion of the male chloroplast genome differs among zygotes but usually occurs during 50-120min. The degrees of the preferential destruction of male chloroplast genomes in young zygotes were quantitatively examined using VIMPCS after 4′-6-diamidino-2-phenyindole (DAPI) staining. The results show that each gamete (mt⁺, mt^-) has a cu-pshaped chloroplast including 7-8 chloroplast nuclei (ct-nuclei). Each ct-nucleus (mt^-, mt⁺) is composed of 1-43 copies of the chloroplast genome (mean number, 10 copies) and female and male chloroplasts contain about 83 and 86 copies, respectively. In most zygotes, all copies of the chloroplast genome from the male gamete are completely digested during the first 40-180min after mating, while all copies from the female gamete persit till at least 4 days later when 7ct-nuclei have fused together to form one large ct-nucleus. This suggests that the preferential digestion of the chloroplast genome of male origin does not affect the chloroplast genome of female origin in zygotes.

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By using anionic and cationic iron colloid particles, which are Prussian blue reaction positive and 5-250nm in diameter, the surface charge-related endocytosis of rat macrophages was observed. The anionic particles were solely adhered to the surface of peritoneal and liver macrophages by exposing the cells for a short time period to the particles in vitro as well as in vivo by perfusion. The particles then were taken up selectively by macrophages, but not by neutrophils, eosinophils, lymphocytes, mast cells, fibroblasts and vascular endothelial cells, as observed by light and electron microscopy after intraperitoneal, subcutaneous and intravenous injections of the particles. The anionic iron colloid particles were taken up by macrophages, delivered to lysosomes and disintegrated quickly. Ferritin was first synthesized there and then dispersed into the cytoplasmic matrix. In contrast, cationic iron colloid particles were adhered to the surfaces of all kind of somatic cells and internalized by the process similar to adsorptive endocytosis. The ingested particles were found in thin canalicular structure or tiny vesicles being adhered to their luminal surfaces. The morphology of these compartment was much different from the phagosomes of macrophages formed by ingesting anionic iron colloid particles, and showed no deliverly of the particles to lysosomes as long as 4hr after the incubation. Some particles were found in lysosomes by another 5hr of chase incubation. The results indicate that the “non-specific receptor mediated phagocytosis” by macrophages is induced by the binding of anionic macromolecules to the cationic receptor on the cell surface. And the adsorptive endocytosis will induce two kinds of different mechanism, one is currently accepted concept, the receptor mediated endocytosis and another one is the surface charge-mediated endocytosis.

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Neutral α-glucosidase in mouse peritoneal macrophages was shown to be associated with granules distinct from lysosomes. When post-nuclear supernatants were centrifuged in a Percoll density gradient containing 20units/ml of heparin and 10mM Tris-HCl (pH 7.2), neutral α-glucosidase activity was clearly separated from β-glucuronidase activity. Activity peaks for alkaline phosphodiesterase, a plasma membrane enzyme, and for UDP-galactosyl transferase activity, a Golgi enzyme, were detected in different fractions. Release of the enzyme activities into extracellular medium depending on phagocytosis or pharmacological stimuli, was also different. These observations suggested that neutral α-glucosidase localize in nonlysosomal granules in macrophages.

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In order to confirm the relationship between prostaglandin synthesis and lipid peroxidation in the rat peritoneal macrophages, we have studied the immunocytochemical localization of glutathione-peroxidase (GSH-PO) in the rat peritoneal macrophages under zymosan, phorbol myristate acetate (PMA), ionophore A23187, testosterone, parabromophenacyl bromide (PBPB) and BW 755C treatment. In the untreated peritoneal macrophages, GSH-PO was predominantly observed in cytosol. In zymosan, PMA, ionophore A23187 and testosterone-elicited peritoneal macrophages, GSH-PO was mainly detected in cytosol as large granules. In contrast, the intensity of the GSH-PO staining in PBPB-elicited peritoneal macrophages were weaker than that of other experimental groups. In BW 755C-elicited peritoneal macrophages, GSH-PO was clearly observed in cytosol. Based on our findings, it is strongly suggested that GSH-PO in peritoneal macrophages may be playing an important role in reduction of lipid peroxides such as arachidonic acid hydroperoxides induced in the process of arachidonic acid cascade.

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