To examine charged sites on the peritoneal free surface, cationic and anionic colloidal iron methods were applied. Light microscopy of the human and mouse specimens using cationic colloidal iron staining at pH 1.5-7.3 and successive ferrocyanide treatment resulted in a distinct Prussian blue reaction on the free surface of the parietal, mesenteric and visceral peritonea at all the examined pH values. In transmission electron microscopy of specimens stained with cationic colloidal iron at pH 1.5, colloidal particles accumulated in a dotted fashion on the free surface of the mesothelial cells. At pH 7.3, colloidal particles were deposited as numerous fine strands (100-300nm in length), whose ends often attached to the luminal aspect of the mesothelial cell membrane. Anionic colloidal iron stain at pH 7.2 gave no deposition of colloidal particles on the mesothelial free surface. Priorly methylated samples lost stainability of the peritoneal free surface to cationic colloidal iron staining at pH 1.5 or 2.5, while methylated-saponified sections recovered it. Pretreatment with neuraminidase inhibited the cationic colloidal iron staining at pH 1.5 on the mesothelial free surface. These results indicate that the mesothelial surface anionic sites ionizing at pH 1.5 are mostly due to the carboxyl group of sialic acid. It is suggested that the peritoneal free surface substance stained with the cationic colloidal iron may be membrane-associated sialomucin, whose rich negative-charged sites may repulse each other to prevent peritoneal adhesion or to maintain peritoneal cavity.

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Normal intact and regenerating axon terminals up to 30 days after nerve crushing were studied in the rat flexor carpi ulnaris muscle by confocal laser scanning microscopy (CLSM) and electron microscopy using immunohistochemistry for protein gene product 9.5 (PGP 9.5).
The motor axons were intensely and homogeneously stained along their entire length. “Three dimensional” organizations of elaborate axon terminals were clearly demonstrated by reconstructing serial optical images obtained by CLSM in normal neuromuscular junctions. In the injured nerve, the earliest regenerating axons could be identified at endplate regions six days after nerve crushing as intensely immunoreactive thin processes which bifurcated in T-shape and formed delicate lace-like terminals. Such lace-like terminals were composed of fine thread-like portions 0.2-0.8μm in diameter and expanded portions of 2-3μm in diameter.
Electron microscopy revealed that all the axon terminals in the cytoplasm were stained almost homogeneously by PGP 9.5 immunohistochemistry up to their extreme tips. Axon terminals were in direct contact with the basal lamina of the postsynaptic folds, and showed occasional branching. The thin thread-like portions contained no mitochondria but only a few vesicles, where as the expanded portions, abundant mitochondria. And preterminal axons and some expanded portions were abutted by Schwann cells, while thin thread-like portions were exposed with no association with Schwann cells. Twenty to 30 days after crushing injury, regenerating motor axon terminals resumed their mature form in terms of branching elaborations and ultrastructural features.
Thus, CLSM of PGP 9.5 immunocytochemistry combined with electron microscopy was able to demonstrate the “three-dimensional” organization of elaborate axon terminals at high resolution in the normal and regenerating neuromuscular junctions. Using this technique, extremely fine processes of axonal terminals were identifiable at the earliest stage of reinnervation.

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Three-dimensional cytoarchitectures of the periendothelial cells of human cerebral venous vessels as well as a scalp vein were studied by scanning electron microscopy after removal of extra-cellular connective tissue matrices with a KOH-collagenase digestion method. Postcapillary venules appeared covered with pericytes, while collecting venules had stellate periendothelial cells which formed a basket-like network around the vessel wall. As the size of the venous vessels increased, the stellate periendothelial cells became flat in shape and dense in arrangement. Although they had some characteristics similar to the smooth muscle cells in surface structure, no spindle-shaped smooth muscle cells were recognizable even in the superficial cerebral veins. On the other hand, the scalp vein was densely covered with spindle-shaped smooth muscle cells running circularly. These findings suggest that the cerebral venous vessels cannot constrict strongly as compared with the scalp vein, but probably regulate blood volume by mildly changing the caliber of the vessels.

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The three-dimensional arrangement of the medial smooth muscle cells of human major intracranial arteries was studied by scanning electron microscopy after removal of extracellular connective tissue matrices with a KOH-collagenase digestion method. In the straight portion of the major arteries, the smooth muscle cells were arranged roughly circularly, whereas the arrangement was somewhat random in the vertebral, basilar, and internal carotid arteries. Groups of the longitudinal muscle cells were also found in the vertebral, basilar, and internal carotid arteries, but were absent in the anterior, middle, and posterior cerebral arteries. At a higher magnification, smooth muscle cells in these arteries formed anastomosed bundles about 5-30μm in diameter.
The smooth muscle cells in the dichotomous branching and the uniting portions were arranged circularly, but multidirectionally and longitudinally oriented smooth muscle cell groups were present in the facial and dorsal walls of the forked vessels. These multidirectional muscle cell groups were small in number in the portion where the major arteries gave off lateral branches at a right angle. So called “medial defects” were found at the crotch of the bifurcating region in two cases out of four. Smooth muscle cells near the defects tapered off toward the center of the defect where internal elastic lamina with oval fenestrations were exposed. However, no special arrangement of the smooth muscle cells was observed around the “medial defects” as compared with that in the ordinary bifurcating region.

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The in vitro cytotoxic reaction of leucocytes from the seawater teleosts gilthead seabream (Sparus aurata) and sea bass (Dicentrarchus labrax) against tumor target cells was studied by transmission and scanning electron microscopy. Head-kidney, blood or peritoneal exudate leucocytes were incubated with HeLa or B16 melanoma cells. After incubation, conjugates of leucocytes (effectors) and tumor cells (targets) were observed. Both the effector-target cell binding and target cell lysis steps of the cytotoxic process were characterized. Usually more than one effector was bound to the same target. The effectors exhibited ultrastructural features of either monocyte or lymphocyte cells. Monocyte-like effectors possessed an oval or kidney-shaped heterochromatinic nucleus and a few granules. They were flattened against the targets over a broad area or, occasionally, made several punctuated contacts. Lymphocyte-like effectors had a large, rounded or indented nucleus, numerous free ribosomes and occasional cytoplasmic granules. These effectors established spot contacts with the target. Contacts between monocyte-like and lymphocyte-like effectors were also recognizable. After incubation, some targets appeared virtually intact, while others had a smooth surface or showed cell processes and surface blebs. These ultrastructural changes in the targets are similar to those described as mediated by mammalian cytotoxic cells.

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The novel pituitary protein 7B2 has a widespread distribution in neurons and paraneurons and is known to be associated with endocrine disorders. In the mammalian lung, solitary neuroendocrine cells (NECs) and their innervated aggregates, termed neuroepithelial bodies (NEBs), are scattered along the bronchopulmonary tree; however, presence of 7B2 in these structures has not previonsly been reported.
Bouin fixed, paraffin embedded rat lungs were cut and stained for 7B2, using a polyclonal antibody and a streptavidin biotin immunoperoxidase method. The identity of NEBs was confirmed on adjacent serial sections by the immunocytochemical reaction for calcitonin generelated peptide (CGRP).
NEBs selectively labelled for 7B2 are distributed throughout bronchi, bronchioli and alveolar ducts, whereas NECs are always negative. Likewise, 7B2 immunoreactivity is not detected in intrapulmonary ganglion cells nor in nerve fibers. Immunoreactivity for CGRP, on the other hand, occurs in NECs, NEBs, nerve fibers and neuronal somata. Colocalization of both peptides is demonstrated in almost all NEBs.
This study presents the first immunohistochemical demonstration and morphological mapping of 7B2 in the rat lung. The finding that only NEBs are stained suggests biochemical and functional variations between NEBs and NECs as well as the absence of a role for 7B2 in the pulmonary nervous system.

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Morphodynamics of oocyte follicle cells association during the development of human ovarian follicles were studied by transmission electron microscopy and high resolution scanning electron microscopy including the ODO method.
For this study primordial, primary, growing preantral and antral follicles were systematically analysed in a total of 20 adult and fetal (3-8 months and at term) ovaries.
In early stages of follicle development (primordial and primary stages) the flattened and/or polyhedral cells, closely associated with the growing oocyte, project an increasing number of microvillous processes. These are in apposition with the oolemma, and form bulbous terminals presenting attachment zones such as zonula adherens, desmosomes and communicating junctions (gap junctions). “Focal contacts” between oolemma, and lateral microvillous extensions of follicle cells were also present.
Unusual forms of contact between follicle cell microvilli and oocytes in the early stages of growing primordial and primary follicles were also observed. These consist of long, thin extensions penetrating into the oocyte through deep invaginations of the oolemma. The aid of high resolution SEM of specimens subjected to the ODO method clearly reveals their 3-D arrangement within the ooplasm. They appear as long tortuous microvilli coming very close to the nucleus, and in their course are closely associated with a variety of organelles such as Golgi vesicles, endoplasmic reticulum membranes and nascent forms of smooth endoplasmic reticulum.
Using integrated observations by TEM and SEM, there may be as many as 3-5 “intraooplasmic processes” even in only one plane of fracture of an oocyte. Therefore, if the total volume of the oocyte and associated cells is considered, their amounts appear to be higher than previously reported. Thus, they have to be considered as normal devices of deep contact between the ooplasm and associated follicle cell extensions.
The presence of such structures within the ooplasm in early developing follicles well coincides with the great increase in volume of the oocyte. Although it is commonly believed that the activation of the growing oocyte may depend on the numerous contacts between the oolemma and follicle cells (mostly via gap junctions), the finding of these additional intraoocytic extensions suggests that they may in someway contribute to the initiation of growth in the human. In fact, these microvilli penetrate deep into the ooplasm, much like a sword in its sheath. After contacting numerous oocytes' organelles, they come close to the nucleus, where they might transfer, more extensively and easily, a variety of ions or molecules (as signals), including nutrients. In turn, they may mediate or integrate a parallel activation on specific oocyte organelles and their cohort of enzymes.
In later stages of follicle development (pluristratified and antral follicles provided with a thick and complete zona pellucida), these intraooplasmic microvilli were very rarely observed. In large antral follicles close to ovulation, the bulbous processes of follicle cells contacting the oolemma were noted, and the attachement zones (zonula adherens, desmosome) and gap junctions appeared very numerous. All these junctions disappear by being disrupted, at the time of ovulation, by active retraction of follicle cell extensions.
The long exposure of specimens to the ODO maceration method allowed full observation of the real 3-D surface pattern of follicular cells and their extensions, including those of the so-called corona radiata. In fact, through the chemical dissolution of liquor folliculi and zona pellucida, these cells, which were mainly pear shaped, showed a characteristic apical polarization of their numerous microvilli toward the oocyte. As a rule, these unusual microvilli measured 7-10μm in length and the apical surface of a single corona cell toward the zone/oocyte formed up to

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The effects of kanamycin, an antibiotic of the aminoglycoside group, on the auditory sensory epithelium of the budgerigar, Melopsittacus undulatus, were examined using both scanning and transmission electron microscopes. Results show that the threshold of the auditory brainstem response increased in birds treated with kanamycin 200mg/kg for 49 days. While the auditory sensory epithelium of the normal budgerigar consists of short and tall hair cells, and supporting cells, following kanamycin administration, the middle to proximal region of the epithelium of the inner ear showed degenerative changes, with the other parts remaining apparently intact. In the damaged region, the short hair cells were flattened, and the tall ones became heterogeneous in shape. Both types of cells contained many dense bodies in their cytoplasm; they were rounded in shape and homogeneously dense. Severely degenerated tall hair cells also contained many large vacuoles with heterogeneous contents. Because the dense bodies and large vacuoles were positive for acid phosphatase reaction, they were respectively judged to be primary lysosomes and secondary lysosomes containing degenerating cell debris. Most supporting cells in the impaired region were lower in cytoplasmic electron density, and their apical surface became enlarged in area. Some flattened short hair cells were situated on the apical part of the swollen supporting cells. This finding suggests that the short hair cells are pressed toward the scala media by the supporting cells.

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Major nerve trunks that supply abdominal viscera contain axons of postganglionic neurons that originate in the coeliac ganglion, superior mesenteric ganglion, inferior mesenteric ganglia, hypogastric nerve ganglia and the sympathetic chain ganglia. Using the retrogradely transported neuronal marker Fast Blue, we mapped the distribution of labelled nerve cells after application of the dye to either the superior coeliac nerves, inferior coeliac nerves, superior mesenteric nerve, colonic nerves or hypogastric nerves.
Distinctive patterns of nerve cell locations were associated with each nerve trunk. Within the coeliac ganglion, nerve cells that projected into the superior coeliac nerves were almost exclusively lateral, whilst neurons in the medial part of the coeliac ganglion and in the rostral pole of the superior mesenteric ganglion tended to project into the inferior coeliac nerves.
Nerve cells located around the emerging superior mesenteric nerve in the superior mesenteric ganglion contributed the majority of axons to that nerve. Cells in both poles of the inferior mesenteric ganglia contributed the majority of postganglionic axons to the colonic nerves, but some cells in the caudal pole of the superior mesenteric ganglion also projected into the colonic nerves.
Postganglionic axons in the hypogastric nerves originated from cells predominantly located in the caudal pole of the inferior mesenteric ganglion; however, cells in the rostral pole of the inferior mesenteric ganglia, the superior mesenteric, coeliac and hypogastric nerve ganglia also contributed some axons.
Nerve cells of sympathetic chain ganglia projected into each of the nerve trunks; they were distributed rostro-caudally according to the nerve injected.
The more rostrally located coeliac nerves contained axons mainly from the lower thoracic and upper lumbar ganglia, whilst the more caudal colonic and hypogastric nerves contained axons from the mid-lumbar ganglia. Thus a widespread distribution of sympathetic neurons projects into each of the major abdominal nerve trunks, with the ganglion from which the nerve emerges being the greatest contributor, and the region around the emerging nerve comprising the preferred location within the ganglion.

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Comparatively little is known about hostdefense activities in the fish heart. Investigations showed that intraperitoneally injected carbon particles are actively taken up by the cardiac endothelial cells of the medaka Oryzias latipes, but less so by those of the goldfish Carassius auratus and lemon tetra Hyphessobrycon pulchripinnis. (In vitro experiments confirmed these species differences in endocytic activities by these cells.) Electron microscopy revealed that endothelial cells of the medaka atrium have large cytoplasm with many organelles, and ingested carbon particles were observed within phagosomes of cardiac endothelial cells even at 4°C. Phagocytic cells, which apparently reside in the heart, were found in all the species examined. These cells were located on the endothelial cells and developed cytoplasmic processes extending toward the heart lumen and/or the intercellular spaces of the endothelial cells. The heart with its resident phagocytes is proposed to function as a host defense organ—at least in certain fish species.

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The atomic force microscope (AFM) was applied for the first time to embedment-free biological sections. Aldehyde-fixed tissues (kidney, liver etc.) of mice were postfixed with osmium tetroxide and cut into 500-700nm thick sections after embedding of the tissue block in polyethylene glycol (PEG); the sections mounted on glass slides were deembedded and critical point-dried.
The AFM images were collected in air from those tissue sections in a dynamic force mode. Solid-heightmode images, which were comparable to transmission electron microscope (TEM) images, seemed to provide us more useful information than the solid-mode images which resembled scanning electron microscope (SEM) images. Internal structures including chromatin fibers in the nuclei and cytoplasmic organelles were clearly recognizable by AFM. Minute surface structures including the end-feet of renal podocytes were demonstrated.
We confirm that a vertical resolution of 0.1nm and a lateral resolution of 5-10nm are attainable with the dynamic force mode of AFM using thin sections of tissues. The present paper proposes the usefulness of AFM for observation of biological materials without metal coating in a non-vacuous environment.

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