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The three-dimensional architecture of the autonomic nerve terminals in the rat exocrine pancreas was investigated by scanning electron microscopy using the HCl-digestion method as well as by transmission electron microscopy. Unmyelinated nerves presumed to be autonomic in nature were found in networks covering the outer layers of arterioles and their capillary extensions, with nerve fibers often leaving the capillaries to surround acini in the interacinar spaces. Schwann cells formed scaffolds for axons of the networks. No other distinct type of cells such as the so-called interstitial cells of Cajal were found to be associated with the formation of the networks. Although nerve fibers of the networks were locally in close association with the walls of the blood vessels or with the bases of acinar cells, no specialized axonal contacts with these tissues were found. However, local swellings, presumably varicosities, were observed by scanning electron microscopy on the surface of nerves. These findings suggest that the networks of unmyelinated nerves represent terminal apparatuses of the autonomic nerves in the pancreas. Schwann cells in the terminal networks were characterized by an unusual abundance of cell organelles. These "terminal Schwann cells" well correspond in location and reticular extension to the "interstitial cells of Cajal" as demonstrated by silver impregnation and vital methylene blue staining. The occurrence of well developed Golgi apparatuses, rough endoplasmic reticulum, and numerous ribosomes suggests that the cells are specialized Schwann cells which most likely require high levels of cellular activity in order to maintain their elaborate cytoplasmic processes extending along the terminal networks, and also to sustain the specific functions of axon terminals.

The three-dimensional morphology of the surface of myelinated nerve fibres in the mouse sciatic nerve was studied by scanning electron microscopy after combined potassium hydroxide treatment and collagenase digestion (to remove the surrounding collagen fibrils and basal laminae from nerve fibres) as well as by transmission electron microscopy. The myelinated nerve fibre appeared as a long cylinder with sporadic annular constrictions corresponding to the nodes of Ranvier. Slight swellings of the surface due to Schwann cell nuclei were usually found at the middle of each internode. The surface of the nerve fibre clearly exhibited a network of bulges, which consisted of longitudinal bands extending from the nuclear swelling to the nodes of Ranvier through the internode, and transverse trabeculae bridging between these longitudinal bands. These bulges on the surface of nerve fibres were the site of the retained Schwann cell cytoplasm external to the myelin lamellae. These cytoplasmic networks on myelinated fibres presumably corresponded to the networks described by Cajal following silver impregnation. In addition, other thin elevations and focal round swellings were also found associated with these longitudinal bands and transverse trabeculae. These networks of Schwann cell cytoplasm are considered to be cytoplasmic channels for nutrition. The two apposing paranodal bulbs of nodes of Ranvier were often asymmetrical in their structure. The networks of the paranodal region were more complicated than those in the internode. The networks of Schwann cell cytoplasm converged into a continuous circumferential collar toward the node, which in turn gave rise to finger-like projections into the nodal gap.

Mechanoreceptive sensory corpuscles (murine Meissner corpuscles) in the toe pad skin of mice, consisting of axon terminals and lamellar cells, were studied following freeze-substitution in order to clarify the plasma membrane relationships between axon terminals and lamellar cells. Tissue preservation of corpuscles was excellent when the corpuscle was located within 10 microns from the contact surface with the precooled metal block. The axolemmata appeared more electron-opaque than did plasma membranes of lamellar cells. The inner leaflet of the unit membranes was thicker than the outer leaflet in the axolemma, and the contour of cell plasma membranes was relatively smooth and straight. Characteristic focal or regional approximations of plasma membranes were noted between the axon and abutting lamellae. Such membrane appositions resembled gap junctions, although no gap junctions were found between the axon and lamellae in chemically fixed materials. Similar gap junction-like close appositions of plasma membranes also were found between neighboring lamellae. These approximations occurred more frequently than typical gap junctions seen in chemically fixed materials. These findings indicate that there may be a relationship of the plasma membranes in the axon terminals and in abutting lamellae as well as between neighboring lamellae that have not been identified as yet in conventional chemically fixed material. Another striking finding was that basal laminae on lamellar cells exhibited the same electron opacity as the surrounding connective tissue matrix and thus the two are indistinguishable from one another. Furthermore, the lamina lucida was not evident, and basal lamina material was directly contiguous with the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

A cell-maceration/scanning electron microscope (SEM) method was employed to demonstrate the arrangement of the collagen fibrillar network of various tissues. Immersion of fixed tissues in NaOH (25 degrees C) for 3-7 days, followed by rinsing in distilled water successfully removed the cellular elements, exposing collagen fibrils which were identified as such by transmission electron microscopy in their natural locations. SEM observations of the preparations are able to demonstrate the three-dimensional architecture of collagen fibrils much more precisely than other methods, including the silver impregnation method. Collagen fibrils, forming sheaths for housing individual cardiac myocytes, fused together, thus ensuring an equal stretch of contiguous myocytes and preventing the slippage of adjacent cells. Individual skeletal muscle fibers and nerve fibers were ensheathed by the meshwork of collagen fibrils running in two opposite helices. Such structures seem to play an important role in resisting the stretching impetus. At the epithelial-connective tissue junction of the tongue and fingertip skin, interwoven collagen fibrils formed numerous microridges which probably provide a broad anchorage for the epithelium. In the intestinal mucosa, the collagen fibrillar network immediately below the basal laminae of the villous epithelium possessed heterogeneous pores. As the collagen fibrillar network shows morphological features specific to individual organs and tissues, it is suggested that such formations not only constitute the skeletal framework but also provide those cells which are housed there with a microenvironment suitable for their activities.

The present study deals with the localization and ultrastructure of S-100-immunoreactive cells in the human thymus. These immunoreactive cells are distributed mainly in the medulla with some scattered elements in the cortex. Electron-microscopic observation revealed that the cells are characterized by an irregularly shaped nucleus, tubulovesicular structures in the cytoplasm and characteristic interdigitations of the plasma membrane. The cells often embrace lymphocytes with their branched processes. On the basis of these morphological features, the immunostained elements were identified as interdigitating cells (IDCs). The immunocytochemistry for S-100 visualizes the precise distribution and extension of the IDCs under the light microscope and indicates that the IDCs form no structural networks such as those established by the thymic epithelial cells. Since the IDCs in human lymph nodes have also been reported to contain S-100-like immunoreactivity, S-100 protein can be regarded as a useful marker for identifying the IDCs in the human thymus and other lymphoid organs.

The fine structure of the stratified lamina fenestrata of the mouse glomerulus was described in detail in both transmission and scanning electron microscopy. We propose to name the extremely developed structure of the stratified lamina fenestrata as the alveolus endothelialis. It consists of numerous small irregular spaces partitioned by thin cytoplasmic processes. Individual spaces communicate with each other through the fenestra surrounded by the cytoplasmic partitions. This structure occurs not only on the basal lamina but also on the perikaryal portion of the endothelium. The constant appearance of the alveolus fenestratus without association of any pathological or degenerative changes suggests that this structure represents a certain general and fundamental feature of the fenestrated endothelial cell. Possible mechanisms for the genesis of this structure are briefly discussed.

The microvascular architecture of the human corpus cavernosum penis was studied by scanning electron microscopy of vascular corrosion casts. The corpus cavernosum was supplied by the penile deep artery. It gave off branches to become either arteries distributed within the corpus cavernosum or those directly supplying the corpus spongiosum urethrae. The former arteries further divided into small arteries which fell into two categories: 1) arteries breaking up into capillaries, and 2) arteries draining directly into the cavernous sinuses. The capillaries were collected into venular networks just beneath the tunica albuginea (the subalbugineal venular plexus), while the cavernous sinuses were collected into venules at the periphery of the corpus cavernosum. These postcavernous venules also received venules from the subalbugineal venular plexus, and left the corpus cavernosum. Thus, two circulatory routes are evident within the corpus cavernosum. These findings suggested that the penile erectile cycle is controlled by hemodynamic changes between these two routes within the corpus cavernosum.

Microcirculation of the pancreas in the rat and rabbit with special reference to the islets was studied by scanning electron microscopy (SEM) of vascular corrosion casts, light microscopy (LM) of India ink-injected/cleared tissues, and intravital microscopy of in situ organs. The following observations were made: Approximately 10-20% of the total terminal arterioles supplied the islets, while the remainder directly supplied the exocrine pancreas. The vas afferens of the islets divided into sinusoidal capillaries with frequent U-shaped turns in the cortical A and D cell area of the islets, and their secondary branches supplied the core B cell area. Intravital microscopy confirmed that blood irrigated the cortex of the islets first and the core portion second. All islets observed possessed insulo-acinar portal vessels. About 60% of the islets in the rat possessed emissary veins leading directly into the systemic circulation, while in the rabbit, less than 5% of islets possessed emissary venules of small diameter. Thus, the well-developed emissary veins of the islets seemed characteristic of the rat, as compared with the rabbit and several other mammals examined previously. The insulo-acinar portal system seems to represent a short vascular route through which islet secretions are transported in high concentrations to the exocrine pancreas, there to exert their actions. The emissary veins of the islet seem to serve for the quick conveyance of insular secretions into general circulation. It is suggested that the pancreatic lobule is made up of subdivisions or microcirculatory units, each of which is supplied centrally by the insulo-acinar portal system, while peripherally the unit also receives direct branches of intralobular arterioles. The veins run the periphery of the unit. The occurrence of sphincters in the vas afferens and the emissary veins of the islets is suggested as being involved in the regulation of the islet blood flow.

The three-dimensional structure of the rat thymus was studied by combined scanning- and transmission electron microscopy. The thymus consists mainly of four types of cells: epithelial cells, lymphocytes, macrophages, and interdigitating cells (IDCs). The epithelial cells form a meshwork in the thymus parenchyma. Cortical epithelial cells are stellate in shape, while the medullary cells comprise two types: stellate and large vacuolated elements. A continuous single layer of epithelial cells separates the parenchyma from connective tissue formations of the capsule, septa and vessels. Surrounding the blood vessels, this epithelial sheath is continuous in the cortex, while it is partly interrupted in the medulla, suggesting that the blood-thymus barrier might function more completely in the cortex. Cortical lymphocytes are round and vary in size, whereas medullary lymphocytes are mainly small, although they vary considerably in surface morphology. Two types of large wandering cells, macrophages and IDCs, could be distinguished, as well as intermediate forms. IDCs sometimes embraced or contacted lymphocytes, suggesting their role in the differentiation of the latter cells. Perivascular channels were present around venules and some arterioles in the cortico-medullary region and in the medulla. A few lymphatic vessels were present in extended perivascular spaces. The present study suggests the possible existence of two routes of passage of lymphocytes into the general circulation. One is via the lymphatics, while the other is through the postcapillary venules into the blood circulation. Our SEM images give evidence that lymphocytes use an intracellular route, i.e., the endothelium of venules.

A backscattered electron (BSE) image was applied to the observation of freeze-cracked tissue blocks stained with the indirect immunoperoxidase method. On the cracked surface of the pancreas, the BSE image clearly revealed the immunostained B cell granules as bright spots. A pair of secondary electron and BSE images on a fractured surface are useful for the three-dimensional localization of antigens in cells.

Certain parts of the peripheral nervous system were observed by a modification of the KOH-collagenase method by Miller et al. (1982). The sciatic nerve of the mouse, and dorsal root ganglion, lingual muscle and jejunum of the rat were fixed over one day at room temperature in a fixative containing paraformaldehyde and glutaraldehyde. After fixation, specimens were treated with 5N KOH solution for 5-10 min at 60 degrees C, immersed in a collagenase solution for 3-5 h at 37 degrees C and processed for scanning electron microscopy (SEM). In adequately treated specimens, connective tissue matrices and basal laminae were completely removed without causing any severe tissue damage. The three-dimensional visualization of cellular elements of peripheral nerves was enabled with the following results: 1) Individual nerve fibers of the sciatic nerve were clearly exposed. Myelinated fibers were non-branching cords with local annular constrictions at the node of Ranvier, while unmyelinated ones branched and anastomosed with one another to form loose networks. The sites of accumulation of the Schwann cell cytoplasm swelled on the surface of the myelinated fibers. Mesaxons were visualized as longitudinal furrows. 2) Ganglion cells covered by satellite cells were observed in the dorsal root ganglion. The ganglion cells were covered by their own convoluted dendro-axonal processes, thus forming the initial glomeruli of Cajal. Satellite cells at the glomeruli extended many finger-like projections surrounding the dendro-axonal processes. 3) Motor endplates were observed in lingual muscles. Terminal Schwann cells (teloglia) at the endplate were clearly visualized: the round perikaryon extended cytoplasmic processes along the axonal branches within the endplate. The processes issued fine finger-like projections from their margins. 4) Vascular autonomic plexuses were clearly demonstrated in lingual muscles. Unmyelinated nerves branched and anastomosed to form elaborate nerve networks around the vessels. Neuronal processes and associated Schwann cells were identifiable at high magnification. 5) Submucous nerve plexuses in the jejunum consisted of numbers of ganglia and interconnecting strands of fibers which formed very complicated networks. These observations indicate that this modified KOH-digestion method is useful for the SEM study of the three-dimensional cellular organization of nervous elements in various tissues.

The present study demonstrates the applicability of backscattered electron images to the scanning electron microscope (SEM) observation of various biological specimens stained with heavy metals. Techniques of specimen preparation for backscatter imaging were introduced and results obtained by several staining methods were shown: silver staining for argyrophile fibers (Ishii's modification of Bielschowsky-Maresch's stain), Golgi staining for intracellular canaliculi of parietal cells, osmium-staining of intracellular lipid droplets, periodic acid-thiosemicarbazide-osmium staining for the detection of periodate-reactive mucosubstances and polysaccharides, and enzyme immunohistochemistry for bioactive peptides. Observation of stereo-pairs of backscatter images augments information on the three-dimensional localization of stained substances. Moreover, pairs of secondary emission and backscatter images of the same area clearly visualize the correlation between the surface structure and stained substances. Colored images by superimposition of secondary emission and backscatter information are also useful for an understanding of the orientation of the stained substances in three-dimensional structures. It is suggested that backscatter imaging can be widely used for various topochemical studies in three-dimensional extensions of biological specimens.