The age of ultrahigh-resolution scanning electron microscopy (SEM) began in 1985, when the UHS-T1, with a resolution of 0.5nm, was developed. Commercial instruments of the same or similar types followed rapidly. As instrumental resolution progressed, conventional specimen preparation methods became inadequate, and a number of new techniques were devised.
In this paper, detailed procedures for these preparation methods such as the CC plate method and heavy metal impregnation are described, together with precautions recommended for achieving ultrahigh-resolution. Some applications of the method to biological specimens are also reported. Morphological identification of immunoglobulins prepared from human blood was attempted, and although the identification was not completely successful this technique may yet come to be of use in the clinical examination of allergic or infectious diseases.
SEM images of complement, Clq, proteoglycan and the helical structure of double stranded DNA are shown, as also is the visualization of immunolabelled cell-surface receptors.

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The major task of biological high resolution SEM and TEM is to provide structural information for correlating structure and function. It is the only methodology with the inherent power needed to observe structures down to molecular dimensions within the context of complex biological systems. Specimen preparation and imaging techniques should therefore be directed towards the preservation and imaging of the smallest significant details in order to fully exploit this unique, integrating feature of biological electron microscopy, complementing the progress of the techniques used in cell biology, biochemistry, and molecular biology.

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Cell surfaces interface with a variety of environments and, as a consequence, cell surface properties are of considerable functional importance to the biological organism. SEM immunocytochemistry (SEM-IC) is one of a range of techniques used to analyse cell surface properties. A major goal of SEM-IC centres on extended survey or high-magnification morphological analysis of cell and tissue surfaces combined with molecular profiles of these surfaces as established by gold-labelling. The properties of colloidal gold make it the marker of choice for SEM-IC and a representative gold-labelling protocol is outlined. The SEM-IC gold-labelling technique has been applied advantageously to the analysis both of cell surfaces and of cytoskeletal and extracellular matrix elements: a tabulation of the main SEM-IC biomedical applications is given.
Illustrated examples demonstrate how SEM-IC provides a highly effective approach for analysis both of cell and tissue differentiation-maturation sequences, and of pathological change involving not only the entire tissue or cell surface but also minute changes in microdomain characteristics of the individual cell surface.
Steps in exploiting the technique of colloidal gold SEM-IC have been several-fold and include: use of backscattered electron imaging; accurate localization of gold particles by superimposition on topographical maps of the cell surface; and use of small (1-10nm) gold probes followed by silver enhancement in order to minimize steric hindrance. Factors under assessment include: use of low voltage SEM; BE imaging of samples coated with ultrathin metal films; and use of gold-labelled SEM-IC for direct quantification of the numbers of target molecules exposed on cell surfaces by automated image analysis of the digitized BE image.

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Previous investigations have elaborated on the necessity for dimyristoylphosphatidylcholine (DMPC) molecules to conform in a ripple phase at the temperature intermediate between the transition and pretransition in order to protect the hydrophilic-hydrophobic polarity of the bilayer. Present observations, in addition to the asymmetric Pβ phase of 11.1±1.3nm wave-length, demonstrate orthogonal corrugations perpendicular to the main wave direction. Whether this is a prerequisite for maintaining the molecular orderdisorder balance at such specific temperature, or it results from yet unknown source is unclear at present.

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This report introduces practical techniques and applications of complementary scanning electron microscopy (SEM). To identify the complementary structures at high magnification, we first made a montage pair of low magnification micrographs as a guide map. Consulting the map, we took complementary micrographs at high magnification. When taking a picture at high magnification, we drew the outline of the most prominent structures on a transparent plastic plate attached to the cathode-ray tube of a SEM. Then another picture of the complementary structures was taken after adjusting the complementary structures to the reverse image of the plastic plate.
We performed three applications of the complementary SEM: 1) complementary observation of the epithelial underside and lamina propria of the rat urinary bladder; 2) complementary observation of the fractured Golgi apparatus; and 3) evaluation of specimen drying methods.
After proper digestion of the rat urinary bladder using strong alkali, the epithelium was detached from the underlying lamina propria. On the basal side of the epithelium, the meshwork of grooves were visible. The observation of the corresponding lamina propria confirmed that the grooves were occupied by blood capillaries located in the uppermost part of the lamina propria.
The complementary observation of the Golgi apparatus was useful for understanding its three-dimensional architecture. The observation on both complementary fractured surfaces of the Golgi apparatus from the mouse lacrimal gland demonstrated the continuity of the Golgi stacks. It was also effective for observing both the cis and trans side of the same Golgi stack.
Complementary SEM was also useful for evaluating the specimen drying method. One fractured specimen obtained by freeze-cracking was dried by the criticalpoint drying (CPD) method, and another complementary specimen was dried by the t-butyl freeze-drying method (BFD). The precise comparison of the preservation of the endoplasmic reticulum of pancreatic acinar cells indicated that the BFD technique preserved the ultrastructures better than CPD.

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The addition of ethidium bromide during the last 2.5-3h of lymphocyte culturing restricted chromosome contraction and preserved the banding structure in scanning electron microscopy. Treatment of the chromosomes with trypsin and use of impregnation with osmium tetroxide and thiocarbohydrazide resulted in a structural preservation of high resolution quality.

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Results obtained by extracting human fibroblast cells for the study of cytoskeletal structures are compared. Cells grown in culture were treated with digitonin in contrast to other methods of detergent preparation using Triton and Saponin. The three dimensional intracellular network which resulted from digitonin treatment was found to be similar in appearance to the structures observed by high voltage transmission electron microscopy of untreated cells described as a microtrabecular lattice by PORTER and TUCKER (1981). Our results, obtained by high resolution, field emission scanning electron microscopy indicate that the microtrabecular lattice may indeed be one conformation of a dynamic cytoskeleton.

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Intussusceptive capillary growth represents a new principle for microvascular growth as described in the lungs of growing rats. According to this concept, the capillary network expands by the formation of slender transcapillary tissue pillars, which give rise to new vascular meshes. The process was first observed in Mercox casts of the lung microvasculature, which revealed the existence of multiple tiny holes with diameters around 1.5μm. Consecutive transmission electron microscopic investigation of serial sections demonstrated that the holes corresponded to slender tissue pillars (BURRI and TAREK, 1990). The corrosion cast technique thus appears to be an adequate screening method for intussusceptive growth.
In the present investigation, Mercox casts of various vascular systems, namely, those of the eye, submandibular gland, heart, liver, stomach, small and large intestine, trachea, kidney, uterus and ovary were prepared from rats aged between 4 and 9 weeks in order to screen them for the existence of the typical tiny holes representing tissue pillars. In all organs investigated, these structures were observed in various locations to a variable degree. They were mainly encountered within dilated vascular segments or at triple or quadruple branching points of the circulation. Even in capillary networks with a three-dimensional arrangement could these pillars be detected. Intussusception thus appears to be a principle of growth appertaining to many vascular systems.

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Microvascular beds and pericytes in the submandibular gland, thyroid gland and heart were studied by combined scanning electron microscopy and chemical digestion.
The submandibular gland had a relatively loose network of blood capillaries, the thyroid gland posessed a close-meshed network of capillaries, and those in the myocardium ran parallel to the long axis of myocardial cells. The thyroid gland exhibited the largest numbers of pericytes. Three types of pericytes could be distinguished by their shape and localization. Type I pericytes, which were confined to true capillaries, had a fusiform or polygonal cell body, a few long, slender longitudinal processes (primary ones) and short, fine circumferential processes (secondary ones). Type II pericytes, which were found in the arterial side of myocardial capillaries, were characterized by large, circumferential band-like processes completely encircling the vessel. Type III pericytes, which were seen on the venous side of thyroid and myocardial capillaries, had a flattened cell body and short, irregular processes. Type II and III pericytes appear to show an intermediate or transitional form between smooth muscle cells and typical pericytes (Type I).
Judging from the configuration of pericytes suggests that these are related to functions such as contraction as well as mechanical support.

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The three-dimensional ultrastructure of the autonomic nerve terminals in the lamina propria mucosae of the rat large intestine was studied by scanning electron microscopy using the KOH-collagenase digestion method as well as by transmission electron microscopy. Observations showed unmyelinated nerves in the lamina propria mucosae as well developed just above the muscularis mucosae and forming a twodimensional irregular network by anastomosing with one another. Each nerve in the network consisted of axons and Schwann cells. The axons were about 0.1μm in diameter and had local swellings (about 0.2-0.5μm diameter) along their course. Transmission electron microscopy revealed the presence of synaptic vesicles in these varicosities. Although some of the axons left the Schwann cell processes to run separately, most of them twined around or were embedded in the Schwann cell bodies or processes. Blind ends of axons were only occasionally observed in the nerve network. These findings suggest that the network of the unmyelinated nerves itself represents a terminal apparatus acting upon the muscular and glandular cells.

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The portal vein was investigated in male spontaneously hypertensive rats (SHR), from Wistar-Kyoto (WKY) and Wistar strains, in animal 16-20 weeks old. In SHR, the inner circular smooth muscle was unchanged, but the outer longitudinal layer showed marked alterations in shape and size, readily observed in three-dimensional micrographs using scanning electron microscopy. The cells in both Wistar and WKY were elongate and tubular with little variation along their lengths and with a relatively smooth sarcolemma. This applied to both the inner and outer layers of smooth muscle. In contrast, the smooth muscle cells from SHR in the outer layer varied considerably in thickness along their lengths, and had very irregular outlines with numerous pits or depressions of varying sizes. In addition, the cells frequently had major forks or branches. The vasa vasorum running through the muscle layer, fibroblasts and nerve bundles were also indentified. Sectioned material (transmission electron microscopy) showed a change in shape and hypertrophy of the smooth muscle cells from the portal vein of SHR, and also demonstrated a significant increase in paracellular connective tissue in the outer layer of smooth muscle. Such major morphological alterations in the outer layer of smooth muscle in the portal vein from SHR could have profound effects on functional studies.

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This paper reviews scanning electron microscopic observations of cellular elements forming various lymphoid organs.
The reticular cells in the secondary lymphoid organs are stellate, smooth-surfaced forms extending slender processes to comprise a three-dimensional network. The reticular fibers are usually covered by reticular cell processes, though they are naked in certain regions. Other types of reticular cells are observed in certain places: the “retothelial” type in the lymphatic sinus of the lymph nodes, and the “follicular dendritic” type in the germinal center of various lymphoid organs.
The thymic epithelial cells are divided into two main types: stellate cells which form a three-dimensional meshwork throughout the thymus parenchyma; and large vacuolated cells located in the medulla. A continuous single layer consisting of the processes of the stellate epithelial cells separates the parenchyma from the connective tissues of the capsule, septa and vessels.
The M cells in the epithelium of the gut-associated lymphoid tissues (GALTs) are cells with numerous irregular microprojections on the luminal surface. They often attach microorganisms to the luminal surface, reflecting their functions of antigen transport into the underlying lymphoid tissue. Lymphocytes of various shapes often cluster in the intercellular spaces under the M cells, a phenomenon believed to indicate direct stimulation of lymphocytes by certain transported substances.
Macrophages are amoeboid cells independent of and unable to transform into reticular and endothelial cells, at variance with prerequisites of the reticulo-endothelial system concept. Multiple features of macrophages probably reflect the presence of the subpopulations as well as the phases of their activity. The interdigitating cells (IDCs) are clearly distinguished by macrophages since they possess characteristic knob-like cytoplasmic processes gearing with each other. The IDCs often embrace lymphocytes, suggesting that the former cells may either nurse or deliver certain immunological infomation to the latter.
The endothelial cells in the postcapillary venules (high endothelial venules) in the secondary lymphoid organs (except the spleen) are dome-shaped and provide a special route for the circulating lymphocytes entering into the lymphatic parenchyma. The postcapillary venules in the thymus are composed of flattened epithelial cells and provide a specific site for lymphocytes “educated” in the thymus to pass into the general circulation.

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The cytoarchitecture and distribution of myoepithelial (mecs) and basal (bc) cells of intralobular (intercalated, striated) and interlobular (excretory) ducts of human major salivary glands were studied by SEM through a variety of maceration and microdissection techniques. Intercalated ducts are covered by mecs which, unlike the large stellate cells of acini, are spindle shaped. Small star shaped mecs are rarely observed even in the most distal striated ducts, while no such cells are seen in excretory ducts. Basal cells form a more or less continuous row of small, basally placed cells in excretory ducts. Sparse bc are occasionally present in proximal striated ducts as well. Following microdissection, bc exhibit a cup-shaped apex which embraces the convex base of principal cells. This configuration may explain why, in TEM, bc seem to possess lateral processes which may be mistaken for mecs processes. Moreover, the lateral surfaces of be do not exhibit the complex system of plasmalemma folds typical of principal cells.
The present study demonstrates that fusate mecs are present in intercalated ducts and that basal cells are distinct from myoepithelial cells. These results may have some relevance in histogenetic studies of salivary gland neoplasms.

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The celiac disease syndrome is characterized by structural and ultrastructural alterations of the small intestine mucosa. According to criteria by European Society of Paediatric Gastroenterology and Nutrition, the conclusive diagnosis of celiac disease in children depends on the demonstration of histological relapse of the mucosa after reintroduction of gluten in the diet, as this syndrome is a permanent condition of gluten intolerance. Under these diseased conditions, the structure of the intestinal villi has been studied by light microscopy; morphological alterations were revealed only when the gluten challenge induced a clinical relapse.
Scanning electron microscopy analyses of the intestinal mucosa in celiac diseased patients showed a strikingly uniform destruction of the villi with changes in their dimensions and arrangement. At high magnification the enterocytes were irregular in size and shape with a decrease and disruption of the glycocalyx. Reductions in length and density of microvilli were also clearly identified.
Although these scanning electron microscopy findings could not demonstrate a relationship between the degrees of mucosal atrophy and the duration of the gluten challenge, they nevertheless revealed early stages of fine villous alterations that cannot be detected by the presently employed low resolution light microscopic techniques.

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In the rat, the forestomach is separated from the glandular stomach by a fold of the forestomach mucosa which generates the “limiting ridge” on the inner surface of the organ. This ridge overlaps a deep groove which is flanked proximally by the forestomach and distally by the glandular stomach. Light microscopy and scanning electron microscopy reveal that the keratinized squamous epithelium of the forestomach merges into the columnar epithelium of the glandular stomach at the bottom of the groove. Among the columnar cells of the distal wall of the groove are numerous brush cells. A remarkably thick lamina muscularis mucosae extends deep into the ridge. The peculiar architecture of the “limiting ridge” and the presence of numerous brush cells in its distal wall suggest that the region not only represents the transitional zone between forestomach and glandular stomach but that it might have a more specific function.

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The unique ultrastructure of the epithelial cells that line the entire length of the proximal tubule have been described. In particular, scanning electron microscopy has been used to illustrate the varied nature of the apical, lateral and basal surfaces of cells located along P₁, P₂ and P₃. The functional implication of the highly specialized microanatomy of the basal cell surface remains an interesting area of investigation.

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The three-dimensional fine structure of cells composing the renal tissue was demonstrated by SEM after the removal of extracellular matrices by NaOH maceration. This paper focuses on glomerular mesangial cells, Goormaghtigh's cells (extraglomerular mesangial cells), and epithelial cells in the thin limbs of Henle's loop in the rat, rabbit and dog.
Mesangial cells reveal rough surfaces covered with short microvilli. The cells extend long branching processes in close association with the glomerular capillary, suggesting a role for them of regulating the capillary caliber. The mesangial cells interdigitate with each other by their microvilli, forming an intercellular labyrinth. Goormaghtigh's cells at the glomerular hilus are also covered with microvilli, which form narrow labyrinthine spaces between the cells. The labyrinth among the mesangial cells and that among Goormaghtigh's cells connect with each other at the hilus, giving rise to a channel system leading from the periphery of the glomerulus through the hilus to the interstitial space outside the glomerulus.
Renal tubule cells display complicated intra- and intercellular interdigitations on the basal aspect. The pattern of epithelial interdigitation is specific to each tubular segment. The descending and ascending thin limbs of the long loops demonstrate a striking contrast to each other. The former is characterized by moderate intercellular interdigitation and by numerous microvilli on the lateral and basal surfaces; the latter is marked by elaborate, pectineal interdigitations, and by smooth basolateral surfaces.

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We investigated by transmission and scanning election microscopy the interface between the epithelium and lamina propria in the rat urinary bladder. A digestion technique that dissolves the basal laminae and collagen fibrils was effective in cleaving the mucosa at this level; the specimens were then prepared for scanning electron microscopy, thus visualizing the basal epithelial surface and the uppermost surface of the lamina propria. The underside of the epithelium is scored by very numerous grooves which in the intact organ are occupied by a dense network of blood capillaries. These vascular grooves allow a large number of capillaries (epithelial capillaries) to run at a distance of a few tenths of a micron from the epithelium. On the side of the lamina propria, after collagen and other extracellular materials had been removed, the capillary network itself is visible in the uppermost region. The network is complementary to that of vascular grooves. Other smaller grooves on the basal surface of the epithelium correspond to nerve fibres which run within a few tenths of a micron from the epithelium.

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Atrophic changes in the basal portion of the seminiferous tubules caused by daily injections of estradiol have been found by scanning electron microscopy disclosed after a digestion technique using collagenase and trypsin
In the control rat, the smooth basal portion of the epithelium was composed of polygonal Sertoli cells and reticularly-arranged ovoidal spermatogonia. After estradiol treatment, there appeared wavy furrows and ridges, and many folded micro-wrinkles in the complicated basal portion of the seminiferous tubules. The wavy furrows and ridges were repeatedly formed and appeared just like a cornice. These structures ran in an orderly manner perpendicular to the longitudinal axis of the tubules. In contrast, many micro-wrinkles ran in parallel to the longitudinal axis. These two structures had a characteristically similar size and orderly arrangement. They are discussed in relation to the mechanism by which the seminiferous tubules shrink by injections of estradiol.

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Direct visualization of the three-dimensional architecture of the tubal musculature (myosalpinx) was made possible by a technique involving chemical digestion of interstitial connective tissue, followed by ultrasonic microdissection and final observations under the scanning electron microscope. The isthmic myosalpinx in the guinea pig, rabbit and in humans consists of muscular bundles that tend to lie longitudinally, circularly or oblique. The muscular bundles along the tubal wall change direction, branch and intermingle with one another, giving rise to an irregular network in which distinct layers are not readily distinguishable. In the ampulla, the muscle bundles form a very irregular three-dimensional network of fibres that follow different orientations. The authors suggest a primary role for such a structure in the random pendular transport of the gametes as well as in the denudation of the egg by deformation of the myotubal wall.

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The ultrastructure of the follicle-oocyte complex in rodents and humans was revealed by high resolution scanning electron microscopy (SEM) following the Osmium-DMSO-Osmium maceration method (TANAKA and NAGURO, 1981).
In primary follicles, the majority of oocyte organelles such as mitochondria and Golgi complex components are concentrated in a juxtanuclear area. In particular, many spherical mitochondria are oriented all around the nucleus. After maceration of the ooplasm matrix, most of these mitochondria appear intermingled with numerous microtubules (MT) and associated with many Golgi vesicles. Such a nuclear polarization of organelles, essential to the oocyte metabolism, might depend upon a MT activity. MT might guide mitochondria to gather in the perinuclear region and further maintain their close associated to the nuclear envelope. A similar relationship among microtubules, vesicular Golgi complex and mitochondria has been also observed when, in maturing oocytes, these organelles migrate and gather in other areas of the ooplasm.
A pattern common only to human developing follicles appears in the occurrence of long microvilli projected from follicle cells deep into the oocyte. These unusual microvilli running within the ooplasm are surrounded by several vesicles of the Golgi complex and endoplasmic reticulum, and often end close to the nucleus.
In the antral follicle, the microvilli of corona cells, directed toward the oocyte (after their full exposure through the chemical dissolution of the zona pellucida matrix) are extremely numerous (up to 70/cell), long (up to 7/10μm) and tortuous. They resemble epidydimal stereocilia, may be ramified and possess bulbous tips. In contrast, oocyte microvilli are thin and short. The“curly hair-like microvilli” of corona follicle cells and especially those terminating deep in the oocyte cytoplasm, besides being the expression of active exchange of nutrients, may serve to finely modulate oocyte maturation.

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In order to study the three-dimensional topographic arrangement of the oocyte and zona pellucida, follicular cells and follicle microvasculature, this study applied alcali maceration methods for tissue exposure, the ruthemium red-detergent method for the extracellular matrix visualization, and the vascular corrosion cast technique to rabbits, guinea-pigs and mice ovaries at different stages of follicular development.
Macerated samples showed a gradual differentiation of the oocyte surface. This, in primordial follicles, appeared rather smooth, but, with the follicular development, displayed a gradual increase of blebs and microvilli. The latter widely covered the surface of oocytes contained in large or mature follicles.
The outer surface of the zona pellucida showed numerous fenestrations, whereas the inner one was smooth. The ruthenium red-detergent method permitted a well detailed view of the filamentous texture of the zona pellucida.
The three-dimensional distribution of the contacts between oocyte and neighbouring follicular cells was clearly evaluated in macerated samples. Follicular cells of primary follicles were characterized by their short cytoplasmic processes reaching the oocyte surface. In secondary follicles, these processes issued secondary processes. In larger follicles, the secondary processes of the corona cells were much longer and thinner, and took a tortuous course to reach the oocyte surface, which ran among the numerous oocyte microvilli. This microvillous arrangement greatly increases contact between the oocyte and corona cells, and suggests a coordinated reciprocal control of the activities of both cell types. These data also showed that the spongy and filamentous nature of the zona pellucida is closely dependent upon the temporal differentiation and enormous increase in number of follicular cell projections and their ramifications.
Maceration revealed the theca cells surface. In smaller follicles these appeared as fusiform cells which resembled fibroblasts. In larger or mature follicles, many theca cells differentiated to possess morphological features of steroidogenic cells. In addition, these cells delimited a series of intercellular communicating lacunae, continuous with wide pericapillary spaces.
The gradual differentiation of the follicle towards a structure having an endocrinal role was further emphasized in vascular corrosion casts. A simple microvascular net made of thin capillaries supplying primary follicles was seen to transform into an elaborate sinusoidal network made of thick permeable capillaries, supplying mature follicles.

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The present scanning electron microscope investigation describes the morphological changes occurring on the apical surface of the guinea pig vaginal epithelium during the estrous cycle. Dramatic changes can be observed at estrus in which the surface of the vaginal epithelium is covered by a distinct layer of mucous cells.

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The manageability of the hair is dependent on the surface composition of the hair shaft. Exogenously caused damage leads to unmanageability by massive superficial defects in the hair cuticle. The symptoms of trichorrhexis nodosa and trichoptilosis are present, and superficial horny scales are bent up and curve under in tunnel-like fashions. Appropriate hair care can improve these defects. Endogenous damage to the hair leading to unmanageability is characterized by spiral furrows along the shaft. It can not usually be improved by the use of care products.
The examples presented in the paper demonstrate that exogenous and endogenous causes of unmanageability are characterized by different alterations in the hair shaft.

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The development of preparation techniques that include freeze fracturing provide an ideal method for studying the differentiation of plant tissues in the scanning electron microscope. This is illustrated with reference to tapetal development in Catananche caerulea, which has a plasmodial tapetum, and in Lolium perenne, which has a secretory tapetum.

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This paper reviews the cell-maceration/scanning electron microscopic (SEM) technique and its application in the study of human livers. The maceration of glutaraldehyde-fixed tissues with 2N-NaOH and water at room temperature effectively and consistently removes all the cells, thus exposing collagen fiber networks. SEM of the macerated tissues shows three-dimensional arrangements of collagen fibers more clearly than previously reported methods. High resolution SEM observations of macerated and non-macerated collagen fibrils of the rat tail tendon have revealed that both show similar cross-striated bandings that are determined by an alternate succession of elevated and depressed segments along the collagen fibrils, with a period of approximately 65nm. Three ridges have been observed in the nonmacerated collagen fibrils: two on the margins of the elevated segments and one at an intermediate point of the depressed segment. The macerated collagen fibrils show a straight arrangement with slightly wavy microfibrils.
The subendothelial spaces of Disse in the human liver contain abundant collagen fibers. There are some collagen fibers that stretch between adjacent collagen fiber sheaths in the subendothelial spaces of Disse, either forming a mono-layered network or coursing individually. The collagen fibers in the spaces of Disse are continuous with those in the liver capsule and in the Glisson's sheaths and with those around the central and sublobular veins. The collagen fibers in human livers form a network of the liver as a whole, thus constituting a hepatoskeletal system.

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Monomeric methyl methacrylate resin, supplemented with 1.0% benzoyl peroxide and 1.0% N, N-dimethyaniline, enables good vascular casts useful for scanning electron microscopy when the injected organs or tissues are promptly heated in a microwave processor. The details of this vascular casting and scanning method are described with a clear demonstration of the rat pancreatic blood vascular bed, in particular, its intralobular, extralobular and translobular insulo-acinar portal systems and other vascular routes, including the insulo-venous vessels, which are sinusoidal in nature.

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