Contact guidance in vitro : A light, transmission, and scanning electron microscopic study

Contact guidance was studied in cultures of chick heart fibroblasts and kidney epithelium by observing the relation of these cells to fine grooves ruled in plastic culture dishes, and also to ridges or grooves in plastic replicas moulded from rulings made in metal. The relation of the cells to the regularly arranged collagen fibers of fish scales was also studied by scanning and transmission electron microscopy (SEM and TEM). On the rulings with groove periodicity in the range of 5 μm about 75% of the cells were aligned, but on grooves separated about 30 μm only 60% of cells were aligned. Cytoplasmic components of the cells such as microfilaments maintained a constant relation to the axis of the cell as a whole, but they, and also any cytoplasmic extensions, such as filopodia, bore no consistent relation to any features of the substratum, whether or not the cells were aligned. The cells were not guided to become aligned by filopodia or lamellipodia. The most remarkable and consistent finding was that cells bridged over grooves without contacting their surfaces, whether the grooves were 2 or 10 μm wide. The bridging was a characteristic of cells growing on any of the substrates, including those with grooves or ridges, and also of collagen substrates made from fish scales. A hypothesis is proposed to explain the contact guidance seen on ridged or grooved substrata and on the orientated collagen fibers involving the observed cell bridging and the fact that linear cell-to-substrate contacts (focal contacts) are known to be vital for cell movement. The cell is considered to be stiff so that as it bridges over much of the substratum there is only a limited area available for contact. Assuming that focal contacts need to be of a certain length to provide adhesion, a cell orientation that presents the maximum linear contact would be favoured. An examination of the results of this study and of the reports in the literature shows that cells on these types of substrata take on an orientation such that linear contacts would be expected to predominate.     

Tumor-basophil interactions in vitro--a scanning and transmission electron microscopic study.

Purified guinea pig basophils, or basophils either specifically degranulated with antigen or nonspecifically degranulated with lectin, were cultured with guinea pig line 1 hepatoma cells for 1 to 24 hr and studied ultrastructurally. As early as 1 hr of culture, degranulated or nongranulated basophils and tumor cells formed close contacts by mutually intertwined elongated cell processes and also in cultures containing degranulated basophils, extruded membrane-free basophil cytoplasmic granules became firmly attached to tumor cells. At later intervals, some tumor cells cultured with basophils exhibited cytostatic and cytopathic changes, including dense mitochondria, centralization of organelles, dilated perinuclear and rough endoplasmic cisternae, cell swelling and cytoplasmic lucency, disrupted cytoplasmic organelle and plasma membranes, nuclear pyknosis and fragmentation. Some tumor cell specialized surface attachments were either disrupted or damaged at points of basophil or basophil granule adhesion. Tumor damage was most extensive in cultures containing degranulated basophils, although only a minority of tumor cells (less than 10%) was affected. Tumor injury was seen much less frequently in the presence of nondegranulated basophils, and was absent in control cultures of tumor alone. The occasional viable tumor cells that phagocytosed basophil granules were apparently unharmed, suggesting that internalization of basophil granules by tumor cells is not cytotoxic.     

The vasculature of the rat penis: a scanning electron microscopic and histologic study.

As a basis for understanding the mechanism of erection in an animal model frequently used in research in reproductive biology, the angioarchitecture of the penis of the rat has been described using scanning electron microscopy. Study of the penile vasculature of the rat indicates that the corpora cavernosa penis and the corpus spongiosum are independent erectile tissues, each with its own arterial and venous vessels. The large vascular spaces and abundant smooth muscle of the penile crura are compatible with its role in regulating blood flow to more distal penile tissues. Helicine arteries of the crura, but not the parent deep penile artery or arteries elsewhere, have muscular cushions in their walls. The venous drainage of the penile crura is via subtunical veins which are thought to be compressed during erection to elevate pressure within the penis. Large, paired cavernous veins drain the shaft of the penis. A unique method for inhibiting blood flow from the penis is indicated by the division of the cavernous veins into smaller channels prior to joining the subtunical venous plexus. Erectile tissue in the bifid origins of the corpus spongiosum has abundant cavernous muscle, while in the remainder of the corpus spongiosum little smooth muscle lines the cavernous spaces. The cavernous spaces on either side of the urethra coalesce to form vessels, each of which communicates with cavernous spaces in the glans. In addition, a bypass of the glans is effected by communication of these vessels directly with the deep dorsal vein. The apparent absence of muscular pads in vessels of the spongiosum, the relative paucity of cavernous smooth muscle, and the ample venous drainage provided by the deep dorsal vein may account for the lack of a venous occlusive mechanism similar to that of the corpora cavernosa penis.     

A scanning electron microscopic study of rat Masugi nephritis.

Changes in the glomerular capillaries in the first phase of rat Masugi nephritis were studied by scanning electron microscopy. The changes developed immediately after the injection of nephrotoxic rabbit IgG and early endothelial lesions (2 to 6 h) were characterized by an increase in microvilli and a decrease in endothelial pores. The microvilli were fused and produced abundant pored projections (cytofolds). The peripheral endothelium was then lifted off from the glomerular basement membrane (GBM), leaving scattered endothelial fragments on the GBM. The denuded GBM exhibited a rather uniform, thick carpet-like appearance with occasional crater formation. Depositon of fibrin strands was seen associated with endothelial exfoliation. These later dissolved and were converted to a fibrinoid material, consisting of a complex of fragmented, thin fibrils. A parallel study using the electron microscope revealed that the fibrinoid material was removed by emigrating monocytic macrophages. At the stage of resolution (24 to 72 h), the denuded GBM was covered mostly with a regenerating endothelial layer. A possible process of reorganization of the endothelial pores is discussed.     

Lens fiber organization in four avian species: a scanning electron microscopic study

The three-dimensional organization of the eye lenses of the chicken, the canary, the song-thrush and the kestrel was studied using light and scanning electron microscopy. The lenses of birds are characterized by the presence of two distinct compartments: the annular pad and the main lens body, separated by a cavum lenticuli. The annular pad fibers had a hexagonal circumference all contained a round nucleus and except for the canary were smooth-surfaced and lacking anchoring devices. In the canary, however, the annular pad fibers were studded with edge protrusions and ball-and-socket junctions. The semicircular main lens body fibers of all four species were studded with ball-and-socket junctions and edge protrusions. In contrast with mammals these anchoring devices were present throughout the lens up to the embryonal nucleus. Superficially the main lens body fibers were extremely flat. Additionally membrane elevations and depressions and globular elements were found on these central fibers in three species, the kestrel being the exception. At the transition between annular pad and main lens body the fibers turned their course and the nuclei became oval and disappeared in the deeper aspect of the main lens body. The cavum lenticuli was filled with globules tied off from the annular pad fibers. It seems attractive to assume that the presence of a separated annular pad, a cavum lenticuli filled with globular elements, the extreme flatness of the superficial central fibers and the studding of these central fibers with anchoring devices up to the embryonal nucleus are morphological expressions of the mouldability of the bird's eye lenses and consequently would explain their efficient accommodative mechanism including formation of a lenticonus. The presence of nuclei in the annular pad fibers and their typical change at the transitional zone between annular pad and main lens body are suggestive for a two-phased differentiation in bird's lens fibers: differentiation of the germinative epithelial cells to annular pad fibers which migrate to the main lens body after which they differentiate further to main lens body fibers.     

Nasal pit formation in the hamster: a transmission and scanning electron microscopic study

The surface morphology of cells comprising the nasal placode and adjacent body surface was examined by transmission and scanning electron microscopy during nasal pit formation in hamster embryos ranging in age from $\text{8}\text{1}\text{4}$ to $\text{9}\text{1}\text{2}$ days post coitum. A sharp distinction between the apical surface appearance of cells of the nasal epithelium and cells of the surrounding periderm develops at the periphery of the nasal placode. Periderm cells increase in surface area, exhibit a change in the distribution of surface microvilli, and many acquire a single cilium per cell. Cells of the nasal placode retain a dense surface coat of microvilli and exhibit relatively smaller apical surface areas. Olfactory rods can be positively identified on the basis of their ultrastructure at the nasal groove stage. The ventral margin of the nasal groove does not initially depend on the maxillary process, but is bounded by the lateral and medial nasal processes. From their earliest development the oral and nasal cavities appear to be separated in this species.     

Human chromatin from interphase to metaphase: a scanning electron microscopic study

An improved technique for microinjecting individual paramecium cells with fluid, making possible injection of 30 to 40 cells/h, is described. This technique has been used to show that cytoplasmically determined erythromycin resistance in Paramecium aurelia may be transferred to sensitive cells by the injection of mitochondria prepared from resistant clones. The ability of these mitochondria to transmit erythromycin resistance is unaffected by DNAse or RNAse but is completely removed by low concentrations of a non-ionic detergent. This evidence strongly suggests that the erythromycin resistance determinants are located in the mitochondria, probably in the mitochondrial DNA.     

Subchondral bone failure in overload arthrosis: a scanning electron microscopic study in horses

Mechanical overload leads to a common arthrosis in the metacarpal condyle of the fetlock joint of racehorses. This is usually asymptomatic but severe forms can cause lameness. Subchondral bone failure is often present and the predictability of the site provided an opportunity to study of the progression of bone failure from microcracks to actual collapse of subchondral bone. Twenty-five fetlock condyles from racehorses with various stages of disease were selected. Stages ranged from mild through severe subchondral bone sclerosis, to the collapse of bone and indentation or loss of cartilage known as 'traumatic osteochondrosis'. Parasagittal slices were radiographed and examined with scanning electron microscopy. Fine matrix cracks were seen in the subchondral bone layer above the calcified cartilage and suggested loss of water or other non-collagenous components. The earliest microcracks appeared to develop in the sclerotic bone within 1-3 mm of the calcified cartilage layer and extend parallel to it in irregular branching lines. Longer cracks or microfractures appeared to develop gaps as fragmentation occurred along the margins. Occasional osteoclastic resorption sites along the fracture lines indicated activated remodeling may have caused previous weakening. In one sample, smoothly ground fragments were found in a fracture gap. Bone collapse occurred when there was compaction of the fragmented matrix along the microfracture. Bone collapse and fracture lines through the calcified cartilage were associated with indentation of articular cartilage at the site.     

A scanning electron microscopic study of SV40 infected cells.

The surface of the SV40-infected African green monkey kidney (AGMK) cells was studied morphologically by scanning electron microscopy. In 24 hr post infection (p.i.), the cell surface was covered with slightly elongated microvilli. The microvilli increased in number. In 96 hr.p.i. most of the cells showed SV40-specific cytopathic effects (CPE). Nuclear swellings and the elongation of microvilli were eminent. Microvilli were observed projecting with high densities especially on the nuclear portions of the cell surfaces. Features suggesting cytoplasmic vacuolization were also observed in some cells. Spherical particles viewed in some of the cells at the late stage of infection were considered SV40 virions. Their origin was also discussed.     

Surface configuration of mesothelial cells in effusions. A comparative light microscopic and scanning electron microscopic study.

Surface configuration of mesothelial cells identified by light microscopy (LM) has been studied by scanning electron microscopy (SEM). It has been shown that mesothelial cells may have a variable SEM appearance. The surfaces of a small proportion of mesothelial cells are covered by regular microvilli (MV) and show openings of the pinocytotic vesicles. The surfaces of the majority of these cells are covered by vesicles or blebs. An intermediate population of mesothelial cells, i.e., cells displaying side-by-side blebs and MV, has also been observed. The latter cells no longer display pinocytotic vesicles. Occasional mesothelial cells have smooth surfaces. It has been shown by LM and transmission electron microscopy that cells with blebs are viable and capable of mitotic activity. It is concluded that mesothelial cells, detached from their epithelial setting, lose microvilli and pinocytotic vesicles and acquire surface blebs. The possible relationship between mesothelial cells and macrophages based on surface features has been discussed.     

Brown adipose tissue: a scanning electron microscopic study of tissue and cultured adipocytes

The ultrastructure of rat brown adipose tissue (BAT) and of adipocytes cultured from BAT were studied by scanning electron microscopy (SEM). Brown adipocytes in the intact tissue were arranged in lobules with bundles of collagen among them; within each lobule 20- to 40-microns-large adipocytes were packed together. Fibers of reticular collagen enveloped each adipocyte and also connected each cell to vessels and nerves. At the adipocyte surface rounded protrusions were present, which corresponded to the BAT-typical multivacuolar lipid depot. Gradual digestion of the stroma with collagenase disclosed a more delicate, felt-like cover surrounding each adipocyte, probably representing the external lamina of the cell. Complete digestion of the stroma showed a smooth plasmalemma with occasional roundish blebs which varied in size. Cultured (24 h) brown adipocytes from the stromal-vascular fraction of BAT were elongated or polygonal in shape, with a flattened, central nucleus and a number of spherical cytoplasmatic inclusions which have the same dimension and location as lipid droplets. These inclusions were arranged either at cellular poles or around the nucleus; this suggests that brown adipocytes with mature features were present in the culture. Pictures suggesting the detachment of lipid droplets from the cell body were also visible. Lipid droplet extrusion could be a complementary mechanism which might explain the rapid delipidation of brown adipocytes in culture.     

Rhinoglena frontalis (Rotifera,Monogononta): a scanning electron microscopic study

Females and males of Rhinoglena frontalis (Monogononta, Epiphanidae) are observed by SEM and their external morphologies are compared. The two sexes differ in size and shape of the body. The female body is fusiform with a short, conical foot, while the male body is more slender and has a rather long foot. The rotatory apparatus (or corona) of both sexes is similar with only minor differences and consists of rows and tufts of cilia arranged around the mouth opening. The corona is made of two paired lobes lateral to the mouth and of a third prominent dorsal lobe, usually called proboscis. The three lobes are lined externally by dense rows of cilia, which constitute the cingulum, used for swimming. The central surface of the proboscis is covered with numerous longitudinal rows of cilia bent towards the mouth. The lateral lobes show, on their central surfaces, two concentric arcs of cirri (made of tightly packed cilia) bent towards the mouth. The similar organization of the rotatory apparatus of both sexes is related to the fact that the male, in this species, is able to feed and has a developed mastax and digestive system. The trophi of both sexes are illustrated and compared.     

Morphology of mouse egg cylinder development in vitro: a light and electron microscopic study.

The endocrine control of yolk deposition in Drosophila melanogaster was studied by ligation and transplantation techniques. Endocrine events associated with the initiation of vitellogenesis were found to be synchronized with eclosion rather than the completion fo adult development. Decapitation experiments showed that a cephalic event occurring at about the time of eclosion is necessary for each animal to initiate vitellogenesis. The morphogenetic effect of the head could be replaced by a juvenile hormone analog (JHA). In addition to the cephalic event, a thoracic factor is required for each follicle to initiate vitellogenesis, since preparation of isolated abdomens before 16 hours after eclosion prevented vitellogenesis. In abdomens isolated after this time, no early vitellogenic stages were formed. The suppression of vitellogenesis in isolated abdomens was reversed by implanting corpora allata or by treating these preparations with JHA, but not by implanting corpora cardiaca. Ovaries that were artificially induced to mature by treating isolated abdomens with JHA still displayed the normal complement of ovarian proteins after electrophoresis in polyacrylamide gels. These results show that a circadian clock triggers vitellogenesis via a cephalic signal at eclosion, which in turn triggers events in the thorax or abdomen. The cephalic signal can be superseded by juvenile hormone, whose presence is necessary for each follicle to become vitellogenic.     

Collagenous network in cartilage of human femoral condyles. A light microscopic and scanning electron microscopic study

To determine the spatial arrangement of collagen fibrils in articular cartilage of the human femoral head, three healthy femoral heads, obtained at necropsy, were examined by light microscopy and scanning electron microscopy. Light microscopic observations revealed no collagen fibril organization. Scanning electron microscopic observations showed a fine fibrillar texture throughout the articular cartilage. At the articular surface, smooth and fibrillated areas were detectable. Underneath the articular surface, the collagen network in the superficial zone showed a tighter appearance when compared with the homogeneous collagen network of the matrix in the deeper zones. The calcified cartilage zone was well demarcated from the uncalcified cartilage. The arcade model of Benninghoff [Z. Zellforsch. Mikrosk. Anat. 2: 783-862 (1925)] could not be confirmed. It was concluded that the organization of collagen fibrils in hyaline cartilage shows a three-dimensional network of randomly oriented fibrils.     

Fluorescence and scanning electron microscopic study on self-incompatibility in distylous Averrhoa carambola L.

Various combinations of intermorph, selfing and intramorph pollinations were carried out in Averrhoa carambola and the pollinated pistils were observed under fluorescence and scanning electron microscopes in time-course experiments. In both compatible and incompatible pollinations, similar behavior of pollen germination and penetration was observed in the first 4 h after pollination. In compatible intermorph pollination, pollen tubes were found at the base of the transmitting tract of the style at 8 h and 24 h after pollination in both the pin and thrum morphs. With thrum flowers, selfing resulted in pollen tubes being uniformly arrested at the junction between the stigmatic and stylar tissues. Penetration of pollen tubes into the upper portion of style was observed in thrum intramorph pollination and, when the second member was treated as the gynoecial tissue under the same pollination, penetration of tubes was further enhanced. Pin flowers, on selfing, resulted in pollen tube penetration farther down the style than was the case with thrum selfing. Intramorph pollination of pin morph behaved in a similar manner to selfing and was not affected by genotypes. Beside the stamen-style dimorphism, the receptive surface of the cob stigma was larger in pin than thrum flowers. While pin pollen was round, thrum pollen was oblong in shape with pin to thrum ratio on the polar axis being 1.2 and on the equatorial axis 0.8. The stigma of pin morph belonged to the dry type, while that of the thrum resembled the wet type.