Scanning electron microscopy of chick epiblast expansion on the vitelline membrane. Cell-substrate interactions

Cell-substrate interactions have been studied by examining migrating edge cells of the expanding chick extraembryonic epiblast on their normal substrate and in culture. Scanning electron microscopy shows that the outer face of the vitelline membrane is a random meshwork of fibrils (80 nm diam). The inner face, which is the normal substrate of epiblast expansion, is composed of a random branched system of fibers (400 nm diam) overlain by a network of fibrils (40 nm diam). The epiblast edge in situ has radially oriented filopodia (20 μm long, 200 nm diam.), frequently extending from broad lamellipodia. Blastoderms cultured on the inner face of unincubated vitelline membrane expand at a normal rate but display ruffles as well as filopodia and lamellipodia. When the blastoderm is cultured on the outer membrane face there is no expansion, but cells leave the edge and migrate across the membrane. In these cultures, ruffles are observed on the ventral epiblast face. Absence of the mass of yolk in culture appears to permit or provoke the observed ruffling. Comparison of dissociated epiblast edge cells and skin epithelial cells, cultured on glass and on the vitelline membrane inner face, indicates that epiblast cells remain flattened and display characteristic filopodia on both substrates, whereas skin cells display ruffles on the vitelline membrane but are flattened on glass. The mode of migration of epiblast edge cells seems to be more dependent on intrinsic factors than that of skin cells.     

Scanning electron microscopy of primary membrane bone

Summary The organization and the development of primary bone were studied in a number of newborn or foetal mammals. Specimens were derived from long bones, calvaria and mandibles. The surface of the mineral component was exposed by treatment with an organic solvent (hot 1,2 ethane diamine or cold sodium hypochlorite solution) and in the case of some internal surfaces (endosteal surfaces of long bone shafts, endocranial aspects of calvaria and the lining of the dental groove or crypts of mandibles) some success was achieved in dissecting the soft tissues from the bone matrix surface. These surface were studied by scanning electron microscopy. Ground sections of similar material were studied by microradiography.Primary membrane bone was found to be most different from adult lamellar bone on rapidly growing (periosteal) surfaces and in the following respects. 1) Mineralization of both the ground substance and collagen occured close to the mineralizing front, so that a higher level was reached rapidly and without a maturation phase. 2) The mineral clusters at the mineralizing front did not reflect the collagen fibre distribution. 3) The collagen fibre bundles at the matrix surface, at least at internal surfaces, were irregular in size and random in orientation.Mineralizing fronts intermediate in character between those of rapidly forming, periosteal primary bone and adult lamellar bone were found on the uncommon internal forming surfaces and in the walls of vascular channels.Most internal surfaces were resorbing. There was a wide range in the size of the resorption lacunae — the many which were only as large as the osteocyte lacunae exposed by the resorption process may have been caused by uninucleated cells.This work has been supported by a grant from the Medical Research Council. The Stereoscan scannning electron microscope was provided by the Science Research Council, and the microradiographic equipment by Mr. R. V. Ely and the Ely-Webster trust. We would like to thank Mr. P. S. Reynolds, Miss A. J. Sutton and Mr. P. F. Farmer for technical and photographic assistance and Mrs. Jeanne Mills for secretarial assistance.     

Scanning electron microscopy of the glomerular filtration membrane in the rat kidney

The rat kidney was perfused with saline and glutaraldehyde, treated with Murakami's tannin-osmium impregnation method, ethanol-freeze cracked and dried by the critical point method. Gold-palladium evaporated specimens were observed in a field-emission scanning electron microscope. The glomerular filtration membrane, fractured in different planes was observed with the following results: 1. Adjacent pedicles originate from different podocytes. No interpedicular bridges of apparent cytoplasmic nature could be found. 2. The basement membrane, in grazing fractures shows a horizontally layered architecture. 3. The attenuated endothelial sheet (lamina fenestrata) is divided into compartments, which we suggest should be called "areolae fenestratae", by cytoplasmic crests radiating from the nucleated portion of the endothelial cell. A crest also occurs along the cell margin, which contacts a similar crest at the margin of the adjacent cell. 4. The pores in the areolae fenestratae are variable in size (30-150 nm diameter). A knob-like projection from the apparently naked basement membrane is found in a portion of the pores. 5. Numerous microvilli may occur on the endothelium. Some of them anastomose and fuse with one another to form a net whose meshes appear identical with the endothelial pores. Domes and shelves formed of a fenestrated cytoplasmic sheet also occur above the ordinary level of the endothelial lining. A hypothesis implicating microvilli in the partial renewal of the endothelial sheet is proposed.     

Scanning electron microscopy ofMycoplasma pneumoniae on the membrane of individual ciliated tracheal cells

Summary Cell monolayer cultures were prepared from hamster tracheal explants by a collagenase exposure and subsequent incubation in Waymouth’s MAB 87/3 medium. The epithelial outgrowth occurred on glass cover slips. Cilia on the monolayers continued to beat normally after the “parent” explant was removed. Monolayer cultures infected withMycoplasma pneumoniae had significant amounts of attachment. A morphological analysis of the attachment was conducted with scanning electron microscopy. Clusters, cocci, and filaments ofM. pneumoniae all attached to the epithelial cells, but the filaments were especially common. Mycoplasmas were seen in association with both ciliated and nonciliated cell membranes. On ciliated cells, mycoplasmas were on the ciliary strands and on the cell membrane. When located immediately adjacent to or in between cilia, mycoplasmas were oriented vertically with the constricted attachment tip oriented down toward the host cell membrane. When located more than a micron away from the ciliary fibers, mycoplasmas lay horizontally along the epithelial cell membrane. The photographic data suggest that clusters or “sperules” of mycoplasmas may liberate individual mycoplasmas that attach to the cell membrane. It appears that the receptor sites forM. pneumoniae are rather uniformly distributed along the ciliated cell membrane, and are not restricted to the interciliary areas. Electron microscopy was done with the cooperation of Dr. R. Macleod and the staff of the Center for Electron Microscopy at the University of Illinois. Critical editorial review was provided by C. Dayton. This investigation was supported in part by grants to M. G. G. from the National Institute of Allergy and Infectious Diseases (AI 12559) and the National Heart, Lung, and Blood Institute (HL 23806), Bethesda, Maryland.     

Scanning electron microscopy of the chick embryo

Internal anatomical features of developing chick embryos can be examined using the scanning electron microscope in embryos that have been sectioned at varying levels. Standard paraffin sectioning techniques are used. After removal of paraffin, embryos are dried by the critical point method and examined. The resulting scanning electron micrographs have proved to be exceptionally useful in teaching anatomy of embryos.     

Scanning electron microscopy of the rat retina

Summary Fixed and unfixed, freeze-dried pieces of isolated retina and the posterior part of the eye bulb from adult rats were examined in a scanning electron microscope.The inner limiting membrane shows distinct cell borders, protrusions, and scattered microvilli-like structures. Different types of nerve cells are observed in the ganglion cell layer and the inner nuclear layer. They all lack synaptic boutons on the surface of their perikarya. There is an intercellular space between the processes in the nerve fiber layer.The inner and outer segments are surrounded by a space with extracellular material. Their surface is smooth or slightly undulated. There is no evidence indicating the existence of basal infoldings continuous with the membraneous structures inside the rod outer segments. The connecting piece between the inner and outer segments resemble a symmetrically shaped hour-glass. The surface of the epithelial cells is covered by microvilli forming a honeycomb-like structure and each outer segment is surrounded by several microvilli.The results obtained are discussed in relation to those obtained by transmission electron microscopy. The probable existence of a significant extracellular space and the distribution of extracellular material between the segments and the microvilli are discussed.Supported by grants from H. Jeanssons Stiftelse Riksföreningen mot Cancer (265-B69-OIX) and the Swedish Medical Research Council (B70-12X-2534-02). I would like to thank the Swedish Silicate Research Institute, Göteborg, for using their scanning electron microscope, and Miss. M. Persson for skilful technical assistance.     

Scanning electron microscopy of Mycoplasma pneumoniae on the membrane of individual ciliated tracheal cells

Cell monolayer cultures were prepared from hamster tracheal explants by a collagenase exposure and subsequent incubation in Waymouth's MAB 87/3 medium. The epithelial outgrowth occurred on glass cover slips. Cilia on the monolayers continued to beat normally after the "parent" explant was removed. Monolayer cultures infected with Mycoplasma pneumoniae had significant amounts of attachment. A morphological analysis of the attachment was conducted with scanning electron microscopy. Clusters, cocci, and filaments of M. pneumoniae all attached to the epithelial cells, but the filaments were especially common. Mycoplasmas were seen in association with both ciliated and nonciliated cell membranes. On ciliated cells, mycoplasmas were on the ciliary strands and on the cell membrane. When located immediately adjacent to or in between cilia, mycoplasmas were oriented vertically with the constricted attachment tip oriented down toward the host cell membrane. When located more than a micron away from the ciliary fibers, mycoplasmas lay horizontally along the epithelial cell membrane. The photographic data suggest that clusters or "sperules" of mycoplasmas may liberate individual mycoplasmas that attach to the cell membrane. It appears that the receptor sites for M. pneumoniae are rather uniformly distributed along the ciliated cell membrane, and are not restricted to the interciliary areas.     

Scanning electron microscopy of chloroplast ultrastructure

A range of fracturing and sectioning techniques are now available which permit intracellular structures to be observed in the scanning electron microscope. One such technique, based on the method of Tanaka (1981), has been used to study chloroplast ultrastructure in Japan laurel, Aucuba japonica. Small pieces of leaves were fixed, fractured whilst frozen and transferred to a dilute solution of osmium tetroxide in which cytoplasmic maceration took place. Specimens were dehydrated, critical point dried and examined was required to remove the stroma from fractured chloroplasts. Following this treatment details of the chloroplast envelope, frets, grana and plastoglobuli could be observed. The results were compared with conventionally prepared thin sections examined in the transmission electron microscope and with the three dimensional reconstructions described in the literature.     

Scanning electron microscopy of Penicillium conidia

The morphology of conidia in 211 species and 12 varieties belonging to the genus Penicillium Link ex Gray have been studied and compared.According to surface ornamentation, conidia have been classified into six groups: A, smooth-walled (7% of the species); B, delicately roughened (13%); C, warty (28%); D, echinate (10%); E, striate with low irregular ridges (36%); and F, striate with scarce high ridges or bars (6%). Whereas the first two groups are closely related in both shape and average size, a gradual reduction was observed in size and in the length/width (l/w) ratio in the remaining groups. Echinate conidia were globose, having the largest average size. Only four species produced conidia not surpassing 2 m in diameter. Maximum length observed was 8 m, and most elongated conidia had a l/w ratio of 3.5. Forty per cent of the species studied had globose conidia.Conidia of the monoverticillate species were generally smaller, more globose and frequently with ridges. In the Asymmetrica, the conidia were generally larger, and showed ridges in comparatively few species. Conidia of the Symmetrica, which were frequently striate with ridges, presented the most elongated forms. The largest average size was found in the conidia of the Polyverticillata which were generally warty. Finally, we have considered the variations in surface ornamentation of conidia during the evolution of the genus Penicillium and drawn attention to their possible relationship with certain habitats and ways of conidial dispersion.