Scanning electron microscopy of volk absorption in early chick embryos.

Fresh pullet eggs (White Leghorn Strain) were incubated from 6-19 hours. Blastoderms were fixed in situ with aldehyde fixative, post-osmicated in 2% OsO4, dehydrated in acetone, critical-point-dried, mounted ventral side up, coated with palladium-gold wire and observed in a Cambridge Stereoscan S4 scanning electron microscope. Large extracellular yolk granules had a smooth surface and appeared to break up into smaller particles. Similar particles have been observed intracellularly in transmission electron microscopy. Numerous microappendages, mostly ruffles, suggest phagocytosis as a method of absorption of yolk granules into the cells. Absorption of yolk by the cells of the blastoderm involves an initial break up of yolk granules followed by phagocytosis.     

Scanning electron microscopy of the inner surface of small intestine in chick embryos

The inner surface of the midgut of chicken embryos ageing between seven and fifteen days incubation has been examined by scanning electron microscope. In this period the surface of the mucous membrane undergoes significant morphological changes; in fact, while at seven days incubation it appears to be smooth and regular, in the following days it begins to show longitudinal folds increasingly higher, more numerous and complicated. Since eleven days incubation some folds take a zigzag appearance that progressively becomes more evident and extends to all the folds. In the mean time, the enterocytes undergo a gradual specialization and represent the only type of epithelial cells in this stage of development. Their apex until seven days incubation is dome-shaped, provided with short microvilli and separated from the surrounding cells by deep circular grooves. About thirteen days the apex appears to be less swollen, with longer and more numerous microvilli and bounded by microplicae arranged in an hexagonal disposition.     

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.     

cAMP enhanced aggregation of cells from early chick embryos.

Whole chick embryos incubated for 24–36 hr were disaggregated with EDTA. The populations of single cells were incubated both in suspension and after being plated at various densities on agar blocks in a humid environment. In both cases aggregates formed. The aggregation was enhanced by cAMP and 3-isobutyl-1-methylxanthine (IBMX; a phosphodiesterase inhibitor). The density of aggregates which formed on the agar blocks decreased sharply at a critical cell density, suggesting that aggregation was mediated by a relayed signal. The critical density was decreased by IBMX and increased by phosphodiesterase (PDE), suggesting that aggregation was mediated by a cyclic nucleotide, most probably cAMP. Evidence was obtained for the presence of an extracellular PDE.     

Atrial septation: I. Scanning electron microscopy in the chick

Chick hearts were prepared for scanning electron microscopy by standard methods, the purpose being to investigate the surface morphology of the developing atrial septal region. By Day 3, the atrial septum primum appeared as a sickle-shaped structure. During Day 4, the first representation of the foramina secunda occurred in the mid-dorsal portion of the septum. During Day 5, the septum primum fused with the atrioventricular endocardial cushions, thereby occluding the foramen primum. From Days 5 through 8, the secondary perforations (foramina secunda) multiplied and increased in size. The endocardial-covered cords of cells comprising the septum thickened from Days 9 through 15. This resulted in a marked reduction in the dimensions of the perforations from Day 16 to hatching. The atrial septum at hatching occasionally contained a small single orifice. At 3 days posthatching, the atrial septum was a solid sheet covered with flattened endocardial cells. All interatrial communications were occluded. During Days 5 through 9, two distinct cell types became apparent on the endocardial-covered cords. Simultaneously, fenestrations were observed on the cords surrounding the foramina secunda and on the ventral portion of the atrial septum. The integral role which the fenestrations and cellular types play in the development of the formina secunda is discussed.     

Scanning electron microscopy (SEM) of the chick embryo primitive streak

The structure of the cells forming the primitive streak was examined by SEM in a series of embryos at Hamburger and Hamilton's stages 2--5. Specimens were prepared by stripping the endoderm from fresh embryos in New Culture and by fracturing whole fixed embryos along and at right angles to the primitive streak. At all stages of examination the SEM appearance of cells within the privitive streak was quite different from that of ectodermal, endodermal or mesodermal cells away from the streak. Streak cells were closely packed, lay with their long axes directed from ectoderm to endoderm and possessed many flat leaf-like processes. By contrast the ectoderm formed a columnar epithelium, the endoderm a flat epithelium and the mesoderm was a layer of loosely arrangedcells with long. thin processes. Within the streak SEM did not show any differences between cells that could identify them specifically as future endoderm or mesoderm cells. It was concluded that during gastrulation all the cells migrating through the primitive streak have the same appearance regardless of their eventual destination in the embryo. This structure may be attributable to the type of movement made by cells during invagination.     

Scanning electron microscopy of bone cells in culture

Summary Embryonic and young rat bone cells have been grown in culture and examined in the scanning electron microscope (SEM). Compared with cells fixed in situ and taken directly from the animal, the cultured osteoblastic cells were smoother, flatter and more extensive and showed tighter intercellular contacts. Some matrix is formed in culture and undergoes at least partial mineralization as judged by the accumulation of Ca and P measured by energy dispersive x-ray analysis. Findings concerning the morphology of the collagen arrangement were indecisive. Some superficial cells, free of surrounding matrix, resembled osteocytes in normal in vivo bone. This may indicate that a proportion of the extracellular matrix produced by the cultured cells failed to polymerise into recognizable bone matrix, and that osteocytic morphology is not dependent upon the physical characteristics of the bone matrix.     

Glutaraldehyde fixative tonicity for scanning electron microscopy of delicate chick embryos.

Fresh pullet eggs (White Leghorn strain) were incubated from 19-2ldium-gold and observed in a Cambridge S4 scanning electron microscope. Shrunken cells with intracellular yolk granules embossed on the surface are produced by the strongly hypertonic Karnovsky's fixer (Final: 2010 mOsm). Embryos fixed with modified Karnovsky's fixer (Final: 373 mOsm) possess surfaces with irregular microappendages. Swollen cells with few microappendages are observed when embryos are fixed in a hypotonic environment (Final: 250 mOsm or less). Ideal fixatives preserve a relatively flat surface, with cells bordered by smoothsurface microappendages. For adequate SEM fixation, fixative vehicle should be approximately isotonic for tissue, with aldehyde (2% or less) added to vehicle.     

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 cultured cells of Aedes aegypti

Cultured cells of Aedes aegypti were fixed with glutaraldehyde and prepared for scanning electron microscopy by four procedures: air drying, lyophilization, ethanol dehydration and air drying, and ethanol dehydration and critical point drying. Comparison of the resulting electron micrographs with phase contrast photomicrographs of living cells revealed that although cultured insect cells dried by the critical point method are not completely without artifacts, this method of preservation is superior to other techniques currently used.     

Scanning electron microscopy of developing papillae on the tongue of human embryos and fetuses

The appearance and differentiation of papillae on dorsal andlateral surfaces of human embryonic and fetal tongues, at variousdevelopmental ages, were studied by scanning electron microscopy.Formaldehyde and phosphate buffer fixation provided satisfactorypreservation. At 8–9 weeks, the anterior two-thirds ofthe tongue showed no obvious signs of papillae. In contrast,just anterior to the sulcus terminalis rounded elevations wereseen, suggesting initial signs of circumvallate papillae. At10–13 weeks, the distribution and shape of elevationson the anterior two-thirds of the tongue indicated the beginningof fungiform papillae. Openings located on the dorsal surfaceof many of these fungiforms contained an amorphous central structureprojecting out of the papilla. First signs of foliate papillaewere seen at 10 weeks. At 15–18 weeks, fungiform and filiformpapillae were recognized, although sometimes their borders wereobscured by scaling epithelial cells. At 23–26 weeks,all papillae exhibited their adult form. ^*Presented, in part, at the VIth International Symposium onOlfaction and Taste, Gif-sur-Yvette, Paris, France, 15–17thJuly, 1977.     

Scanning electron microscopy of cells and protoplasts ofSaccharomyces rouxii

Cells ofSaccharomyces rouxii from a normal broth culture were subjected to a high osmotic pressure (2 M KCl), fixed in 3% glutaraldehyde fortified with 2 M KCl, and then processed routinely for examination in a scanning electron microscope. Micrographs revealed birth and bud scars typical for the genus and an apparently undamaged surface topography. Protoplasts were prepared from the same material by digestion of cell walls with snail gut enzymes in the presence of 2 M KCl. Naked protoplasts were obtained and these exhibited surface invaginations. In addition, spheroidal protrusions were noted and these structures were equated with the periplasmic bodies previously described by transmission electron microscopy. The propensity for periplasmic body formation inSaccharomyces rouxii is contrasted with otherSaccharomyces species and the circumstantial evidence that relates periplasmic bodies to cryptic β-fructofuranosidase inS. rouxii is briefly discussed.     

Scanning electron microscopy analysis of aged Mycobacterium tuberculosis cells

Most electron microscopy studies of Mycobacterium tuberculosis ultrastructure were performed in the 1950s and 1960s and lack high resolution by modern standards. This study was performed to re-evaluate the fine structure of M. tuberculosis using modern scanning electron microscopy. Bacteria were grown in rich medium with a constant supply of oxygen for several weeks. Results show that surface bleb-like structures accumulate as cultures age. The most unusual feature of aging M. tuberculosis cultures is that they develop extracellular fibrils, which could play roles of adhering cells to surfaces and to one another.     

Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick

Fried B. and Fujino T. 1984. Scanning electron microscopy of Echinostoma revolutum (Trematoda) during development in the chick embryo and the domestic chick. International Journal for Parasitology14: 75–81. Scanning electron microscopy (SEM) was used to study the development of chemically excysted metacercariae of Echinostoma revolutum on the chick chorioallantois. SEM studies were also made on preovigerous adults of E. revolutum grown in the domestic chick. During worm development on the chorioallantois the tegument changed from smooth to granular and sensory papillae on the suckers became well-defined. As worms developed on the chorioallantois the cephalic collar spines became thicker and more curved and the tegumentary spines showed marked changes in shape, size and distribution on both ventral and dorsal aspects of the body. Changes in the surface ultrastructure of worms grown on the chorioallantois were essentially similar to those observed in preovigerous worms from chicks.     

Scanning electron microscopy of cell aggregation: cardiac and mixed retina-cardiac cell suspensions

Reaggregates of cells from 7-day embryonic chick hearts, 10-day neural retinas and respective cell mixtures were examined by scanning electron microscopy (SEM). Cardiac cell aggregates formed during the first 2 hours were composed of rounded cells arranged in linear or branched arrays. By further collection of single cells and accretion of cell clusters, the aggregates increased in size and formed large grape-like masses. By 12 hours, heart aggregates assumed a spherical shape with partial sorting-out of myogenic from non-myogenic cells; the muscle elements occupied the interior of the aggregates, whereas flattened, squamous-like cells, of a non-muscle character, covered the surface in a multilayered epithelium. Single rounded cells were still found on the surface of 12 to 24-hour aggregates; however, they were absent by 48 hours, suggesting that the collection of free cells by the cardiac aggregates ceased during the second day. Early aggregates (2 hours) of retina cells showed initial stages of axonal and dendritic outgrowth characteristic of neural tissue. Examination of heterotypic aggregates of neural retina and myocardial cells after 2 to 6 hours showed small groups of retina cells attached to the surface of the heart cell clusters. The retina cells did not appear to be randomly distributed within the early aggregates but formed small tissue specific clusters even by 2 hours in culture. These results indicate that SEM should be a valuable tool in the further analysis of homo- and heterotypic cellular aggregation.     

Scanning electron microscopy preparation protocol for differentiated stem cells

The lack of an established protocol for scanning electron microscopy (SEM) studies on stem cells differentiating into adipogenic lineage led us to develop a protocol for the preparation of differentiated adult bone marrow-derived mesenchymal stem cells (BMSC) for SEM. This protocol describes the procedure to maintain and preserve the structural organization of cellular components following differentiation, for morphological and physical characterization. The fixation of the differentiated cells was followed by dehydration using methanol, and vacuum desiccation before microscopy. The use of longer chain alcohols as dehydrating agents was avoided in our method to reduce the dissolution of lipid deposits in cells, thus allowing the maintenance of their structural integrity. The time period for the processing of samples was reduced by avoiding the osmium tetroxide postfixation and critical point drying. Thus, this protocol helps in determining the potential, fate, and degree of stem cell differentiation. This may be useful for SEM analysis of differentiated cells, especially those grown on various scaffolds.     

Scanning electron microscopy in nematode-induced giant transfer cells.

A study of giant cells induced by the root-knot nematode, Meloidogyne incognita, in roots of Impatiens balsamina was made by scanning electron microscopy. The cytoplasmic contents of giant cells were removed by a procedure based on KOH digestion, to reveal inner wall structure. Wall ingrowths typical of transfer cells are present in giant cells from six days onwards after induction. They develop on walls adjacent to vascular tissues, and their distribution and development was examined. Pit fields contianing plasmodesmata become elaborated in walls between giant cells, but pit fields are lost between giant cells and cells outside them. The distribution of plasmodesmata in pit fields suggests that de novo formation of plasmodesmata occurs in walls between giant cells. Various aspects of giant cell formation and function are discussed and wall ingrowth development is compared in giant cells and normal transfer cells.