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.     

Cell surface changes during mitosis and cytokinesis of epithelial cells

Summary PtK2 cells were studied with scanning electron microscopy to record changes on the cell surface during mitosis and cytokinesis. During prophase, prometaphase and metaphase, the cells remain very flat with few microvilli on their surfaces. In anaphase cells, there is a marked increase in the number of microvilli, most of which are clumped over the separating chromosomes and polar regions of the mitotic spindle leaving the surface of the interzonal spindle region relatively smooth. Microvilli appear over the interzonal spindle region in telophase and the cells also increase in height. At the beginning of cleavage, the distribution of microvilli is roughly uniform over the surface but it becomes asymmetric at the completion of cleav-age when the daughter cells begin to spread. At this time most microvilli are over the daughter nuclei and the surfaces that border the former cleavage furrow. The regions of the daughter cells distal to the furrow are the first to spread and their surfaces have very few microvilli. When chromosome movement is inhibited by either Nocodazole or Taxol, microvilli formation is inhibited on the arrested cells. Nevertheless cell rounding still takes place in the normal time period. It is concluded from these observations that the signal for the onset of chromosome movement in anaphase is accompanied by a signal for the formation of microvilli. It is suggested that there is also a separate signal for the cell-rounding event in mitosis and that microvilli do not play a role in this contractile process.     

Scanning electron microscopy of the cleft of the rat pituitary

Summary Scanning electron microscopy of the lining of the pituitary cleft was carried out in normal, lactating, castrated, adrenalectomized, and cyproterone-treated adult rats. Four cell types could be differentiated in the posterior wall in control and experimental animals: (1) cells with a smooth surface, (2) cells with microvilli located at the cellular borders, (3) ciliated cells, and (4) cells with evenly distributed microvilli. The anterior wall showed mainly cells with few microvilli located at their margins, and clusters of ciliated cells. In normal, and more frequently in experimental animals, the anterior wall showed shriveled cells, and variously sized cavities. Colloid appeared either as a network of finely granular material or as compact bodies adhering to the epithelial surface. These observations suggest that a compact component of the colloid is derived at least in part from degraded cells.Fellow of the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET), Argentina. The author owes many thanks to Professor I. Nouzeillez and Dr. J.C. Cavicchia for their assistance in translating this paper     

Scanning electron microscopy of dissociated pancreatic acinar cell surfaces

Summary The pancreatic acinar cell surfaces have been studied by SEM with a dissection technique and correlated with results obtained by TEM. The SEM results demonstrate characteristic arrangement of microplicae which in some areas are densely packed.In many areas, the microplicae are distributed in such a manner that they create zones with typical geometrical shapes and show a relatively smooth surface.These smooth areas may coincide, as indicated by correlated TEM results, with the limits of intimate contact between adjacent acinar cells which, in turn, represent part of the junctional complex. Another aspect revealed by these SEM preparations concerns the presence of groups of densely packed microplicae, arranged in regular rows and distributed along some grooves and/or infoldings of the cellular surface. On the basis of SEM and TEM information, it is likely that these structures correspond to intercellular (and possibly, in some cases, intracellular) canaliculi which topographically form a kind of extensive microlabyrinthine arrangement running along all the cell sides.One final point revealed by fractured samples concerns the finding of spherical zymogen droplets within the vesicles of the Golgi complex. Because in many scanning images these vesicles appear connected by small openings, it is suggested that they may represent a system of intercommunicating chambers (vacuoles) through which the zymogen droplets can be continuously accumulated and discharged into the acinar lumen.     

Some morphological observations on osteoclasts

Summary Osteoclasts of the peripheral portions of the endocranial aspect of young rat parietal and frontal bones were studied by scanning electron microscopy of glutaraldehyde fixed, critical point dried specimens. These studies show Osteoclasts to have a much more complicated form than has previously been realised. Extensively branching, elongated, smooth-surfaced cells, which are for the most part elevated above the level of the surrounding bone matrix surface and sometimes above portions of osteoblasts or other osteoclasts, were identified as motile non-resorbing cells. Portions of the former and other entire cells may be embowered in Howship's lacunae, have microvilli on their dorsal surface, and are surrounded by a serrated border of microprojections which have an apparently firm attachment to the matrix surface. Osteoclasts in short term culture show additional free surface ruffles which are not encountered in specimens taken fresh from the animal. No evidence of recruitment of osteoblasts or osteocytes into osteoclasts was found. Disinterred osteocytes retained an ability to migrate from their lacunae on to surrounding bone matrix surface.We would like to thank Elaine Maconnachie for expert technical assistance and Dr. Martin J. Evans for the use of his tissue culture laboratory. These studies have been supported in part by a grant from the Medical Research Council     

SEM analysis of amphibian mesodermal migration

Summary At the end of gastrulation, the lateral mesoderm of amphibian embryos migrates ventrally between the ectoderm and the endoderm. The present study is an examination of the morphology of the leading cells of the mesodermal sheet and of the substratum over which they move (the inner surface of the ectoderm). The cells of the leading edge of the mesoderm are generally round, with very short and narrow flattened projections in the forward direction. These projections do not have a ruffled morphology, regardless of whether fixation is carried out before or after the ectoderm and mesoderm are dissected away from the endoderm. The inner surface of the ectoderm is covered with fine (450–500A) filamentous extracellular material and the ectoderm cells sometimes extend cytoplasmic processes (approx. 0.1 wide) onto the leading surface of the mesoderm or onto adjacent ectoderm cells. These studies indicate that the morphology of cell migration in amphibians is closer to that seen inFundulus than to that characteristic of chick or mammalian cells.This paper is dedicated to the memory of Mac V. Edds, Jr., who warmly encouraged the developmental biologists of the Pioneer Valley     

Tribolium embryogenesis: a SEM study of cell shapes and movements from blastoderm to serosal closure

Embryogenesis in the beetle Tribolium is of increasing interest to both molecular and evolutionary biology because it differs from the Drosophila paradigm by its type of segment specification (short- vs. long-germ) and by the extensive epithelial envelopes – amnion and serosa – that are typical of most insects but not of higher dipterans. Using scanning electron microscopy of DAPI staged embryos we document development in Tribolium castaneum from blastoderm to completion of the envelopes, recording many details not otherwise accessible; we also provide a time table of the respective stages at 30°C. The nascent blastoderm cells remain basally confluent with the yolksac until after the 13th (=last synchronous) mitotic cycle. The cells in the prospective serosa – the first domain to segregate visibly from the uniform blastoderm – carry surface protrusions likely to contact the overlying vitelline envelope. The embryonic rudiment, the other (and larger) blastodermal domain, gives rise to amnion and germ anlage. In the latter, visible differentiation begins with a ”primitive pit” reminiscent of the posterior midgut rudiment of Drosophila. The subsequent invagination of the mesoderm resembles Drosophila gastrulation, except in the head region where the median groove extends through the entire preoral region. The prospective amnion starts differing visibly from the germ anlage during early gastrulation. It then folds underneath the spreading serosa and, advancing with the latter, closes the amniotic cavity at the ventral face of the germband. The largest (=posterior) amniotic fold covers a crestlike protrusion of the yolksac. Together with marked changes in the shape and arrangement of the amnion cells, this protrusion may contribute to the fold’s elevation and early progress. Received: 12 August 1999 / Accepted: 4 November 1999     

Structure of the cell surface of the yeast Candida tropicalis and its relation to hydrocarbon transport

The surface structure of the hypdrocarbon-utilizing yeast Candida tropicalis was investigated by scanning and transmission electron microscopy (SEM and TEM respectively). The sample preparation technique was based on a rapid cryofixation without any addition of cryoprotectants. In subsequently freeze-dried samples the surface structure was analysed by scanning electron microscopy. Thin sections were prepared from freeze substituted samples. Both techniques revealed hair-like structures at the surface of hydrocarbon-grown cells. The hairy surface structure of the cells was less expressed in glucose-grown cells and it was absent completely after proteolytic digestion of the cells. When cells were incubated with hexadecane prior to cyryofixation a contrast-rich region occured in the hair fringe of thin sections as revealed by TEM. Since these structures were characteristic for hexadecane-grown cells and could not be detected in glucose-grown or proteasetreated cells it was concluded that they originate from hexadecane adhering to the cell surface and are functionally related to hexadecane transport. The structure of the surface and its relation to hydrocarbon transport are discussed in view of earlier results on the chemical composition of the surface layer of the cell wall.Abbreviations SEM Scanning electron microscopy - TEM transmission electron microscopy     

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.     

Switchgrass (Panicum virgatum L.) cell suspension cultures: Establishment, characterization, and application

Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that has received considerable attention as a potential dedicated biofuel and bioproduct feedstock. Genetic improvement of switchgrass is needed for better cellulosic ethanol production, especially to improve cellulose-to-lignin ratios. Cell suspension cultures offer an in vitro system for mutant selection, mass propagation, gene transfer, and cell biology. Toward this end, switchgrass cell suspension cultures were initiated from embryogenic callus obtained from genotype Alamo 2. They have been established and characterized with different cell type morphologies: sandy, fine milky, and ultrafine cultures. Characterization includes histological analysis using scanning electron microscopy, and utility using protoplast isolation. A high protoplast isolation rate of up to 10⁶ protoplasts/1.0 g of cells was achieved for the fine milky culture, whereas only a few protoplasts were isolated for the sandy and ultrafine cultures. These results indicate that switchgrass cell suspension type sizably impacts the efficiency of protoplast isolation, suggesting its significance in other applications. The establishment of different switchgrass suspension culture cell types provides the opportunity to gain insights into the versatility of the system that would further augment switchgrass biology research.     

The aesthetes of chitons

Summary The shells of the chitons Lepidochitona cinereus, Sypharochiton pelliserpentis, Amaurochiton glaucus and Onithochiton neglectus were examined by scanning electron microscopy. In all species the surface terminations of the megalaesthete and micraesthete organs could be identified lying flush with the shell surface, as well as, lenses of the shell eyes in O. neglectus. Periostracal debris and encrusting diatoms were a usual feature of the shell surfaces. The micraesthete subsidiary caps normally appear featureless, but the megalaesthete apical caps sometimes appear to be perforated. The reasons for this perforate appearance are discussed and it is concluded that it provides no evidence for the normal passage of substances out of or into the megalaesthete.I wish to thank the staffs of the Bio-engineering Unit, University of Strathclyde, Glasgow, and the Macaulay Soil Science Institute, Aberdeen, for help and the provision of scanning electron microscope facilities     

Osteoblasts and collagen orientation

Summary Bone was removed from the calvaria of anaesthetized 70 g rats or freshly killed young monkeys and the fibrous periosteum dissected off the inner, formative surface under 0.15 M cacodylate buffer. The bone and undisturbed osteoblasts were fixed in 3% glutaraldehyde in the same buffer for 24 to 48 hours, critical point dried and coated with evaporated carbon and gold for scanning electron microscopy (SEM). Fields of osteoblasts were photographed and chosen cells dissected off the osteoid using a tungsten needle. The control of the dissection was made possible by the use of a system of real-time stereo TV-speed SEM. The fields were rephotographed and the orientations of the osteoblasts were compared with that of the underlying collagen fibres. 62% of all osteoblasts lay with their long axes within 15° of the collagen fibre orientation below and 80% within 30°. Montages of large areas of osteoblasts were also made, and then compared with ones of the same area after the cells had been stripped off on adhesive tape. In general, the orientation of the collagen tended to be the same as the cell that formed it. Collagen fibres below cells at the periphery of a domain sometimes had the orientation of the cells in the adjacent patch. It is not possible to determine whether the cells controlled the orientation of the collagen, or vice versa, from this experiment, but other SEM evidence suggests that the collagen orientation in hard tissue matrices depends on the freedom of cells to move with respect to the matrix surface.Acknowledgements. This work has been supported by generous grants from the Medical Research Council and the Science Research Council. We are grateful to Elaine Bailey and Mr. P. Reynolds for technical assistance.     

元麦叶肉原生质体细胞壁再生过程中的扫描电镜观察及过氧化物酶活性

元麦叶肉原生质体在MS培养基(附加2,4-D 1mg/L,6-BA 0.25 mg/L)中,进行液体浅层培养。用荧光增白剂(VBL)染色,培养1天出现再生壁。通过扫描电镜观察,发现随着培养时间的延长,原生质体表面逐渐出现短棒状突出物和纤维状结构;培养第5天,原生质体表面覆盖较厚的纤维层,与未脱壁的元麦叶肉细胞表面形态结构相似。用愈创木酚作氢供体测定原生质体胞壁再生过程中过氧化物酶活性,发现随着壁再生率提高,过氧化物酶活性明显下降。用聚丙烯酰胺凝胶电泳分离阳极向过氧化物酶同工酶酶谱,酶带也随着培养时间的延长而减少。由刚分离的原生质体中的8条减少到培养4天的2条,反映胞壁再生和过氧化物酶活性呈负相关。     

A preliminary optical and electron microscopic study of the β<Subscript>1</Subscript> integrin distribution pattern of human osteosarcoma-derived cells

Immunogold labelling was used to study the organisation of the ₁ integrins on osteosarcoma-derived osteoblasts (Saos-2 and MG-63). Monolayers of cells were prepared in multiwell culture plates on both uncovered and collagen-covered coverslips, and ₁ integrins were primarily labelled using mouse monoclonal antibodies to ₁ integrins. Indirect immunofluorescence labels using an anti-mouse fluorescein-conjugated goat antibody showed an even distribution of the ₁ integrins on the cell membranes of all cell types used. A concentration of 2 g/ml of the primary antibodies and a 1:100 dilution of the secondary antibodies were determined as the optimal concentration for labelling to use with indirect localisation of the primary antibodies gold conjugated to goat anti-mouse antibodies and viewed under an electron microscope. Ten nanometre gold particles were used for transmission electron microscopy (TEM) and 40 nm gold particles for scanning electron microscopy. TEM showed that ₁ integrins were mainly clustered on the cell membrane processes with less labelling on the cell membranes themselves. The distribution of ₁ integrins on osteosarcoma cells supports the concept that integrins may function by forming focal adhesions at the site of the cytoplasmic membrane processes.     

Morphological changes of osteoblasts in vitro

Summary The appearance of neonate rat endocranial osteoblasts exposed in situ and fixed immediately was compared with that of similar osteoblasts organ cultured for short periods of up to 48 hours in control medium and serum alone, or with added parathyroid extract (PTE). Normal osteoblasts showed a range of variation in size and shape, degree of elongation and orientation. Culturing the osteoblasts resulted in an overall loss of elongation and ordering of the cells, and the production of dorsal ruffles which were more complex and larger in the longer culture times. PTE added to the culture medium caused an increase in cell elongation and a striking recordering of the osteoblasts into domains of parallel cells. The swirling patterns made by these domains were similar to those of the underlying bone collagen. Ruffles, where present, were small and more often peripheral than dorsal.The results indicate that fully differentiated osteoblasts are able to adapt very rapidly to survival and function in culture conditions, and that the endocytosis necessary for synthetic activity is suppressed by PTE although some cell movement may continue.This work has been supported by grants from the Medical Research Council and the Science Research Council.We would like to thank Elaine Bailey and Philip Reynolds for technical assistance, and Dr. Martin Evans for helpfully providing the facilities of his laboratory.     

培养破骨细胞扫描电镜样本制备方法的探讨

目的：探讨体外培养的破骨细胞在自制牛股骨磨片和细胞爬片中扫描电镜制备方法。方法：实验分两组,一组采用新鲜牛股骨制备成5mm×5mm大小的薄片,作为共培养之需;另一组,采用盖玻片制成5mm×5mm的细胞爬片。分别以5×104种植于骨磨片和爬片,培养5天后进行扫描电镜的制备并观察。结果：破骨细胞在牛骨磨片表面生长良好,充分伸展,有细胞突起伸入到实验组材料深部,并形成骨陷窝;在爬片表面生长的破骨细胞,细胞生长良好,粘附性强,细胞之间相互连接较紧密,细胞表面突起明显。结论：牛股骨磨片与破骨细胞在体外相容良好,材料有利于破骨细胞的生长及细胞功能的表达,而破骨细胞爬片更适于细胞外形的观察。将两种方法结合既能反映破骨细胞的形态结构又能展示其破骨功能。     

培养破骨细胞扫描电镜样本制备方法的探讨

目的:探讨体外培养的破骨细胞在自制牛股骨磨片和细胞爬片中扫描电镜制备方法。方法:实验分两组,一组采用新鲜牛股骨制备成5mm×5mm大小的薄片,作为共培养之需;另一组,采用盖玻片制成5mm×5mm的细胞爬片。分别以5×104种植于骨磨片和爬片,培养5天后进行扫描电镜的制备并观察。结果:破骨细胞在牛骨磨片表面生长良好,充分伸展,有细胞突起伸入到实验组材料深部,并形成骨陷窝;在爬片表面生长的破骨细胞,细胞生长良好,粘附性强,细胞之间相互连接较紧密,细胞表面突起明显。结论:牛股骨磨片与破骨细胞在体外相容良好,材料有利于破骨细胞的生长及细胞功能的表达,而破骨细胞爬片更适于细胞外形的观察。将两种方法结合既能反映破骨细胞的形态结构又能展示其破骨功能。