Expression of an Antigen Associated with Basal Bodies of Human Ciliated Epithelial Cells

The process and regulation of ciliogenesis in human epithelia is little understood and many components of the cilium and associated structures have not been characterised. We have identified a monoclonal antibody, LhS28, which recognises a 44,000–45,000Mr protein specifically associated with human ciliated epithelial cells. Immunoperoxidase labelling of formalin-fixed paraffin wax-embedded human tissues showed that LhS28 was expressed in the sub-apical zone of ciliated epithelial cells of the Fallopian tube and upper respiratory tract, but not ciliated ependyma, non-ciliated epithelia or testis containing developing spermatozoa. Immunoelectron microscopy demonstrated that the antigen recognised by LhS28 was associated with the basal body structure of the cilium and specifically with the 9+0 microtubule arrays. LhS28 should be a useful tool in the identification of ciliated cells in pathological specimens and for investigating mechanisms of ciliogenesis.     

High resolution detection of uncoated metaphase chromosomes by means of field emission scanning electron microscopy

HeLa metaphase chromosomes were exammed by means of in lens field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosomes as a model for comparison we report evidence of how traditional scanning electron microscopy techniques such as metal coating and conductive methods can generate errors in chromosome structure evaluation, since both give rise to morphological artifacts. By comparing the morphology of uncoated chromosomes obtained by two different isolation procedures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cytogenetic method gives rise to a chromosome structure consisting of a flattened network of 10 nm fibers, in which higher order chromatin organization is absent. (b) Chromosomes obtained by the polyamine method show both three-dimensional profile and higher level folding of chromatin fibers, supporting the loop chromosome organization previously suggested by scanning electron microscopy observation of hexylene glycol isolated chromosomes.     

Estimating the age of Antarctic larval fish from otolith microstructure using light and electron microscopy

Larval fish of Antarctica have very narrow rings on their otoliths (<1 m) that may not be resolved with light microscopy. In this study, age data from the otoliths of larval Nototheniidae (Gobionotothen gibberifrons and Lepidonotothen larseni), determined using light and scanning electron microscopy, are compared. Rings 0.4 m wide were observed on otoliths viewed under electron microscopy; however, light microscopy could only resolve rings 0.5 m wide. Scanning electron microscopy is more time consuming and costly than light microscopy but has greater resolving power and is recommended to validate ring counts made using light microscopy in otolith studies with Antarctic larval fish.     

Cluster microvilli in coronary endothelium

Summary Examination of cardiac vascular endothelium with scanning electron microscopy, and transmission electron microscopy of previously scanned tissue, revealed several regions of the coronary venous system that contained cluster microvilli. These consisted of 2–15 microvillous projections that emanated radially from a common base or were grouped into a fan-like arrangement. Although rare, these clusters, when present, were widely distributed over the endothelial cell surface.Dr. Smolich is the recipient of a postgraduate scholarship from the National Health and Medical Research Council of Australia     

Water droplets and ice deposits in leaf intercellular spaces: redistribution of water during cryofixation for scanning electron microscopy

An experimental study is described of the formation of extracellular deposits on the surfaces of cells in freeze-fractured, frozen-hydrated primary leaves of Phaseolus vulgaris examined by low-temperature scanning electron microscopy. The deposits, observed under a range of experimental conditions, consisted of (a) droplets with diameters of 1.5 to 3.0 m, (b) droplets with diameters of 10 to 30 m, (c) crystals with diameters of 1.0 to 6.0 m, and (d) granules with diameters up to 0.15 m. The types of deposit were influenced by specimen cooling rate, and their distribution was influenced by the direction of the thermal gradient during cooling. All deposits were predominantly water ice. The quantities of deposited water (up to 4.0% of the leaf water content) increased as the cooling rate was reduced. It is concluded that the ice deposits were primarily artefacts of cryofixation and do not represent the location of water in vivo, as recently suggested. We propose that the deposits arose in four main ways: (1) displacement of water from underlying cells by a pressure wave resulting from the volume increase of intracellular water as it freezes, (2) evaporation of water from warmer cells and its condensation onto colder cells, (3) withdrawal of water from underlying cells by extracellular ice crystallization, (4) condensation of pre-existing water vapour in the intercellular spaces onto cells. The significance of the findings is discussed in relation to the use of lowtemperature scanning electron microscopy in studies of plant morphology and for localizing water and soluble ions within plant cells and tissues.Abbreviation LTSEM low-temperature scanning electron microscopy     

The organization of microtubules during generative-cell division inConvallaria majalis

Summary The organization of the microtubule cytoskeleton in the generative cell ofConvallaria majalis has been studied during migration of the cell through the pollen tube and its division into the two sperm cells. Analysis by conventional or confocal laser scanning microscopy after tubulin staining was used to investigate changes of the microtubule cytoskeleton during generative-cell migration and division in the pollen tube. Staining of DNA with 4,6-diamidino-2-phenylindole was used to correlate the rearrangement of microtubules with nuclear division during sperm cell formation. Before pollen germination the generative cell is spindle-shaped, with microtubules organized in bundles and distributed in the cell cortex to form a basketlike structure beneath the generative-cell plasma membrane. During generative-cell migration through the pollen tube, the organization of the microtubule bundles changes following nuclear division. A typical metaphase plate is not usually formed. The generative-cell division is characterized by the extension of microtubules concomitant with a significant cell elongation. After karyokinesis, microtubule bundles reorganize to form a phragmoplast between the two sperm nuclei. The microtubule organization during generative-cell division inConvallaria majalis shows some similarities but also differences to that in other members of the Liliaceae.Abbreviations CLSM confocal laser scanning microscopy - EM electron microscopy - GC generative cell - GN generative nucleus - MT microtubule - SC sperm cell - SN sperm nucleus - VN vegetative nucleus     

Innervation and vascular supply of the crayfish opener muscle: Scanning and transmission electron microscopy

Summary Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were employed to study the innervation and vascular supply of crayfish skeletal muscle.Blood vessels and nerve terminals identified by TEM were often closely associated. Synaptic regions of the nerve terminals were always located under sarcolemma and contained both dense-cored and agranular synaptic vesicles. Axo-axonal synapses of several different types were observed. Blood vessels consisted of several vessel cells or supporting cells enclosing a lumen, which was connected to the exterior by fine channels between the supporting cells.SEM of whole freeze-dried muscles revealed two types of ramifying structure, which often ran in parallel over the muscle surface. One, identified as nerve, was more cylindrical and had a smoother surface than the other, which was identified as blood vessel. Fine nerve branches disappeared under the sarcolemma, probably near synaptic regions, but synapses could not be seen. Blood vessels also had fine terminations which merged into the sarcolemma.Supported by grants from the National Research Council of Canada and The Muscular Dystrophy Association of Canada. The technical assistance of Mr. M. Uy is acknowledged. Dr. F. Lang held a Postdoctoral Fellowship from the Muscular Dystrophy Association of Canada. Acknowledgement is made for the use of the scanning electron microscope in the Royal Ontario Museum, established through a grant from N.R.C. to the Department of Zoology, University of Toronto for the development of a program in systematic and evolutionary Zoology.     

Direct plant regeneration from leaf explants of Drosera rotundifolia cultured in vitro

Shoot regeneration was obtained from isolated leaves of Drosera rotundifolia L. cultured on MS media with various concentrations of 6-benzyladenine (BA) and -naphthaleneacetic acid (NAA). The best direct shoot organogenesis was obtained on growth regulator-free medium or medium supplemented with 10^-8 M NAA. Liquid culture medium significantly increased regeneration capacity of leaf tissue. Histological and scanning electron microscopy investigations verify direct plant regeneration without intermediate callus formation. Leaf epidermal cells showed the highest regeneration potential leading to the regeneration of buds. Young shoots with three to seven leaflets rooted spontaneously on the growth regulator-free medium within 38 days of culture and isolated mature plants produced fertile seeds.Abbreviations BA 6-benzyladenine - FAA 40% formalin (5%) +90% acetic acid (5%) +70% ethanol (90%) - ME Murashige and Skoog's (1962) medium - NAA -naphthaleneacetic acid - plumbagin 5-hydroxy-2-methyl-1,4-naphthoquinone - 7-methyljuglone 7-methyl-5-hydroxy-1,4-naphthoquinone - SEM scanning electron microscopy - TEM transmission electron microscopy - PPF photosynthetic photon flux     

Complex relationships between the pineal organ and the medial habenular nucleus-pretectal region of the mouse as revealed by S-antigen immunocytochemistry

Summary S-antigen-immunoreactive pinealocytes located in the deep portion of the pineal organ of inbred and wild pigmented mice give rise to long, beaded processes penetrating into the habenular and pretectal regions. In addition, the medial habenular nuclei and the pretectal area contain S-antigen-immunoreactive perikarya, which resemble pinealocytes in size, shape and immunoreactivity and are considered as pinealocyte-like epithalamic cells. Immunoblotting techniques reveal that a single protein band of approximately 48 kDa molecular weight accounts for this immunoreactivity. As shown with the use of the electron microscope, the majority of the S-antigen-immunoreactive processes is closely apposed to immunonegative neuronal profiles and perikarya of the habenular and pretectal regions. S-antigen-immunoreactive processes and perikarya of both pinealocytes of the deep pineal organ and pinealocyte-like epithalamic cells may form the postsynaptic element in conventional synapses involving axons provided with clear synaptic vesicles. Thus, certain mammalian pinealocytes may receive and transmit signals via point-to-point connections resembling neuro-neuronal contacts. These results challenge the concept that the mammalian pineal organ exerts its influence exclusively via the release of melatonin into the general circulation. Furthermore, they provide evidence (i) that neuronal circuits not involving the sympathetic system participate in the regulation of pineal functions in mammals, and (ii) that intimate histogenetic and functional relationships exist between the pineal organ and the habenular-pretectal nuclei in mammals.     

Bovine olfactory and nasal respiratory epithelium surfaces

Summary High-voltage transmission electron microscopy and cryo-ultramicrotomy together with scanning electron microscopy and some conventional transmission electron microscopy of ultrathin sections have been applied to the mucous surfaces of bovine olfactory and respiratory epithelia. Distal segments of olfactory cilia tend to run in parallel and could be followed over distances up to about 30 m using high-voltage electron microscopy. This technique and scanning electron microscopy showed that on average 12–13 of such cilia could be observed per nerve ending. After correction for obscured cilia this number becomes about 17. High-voltage micrographs and micrographs made from sections prepared with a cryo-ultramicrotome showed the presence of electron-lucent pockets inside the olfactory mucus. The latter technique also showed that the mucus itself is not fibrous, but rather a continuum varying in electron density. The mucus layer contains various granular structures. Ciliary and microvillar membranes appear thicker with cryo-ultramicrotomy than when the sections are prepared with conventional techniques. The cores of the axonemal microtubules in olfactory as well as in respiratory cilia are darkly stained with this technique. Vesicles present inside the nerve endings are also darkly stained. Dimensions and some other numerical values of interest in olfaction are presented.     

A microscopic investigation of enamel in wombat (Vombatus ursinus)

Summary A study of the enamel of continuously growing Vombatus ursinus molars was carried out using the techniques of light microscopy, hardness testing, scanning electron microscopy and transmission electron microscopy. From the erupted end to within 8 mm of the growing end, mature enamel was observed and it was found that between comparable areas there were no significant ultrastructural differences in enamel; however, small (12nm diameter), loosely packed needle-like crystals characteristic of developing enamel were observed near the growing end. Mature enamel was found to consist of three optically-translucent regions interleaved with two opaque regions. Opaque enamel was softer than translucent enamel. The opacity and relative softness characteristic of two of the enamel regions was not related to prism pattern or orientation; it was, however, related to the presence of voids (28 nm diameter) in these regions.     

Fine structure of the dorsal lingual epithelium of the frog, Rana rugosa

Scanning and transmission electron microscopy was employed to investigate the ultrastructure of the lingual dorsal epithelial cells of the frog, Rana rugosa. The specimens for scanning electron microscopy were prepared by a method that involved osmium postfixation and treatment with acid to remove extracellular material that adhered to the surface of the tongue. Over almost the entire dorsal surface, filiform papillae, consisting of a large number of non-ciliated cells with microridges and a very small number of ciliated cells, were compactly distributed. Fungiform papillae were scattered among these filiform papillae. A round sensory disk was located on the top of each fungiform papilla. Each sensory disk was encircled by a band of ciliated cells. Transmission electron microscopy revealed that a large part of the filiform papillar epithelium was composed of cells that contained numerous electron-dense granules. These cells were coincident with the non-ciliated cells observed by scanning electron microscopy. In these cells, the nucleus was located on the basal side, and the ergastoplasm was well-developed on the basal side of the nucleus.     

Effects of microprojectile bombardment on embryogenic suspension cell cultures of maize (Zea mays L.) used for genetic transformation

We have investigated the interaction between tungsten and gold microprojectiles with suspension-culture cells of maize used for genetic transformation. Particle size measurements were evaluated before and after DNA precipitation to determine mean particle size and the effect of DNA precipitation on particle aggregation. Following particle bombardment, metal foils were examined by scanning electron microscopy to visualize dispersion of individual particles and aggregates. Particle penetration into suspension-culture cell clusters was examined in paraffin-embedded bombarded cells serially sectioned and viewed with light microscopy and by energy dispersive X-ray microanalysis. Acridine-orange-stained bombarded cells were examined to observe cellular response to particle penetration. Transient expression of reporter genes C1 and B and GUS, (-glucuronidase) were used to assess effects of particle bombardment on embryogenic cell types. Autoradiographic analysis of the transformable suspension cell culture SC82 (see Gordon-Kamm et al. 1990, Plant Cell 2, 603–618) was conducted to evaluate the S-phase and mitotic indices in embryogenic and nonembryogenic cells throughout a subculture passage and in response to DNA/particle delivery. The results of these investigations are discussed relative to cytodifferentiation of suspension cell clusters and recovery of transformed clonal sectors.Abbreviations GUS -glucuronidase - FAA formaldehyde-acetic acid-alcohol - SEM scanning electron microscopy     

Intraventricular blood vessels associated with the deep pineal gland of the Mongolian gerbil,Meriones unguiculatus

Summary Intraventricular blood vessels and choroidal-like cells were studied using scanning electron microscopy and correlative light microscopy. The intraventricular blood vessels were covered on their ependymal surface with a layer of cells essentially identical to the ependyma of the choroid plexus in the gerbil. Similar choroidal-like cells were seen either singly or in clusters associated with the cerebrospinal fluid-contacting pinealocytes of the suprapineal recess. Processes of the cerebrospinal fluid-contacting pinealocytes were seen extending to and making contact with the choroidal-like cells. The intraventricular blood vessels appeared to be derived from the choroid plexus, and typically took one of three courses in and around the surface of the deep pineal: (1) the vessels or their equivalent were located in the suprapineal recess with no indication of penetration into the substance of the deep pineal; (2) the vessels coursed from the suprapineal recess around the anterior surface of the habenular commissure to enter the ventral surface of the deep pineal; or (3) the vessels entered the parenchyma of the deep pineal from its dorsal surface and could be seen coursing through the substance of the gland. The close association between the choroidal-like cells and the intraventricular blood vessels with the deep pineal gland add morphological support for the possibility of interaction between the cerebrospinal fluid, or perhaps the choroid plexus, and the deep pineal gland.     

Stomata of apple leaves cultured in vitro

Examination by scanning electron microscopy showed abaxial stomata on in vitro cultured apple (Malus pumila Mill.) leaves. With leaf ontogeny, most of these stomata appeared to lose their regulatory ability while developing wide vestibules of up to 20 m in diameter. It is proposed that these deformed stomata may be a possible cause for the excessive transpirational water loss and consequent dehydration associated with transferring plants regenerated in vitro from culture.     

Surface morphology of the subfornical organ in the rabbit's brain

Summary The ventricular surface of the subfornical organ of the rabbit's brain was studied with scanning and transmission electron microscopic techniques. The ependymal covering was found to consist of hexagonal cells with convex apical surfaces. From the center of each cellular surface a single kinocilium up to 6 m in length protrudes into the liquor. It is usually covered with secretory material having the shape of pearlstrings. The surface aspect of the subfornical organ suggests secretion into the liquor by emptying of giant vacuoles which originate below the ependyma in nerve cells, move towards the surface, develop pressure while flattening their ependymal cover and finally erupt, leaving collapsed ependyma- and/or nerve cells bag on the surface of the organ. A second mechanism of more granular secretion by ependymal cells appears possible.We are indebted to Fräulein E. Östermann, Frau L. Schulze and Frau H. Zuther-Witzsch for excellent technical assistance.     

Rose leaf structure in relation to different stages of micropropagation

Summary The Mme Isaac Pereire rose was investigated in an attempt to establish how micropropagated roses might best be weaned into normal growth conditions. Leaves of in vitro grown plants, weaned plants and the stock plant were studied, using light microscopy and different scanning and transmission electron microscopical techniques. Features that varied in the different growing conditions were leaf size and thickness, amount of wax, thickness of cuticle and external epidermal cell wall, number and aperture of the stomata, size of the epidermal cells, number of layers of the palisade cells, and size of the chloroplasts in the mesophyll. The rose in the present study had wax on the in vitro cultured plants; this wax was of similar ultrastructural appearance to that of the stock plant, even though in smaller quantities. Weaned plants had an intermediate amount of wax. The cuticle was thin, ranging from 0.04 m on plants growing in vitro to 0.3-0.6 m on weaned plants and stock plants. Stomata were always wide-open on leaves taken from cultures with a relative humidity of 100%. After four weeks in a humidity lowered to 85% stomata had closed.Abbreviations BAP 6-benzyl-aminopurine - CPD critical point drier - CTEM conventional transmission electron microscopy - NAA a-naphthaleneacetic acid - psi pounds per square inch - SEM scanning electron microscopy - TEM transmission electron microscopy     

Ultrastructure of elastic cartilage in the rat external ear

Summary The structure of elastic cartilage in the external ear of the rat was investigated by transmission and scanning electron microscopy.The narrow subperichondrial, boundary zone contains predominantly ovoid cells rich in cell organelles: mitochondria, Golgi complex, granular endoplasmic reticulum and small (40–100 nm) vesicles. Scarce glycogen granules and bundles of 6–7 nm cytoplasmic filaments are also present. Deeper in the boundary zone, one or more cytoplasmic lipid droplets appear and cytofilaments become more abundant.Fully differentiated chondrocytes in the central zone of the cartilage plate resemble white adipose cells. They are globular and contain a single, large cytoplasmic lipid droplet. The cytoplasm is reduced to a thin peripheral rim; it contains a flattened nucleus, few cytoplasmic organelles and abundant, densely packed, cytoplasmic filaments.The intercellular matrix is very sparse. The pericellular ring consists of collagen fibrils about 20 nm in diameter and a proteoglycan cartilage matrix in the form of a stellate reticulum. The complex of these two structures appears in the scanning electron micrographs as a network of randomly oriented, ca 100 nm thick fibrils. Spaces between pericellular rings of matrix also contain thick elastic fibers or plates, apparently devoid of microfibrils. In scanning electron micrographs elastic fibers could be detected only in a few areas, in which they were not obscured by other constituents of the matrix. Immature forms of elastic fibers, oxytalan (pre-elastic) and elaunin fibers, were found in the perichondrial and boundary zones.     

Appearance and endocytic activity of epithelial cells from human efferent ducts in primary monolayer culture

Summary The culture of epithelial cells lining human efferent ducts, obtained from prostatic carcinoma patients, is described. Ciliated cells were observed to beat for at least one month on plastic. On pervious filters low cuboidal cells characterized the monolayers. Cells comprising monolayers over the filter were 5 to 9 m in height whereas taller cells were found over the original fragments (14 m). Some non-ciliated cells contained dark and light vacuoles, others were found to lack them. Both non-ciliated and ciliated cells maintained tight junctional complexes restricting the paracellular movement of horseradish peroxidase. Both types of cultured cells exhibited fluid-phase and adsorptive endocytosis from both apical and basal surfaces. It is reported for the first time that the monolayers form high resistance barriers (150 cm²) that prevent the apical medium from draining to the basal compartment over 24 h.     

Microstructural analysis of anaerobic granules

Summary Photos of scanning and transmission electron microscopy showed that sludge granules treating sucrose wastewater had a 20–40 m surface layer with diverse morphology, consisting of cocci and bacilli, and a loosely packed interior, mainly consisting of Methanothrix. Light microscopy using epi-fluorescent excitation showed not only the similar microstructure, but also the distribution of various genuses of methanogens.