Nanoscale topography of the basement membrane underlying the corneal epithelium of the rhesus macaque

This paper quantitatively defines the nanoscale topography of the basement membrane underlying the anterior corneal epithelium of the macaque. Excised corneal buttons from macaques were placed in 2.5 mM ethylenediaminetetraacetate (EDTA) for 2.5 h, after which the epithelium was carefully removed to expose the underlying basement membrane. The integrity of the remaining basement membrane was verified using fluorescent microscopy in conjunction with antibody staining directed against laminin and collagen type IV as well as transmission electron microscopy. Characterization of the surface of the basement membrane was performed using transmission electron microscopy, high-resolution, low-voltage scanning electron microscopy, and atomic force microscopy. Quantitative data were obtained with all three imaging techniques and compared. The basement membrane has a complex topography consisting of tightly cross-linked fibers intermingled with pores. The mean elevation of features measured by transmission electron microscopy, scanning electron microscopy, and atomic force microscopy was 149 +/- 60 nm, 191 +/- 72 nm, and 147 +/- 73 nm, respectively. Mean fiber diameter as measured by SEM was 77 +/- 44 nm and pore diameter was 72 +/- 40 nm, with pores occupying approximately 15% of the total surface area. Similar feature types and dimensions were also found for Matrigel, a commercially available basement membrane-like complex, supporting that a minimum of artifact was introduced by corneal preparative procedures to remove the overlying epithelium. Topographic features amplified the surface area over which cell-substratum interactions occur by an estimated 400%. The three-dimensional structure of the basement membrane exhibits a rich complex topography of individual features, consisting of pores and fibers with dimensions ranging from 30 to 400 nm. These nanoscale substratum features may modulate fundamental cell behaviors such as adhesion, migration, proliferation, and differentiation.     

Combined immunofluorescence and field emission scanning electron microscope study of plasma membrane-associated organelles in highly vacuolated suspensor cells of white spruce somatic embryos

Highly vacuolated suspensor cells of spruce somatic embryos were examined by immunofluorescence light microscopy using butyl-methyl-methacrylate (BMM) and polyethylene glycol (PEG) embedded sections, transmission electron microscopy (TEM) and field emission scanning electron microscopy (FESEM). The use of PEG embedded embryos provided a rapid method for light microscope detection of antigens before committing to FESEM analysis. BMM embedded specimens provided well preserved suspensor cells for immunofluorescence. FESEM permitted high resolution observation of large areas of the inner surface of the plasma membrane and associated cell organelles. Suspensor cells contained mostly transversely oriented cortical microtubules linked to the plasma membrane and adjacent microtubules by cross- bridges. Light and electron microscopy revealed numerous clathrin coated structures on the plasma membrane. These included flat patches of clathrin, coated pits and coated vesicles. Many coated vesicles were associated with microtubules. Both tubular and lamellar endoplasmic reticulum were observed on the plasma membrane by FESEM.     

Use of a Technovit 7200 VLC to Facilitate Integrated Determination of Aluminum by Light and Electron Microscopy

Technovit 7200 VLC is an excellent embedding medium for both inorganic histochemistry by light microscopy and X-ray microanalysis by scanning and transmission electron microscopy. Liver samples from rats after intraperitoneal treatment with aluminum chloride were fixed in glutaraldehyde and embedded in the resin. Thick sections were easily cut on an ultramicrotome and stained with aluminon for aluminum (Al). An intense positive reaction with aluminon was observed in the Kupffer cells by light microscopy. The surface structures of the same resin block cut for light microscopy were observed under a scanning electron microscope fitted with an energy dispersive X-ray spectrometer. The Kupffer cells appeared white in the backscattered mode. Localization of Al in the Kupffer cells was confirmed by an X-ray distribution map in the scanning electron microscope. Subcellular localization of Al in the Kupffer cells was performed on the same semithin sections using a transmission electron microscope equipped with an energy dispersive X-ray spectrometer. Most Al was found in lysosomes of the Kupffer cells. The resin was stable in the electron beam and chlorine-free.     

Preparation of chromosome spreads for electron (TEM,SEM, STEM), light and confocal microscopy

In the past, ultrastructural studies on chromosome morphology have been carried out using light microscopy, scanning electron microscopy and transmission electron microscopy of whole mounted or sectioned samples. Until now, however, it has not been possible to use all of these techniques on the same specimen. In this paper we describe a specimen preparation method that allows one to study the same chromosomes by transmission, scanning-transmission and scanning electron microscopy, as well as by standard light microscopy and confocal microscopy. Chromosome plates are obtained on a carbon coated glass slide. The carbon film carrying the chromosomes is then transferred to electron microscopy grids, subjected to various treatments and observed. The results show a consistent morphological correspondence between the different methods. This method could be very useful and important because it makes possible a direct comparison between the various techniques used in chromosome studies such as banding, in situ hybridization, fluorescent probe localization, ultrastructural analysis, and colloidal gold cytochemical reactionsAbbreviations CLSM confocal laser scanning microscope - EM electron microscopy - kV kilovolt(s) - LM light microscope - SEM scanning electron microscope - STEM scanning-transmission electron microscope - TEM transmission electron microscope     

Primary cilia in vertebrate corneal endothelial cells

The presence of primary cilia in corneal endothelial cells of a range of species from six non-mammalian vertebrate classes (Agnatha, Elasmobranchii, Amphibia, Teleostei, Reptilia and Aves) is examined by scanning and transmission electron microscopy. Our aim is to assess whether these non-motile cilia protruding into the anterior chamber of the eye are a consistent phylogenetic feature of the corneal endothelium and if a quantitative comparison of their morphology is able to shed any new light on their function. The length (0.42-3.80 microm) and width (0.12-0.44 microm) of the primary cilia varied but were closely allied with previous studies in mammals. However, interspecific differences such as the presence of a terminal swelling in the Teleostei and Amphibia suggest there are functional differences. Approximately one-third of the endothelial cells possess cilia but the extent of protrusion above the cell surface varies greatly, supporting a dynamic process of retraction and elongation. The absence of primary cilia in primitive vertebrates (Agnatha and Elasmobranchii) that possess other mechanisms to control corneal hydration suggests an osmoregulatory and/or chemosensory function.     

Ponceau-Fuchsin as a Routine Countebstain

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

Pore canals in the cornea of a functionally specialized area of the honey bee's compound eye

Summary The fine structure of the cornea in an anatomically and functionally specialized part of the honey bee's compound eye (dorsal rim area) was examined by light microscopy, transmission electron and scanning electron microscopy. Under incident illumination the cornea appears grey and cloudy, leaving only the centers of the corneal lenses clear. This is due to numerous pore canals that penetrate the cornea from the inside, ending a few m below the outer surface. They consist of (1) a small cylindrical cellular evagination of a pigment cell (proximal), and (2) a rugged-walled, pinetree-shaped extracellular part (distal). The functional significance of these pore canals is discussed. It is concluded that their light scattering properties cause the wide visual fields of the photoreceptor cells measured electrophysiologically in the dorsal rim area, and that this is related to the way this eye region detects polarization in skylight.     

Back-scattered electron imaging of sections through the cochlea: a new technique for studying cochlear morphology

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

Development of alveolar septa and formation of alveolar pores during the early postnatal period in the rat lung

In order to investigate the formation of alveolar pores, lungs of rats, after intratracheal perfusion of glutaraldehyde, are processed at postnatal days 1, 7, 14, 16 and 21 for light and transmission electron microscopy and at days 7 and 16 for scanning electron microscopy. The initial low secondary crests of day 1 rapidly elongate to pleats subdividing the primary saccules. The ledges of some pleats partly grow toward each other as ring like diaphragms, leaving openings whose boundary is composed of alveolar epithelium separated by a basal lamina from a connective tissue sheath with capillaries. At day 7, in scanning electron microscopy the lumina of some rudimentary alveoli communicate by apertures of different sizes, as a result of the outgrowth of curved alveolar pleats which narrow to a ring-like aperture. The interalveolar openings observed in scanning electron microscopy resemble those investigated by light and transmission electron microscopy. The number of interalveolar pores increases from day 7 on; they become more and more frequent at days 14, 16 and 21, respectively. It appears that alveolar multiplication in newborn rats proceeds not only by segmentation of terminal respiratory units but also by compoundment of septa. The difference between genuine pores and transsections of folds in transmission electron microscopy will be given closer attention in this study. Also, the incidence and location of type II pneumocytes during rapid enlargement of the alveolar surface area is discussed.     

Back-Scattered Electron Imaging of Sections Through the Cochlea: A New Technique for Studying Cochlear Morphology

A new technique for studying the morphology of the cochlea is described. The development of back-scattered electron (BSE) detectors has allowed the examination of heavy-metal stained tissues by scanning electron microscopy. Comparison with light microscopy on adjacent resin sections through whole decalcified cochleae demonstrated that the back-scattered electron technique provides equal or superior clarity and resolution throughout the light microscope range of magnification, allows identification of lysosomes, mitochondria and endoplasmic reticulum, and extends useful magnification into the range previously associated only with transmission electron microscopy. Back-scattered electron imaging enables the study of sections of the undissected cochlea at high magnifications and resolution.     

Transmission and scanning electron microscopic study of the same cytologic material

The same cytologic material was successively examined by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). After the SEM examination, the specimens were rehydrated for a long period of time to allow the penetration of Epon 812 into the cells. The TEM examination showed the cell organelles to be comparatively well preserved. These consecutively performed LM-SEM-TEM examinations provided useful information on cytologic subjects, especially concerning the origin of the cells.     

Palynology and systematics of morinaceae

Pollen grains from all thirteen species of the three genera to be recognised in a forthcoming revision of the Morinaceae, Acanthocalyx, Cryptothladia and Morina, have been investigated by light and scanning electron microscopy. Fresh pollen of M. longifolia has also been studied by light, scanning electron and transmission electron microscopy. Three main pollen types are described and a key provided for their identification. Each pollen type occurs throughout a genus. Further division of two of these pollen types enables groups of species to be recognised. Functional aspects of the pollen are discussed, especially their unique pre-germinative processes. A cladistic analysis of pollen characters provides a hypothesis for their evalution and supports the classification proposed in the recent revision.     

Preservation of Tracheal Mucus by Nonaqueous Fixative

Two nonaqueous fixatives, composed of fluorocarbon solvents with dissolved osmium tetroxide, were used to determine the feasibility of preserving the mucous coat in bovine and rat trachea for light and electron microscopy. Aqueous fixatives, while providing excellent cytological preservation, wash away the mucous lining, precluding ultrastructural analysis. Inclusion of ruthenium red or alcian blue within aqueous fixative improved retention of mucus, but provided incomplete, patchy results. Fixation with nonaqueous fluorocarbon solvent and dissolved osmium tetroxide preserved a continuous mucous epiphase layer above a clear hypophase layer. Subcomponents of the mucus included an electron dense surface layer, interrupted patches of mucus above the surface layer and electron dense membrane-like material within the mucus. This method of fixation will preserve mucus for light, scanning and transmission electron microscopy, using either intratracheal or immersion methods of fixation. The latter would enable use of materials from large animal models, autopsy or an abattoir.     

Echinococcus granulosus: cellular territories and morphological regions in mature protoscoleces

Basic aspects of the generation, structure and function of Echinococcus granulosus protoscoleces are unknown. We review the work done on the structure and ultrastructure of the E. granulosus protoscolex and provide new data together with a comprehensive view of this form of the parasite. The surface, as observed by scanning electron microscopy, tightly correlates with five cellular territories characterized in the interior using light and transmission electron microscopy as well as a histochemical technique. Three of these territories are surrounded by a basal lamina that is also present in the internal side of the tegument, suggesting a complex internal organization. These cellular territories correlate with the expression of specific genes and the regionalization of DNA synthesis in protoscoleces. Additionally, a proposal to explain movements of the body of this form of the parasite in relation to the neck or to the germinal layer of the hydatid cyst is provided.     

Non-granulated peripolar cells exist in the rat glomerulus

Summary The peripolar cell is a unique cell type in the mammalian glomerulus. Peripolar cells are said to be identifiable during light microscopy by their cytoplasmic granules and by their position at the vascular pole; and during scanning electron microscopy by their distinctive surface morphology. We used both techniques to count peripolar cells in 6 normal rat kidneys. Scanning microscopy revealed that 55(±5)% of glomeruli contained at least one peripolar cell whereas light microscopy revealed granulated peripolar cells in only 4(±2)% of glomeruli. Vascular poles which contained peripolar cells previously identified by scanning were then examined by light and by transmission electron microscopy. Serial sections through these peripolar cells demonstrated the absence of cytoplasmic granules. Our observations suggest that the majority of peripolar cells in the rat contain no granules.     

Morphology,morphometry and electron microscopy of HeLa cells infected with bovine Mycoplasma

Summary The host-parasite relationship of HeLa M cells artificially infected with a bovine species of Mycoplasma was studied by light microscopy, transmission electron microscopy and scanning electron microscopy. The use of morphometry to quantitate some of the findings was explored. The parasites were seen in locations extracellular to the cell surface. The detection of small numbers of organisms by light microscopy was well demonstrated by use of the fluorescent antibody technique. Scanning electron microscopy proved to be an excellent method for revealing the surface details of cell-parasite morphology. Ultra-thin sections showed that the parasites are aligned mostly parallel to the plasma membrane of the host cell but separated by a gap of 10 nm. Morphometry indicated an average of 69 organisms per cell surface occupying 1.7% of the surface area. An increase of 26% in diameter of the HeLa cells, possibly as a result of infection, was observed.The authors wish to thank Christiana Ulness and Andrea Erickson for expert technical assistance and Arnold Schmidt for the operation of the scanning electron microscope. This work was supported by grants from the U.S.P.H.S.: AI 09586, AI 10743, and AI 06720     

Extracellular compartments in matrix morphogenesis: collagen fibril, bundle, and lamellar formation by corneal fibroblasts

The regulation of collagen fibril, bundle, and lamella formation by the corneal fibroblasts, as well as the organization of these elements into an orthogonal stroma, was studied by transmission electron microscopy and high voltage electron microscopy. Transmission and high voltage electron microscopy of chick embryo corneas each demonstrated a series of unique extracellular compartments. Collagen fibrillogenesis occurred within small surface recesses. These small recesses usually contained between 5 and 12 collagen fibrils with typically mature diameters and constant intrafibrillar spacing. The lateral fusion of the recesses resulted in larger recesses and consequent formation of prominent cell surface foldings. Within these surface foldings, bundles that contained 50-100 collagen fibrils were formed. The surface foldings continued to fuse and the cell surface retracted, forming large surface-associated compartments in which bundles coalesced to form lamellae. High voltage electron microscopy of 0.5 micron sections cut parallel to the corneal surface revealed that the corneal fibroblasts and their processes had two major axes at approximately right angles to one another. The surface compartments involved in the production of the corneal stroma were aligned along the fibroblast axes and the orthogonality of the cell was in register with that of the extracellular matrix. In this manner, corneal fibroblasts formed collagen fibrils, bundles, and lamellae within a controlled environment and thereby determined the architecture of the corneal stroma by the configuration of the cell and its associated compartments.     

Conditions critical for optimal visualization of bacteriophage adsorbed to bacterial surfaces by scanning electron microscopy.

The potential of scanning electron microscopy as a tool for the detection of viruses on cell surfaces has been studied using bacteriophage P1 adsorbed to Shigella dysenteriae as a model system. Viral particles were readily detectable by scanning electron microscopy on the surface of infected cells which were fixed with glutaraldehyde followed by postfixation in OsO4 and prepared by critical point drying. The virus-studded surface of the infected cells differed markedly from the relatively smooth surfaces of uninfected control cells. Examination of the same preparations with transmission electron microscopy revealed numerous viral particles adsorbed to the surfaces of infected cells, whereas the control cells were free of viruses as expected. Glutaraldehyde fixation alone did not preserve the surface detail of infected cells: cells adsorbed with viruses were not distinguishable from control cells by scanning electron microscopy although by transmission electron microscopy viruses could be visualized. Air drying from water or absolute alcohol resulted in unsatisfactory preservation as compared to the appearance of infected cells prepared by the critical point method. Thus, scanning electron microscopy is capable of resolving viral particles on cell surfaces, but detection of these particles is completely dependent both on the method of fixation and on the technique of drying used.     

Morphometric analysis of the surface cells of rabbit corneal epithelium by scanning electron microscopy

The corneal surface of female New Zealand white rabbits (1.9-2.6 kg) was examined at x500 magnification by scanning electron microscopy. A total of 112 micrographs, taken as sequential sets from the center to the edge of the corneal surface from 8 different animals, was analyzed using a digitizer pad. Each cell was identified by the number of immediately bordering cells and by the nature of its electron reflex (light, medium, dark). Analysis of areas of the cells by number of bordering cells (number of cell sides) reveals a wide range of areas and skewed distributions especially when the number of sides is 5 or less. Overall, the cell-surface area increases as the number of cell sides increases. However, analyses of the mean surface areas for cells with different numbers of sides and additionally grouped by electron reflex suggests the existence of three separate populations of cells at the corneal surface. The possible etiology and dynamics of this complex cell mosaic are discussed in relation to circadian rhythms and to resurfacing of the cornea following mechanical trauma, ultraviolet radiation, and toxic chemical exposure.     

Sporangia containing Scylaspora from the Lower Devonian of the Welsh Borderland

ABSTRACT. Bulk maceration of Early Devonian (Lochkovian) deposits from the Welsh Borderland has yielded eight specimens of a new type of sporangium characterized by its elongate shape and distinctive spores. The specimens have been examined using scanning electron, transmission electron and light microscopy. The elongate sporangia occur isolated and are fragmented to varying degrees. They contain trilete spores that possess a proximal surface with shallow murornate ornament and a distal surface that is laevigate. The spores belong to the dispersed spore genus Scylaspora , and this is the first report of such spores in situ . Ultrastructural studies demonstrate that the spore walls are bilayered with a lamellate inner layer overlain by an essentially homogeneous outer layer. There is little or no associated extra-exosporal material. To date this is the earliest reported example of lamellate wall ultrastructure in trilete spores. Models of spore wall development are suggested in the light of evidence provided by spore wall ultrastructure. Detailed comparisons of the characters preserved in the fossils (morphological, anatomical and ultrastructural), with those in other fossil and extant plants, currently shed little light on the phylogenetic relationships of these specimens, primarily due to the paucity of comparable data. It is proposed that the plant is probably of vascular status, but in the absence of evidence for vascular tissue, it must be classified as rhyniophytoid.