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The field emission scanning electron microscope (FE-SEM) allows imaging resolution clown to 0.5-1.5 nm, With this improved resolution, the cryo-preparation technique (Cryo-FE-SEWI) can now compete with freeze-fracture replication techniques for the transmission electron microscope (TEM) and with standard TEM techniques that use ultra-thin sections of biological tissue after chemical fixation. Here we compare cryo-prepared animal tissue with TEM images of the same biological structures that have been chemically fixed, dehydrated in ethanol, embedded in Epon and ultra-thin sectioned.

Non-coating fixation methods, in particular the tannic acid/arginine/osmium tetroxide procedure, are employed for a number of reasons on the guinea-pig organ of Corti hair cell stereocilia glycocalyx and the imprints of the stereocilia at the bottom side of the tectorial membrane, and on the rat and cat intestinal epithelial microvilli glycocalyx and mucus-producing goblet cells. These methods are used firstly to confirm that noncoating prepared specimens can be embedded for TEM observation at 60-100 kV without loss of detail information, and these images can be compared with cryo-FE-SEM images of the same structure/tissue. Secondly, they show that specimens treated according non-coating techniques become optimally preserved and electrically conductive, so that no external conductive coating is required. In this way a comparison of images of subsequent fresh fracture faces is possible without a decrease in information on detail, which otherwise could happen after subsequent coating layers required after standard fixation. Thirdly they show that noncoating methods can be used quite well with low accelerating voltages because the osmium-tannic acid complex in the specimen surface produces a large number of backscattered and secondary electrons in the surface layer, showing in particular surface phenomena. Fourthly, they show that with an optimal non-coating preservation, in combination with a well-balanced pre-fixation mixture, preparation artefacts due to extraction and even dehydration and drying are minimized. This is compared with images of the organ of Corti hair cells treated with a so-called three-aldehyde prefixation mixture, which causes disrupted stereocilia to cling onto the bottom side of the tectorial membrane.

Visualization of structural details of specimens in field emission scanning electron microscopy (FE-SEM) requires optimal conductivity. This paper reports on the differences in conductive layers of Au/Pd, Pt and Cr, with a thickness of 1.5-3.0 nm, deposited by planar magnetron sputtering devices. The coating units were used under standard conditions for source-substrate distance, current, HT and argon pressure. Carbon films, deposited by high-vacuum evaporation on small, freshly cleaved pieces of mica, were used as substrate and mounted on copper grids for TEM and SEM inspection. Au/Pd, Pt and, to a lesser extent, Cr coatings varied in particle density, size and shape. Au/Pd coatings have a slightly more granular appearance than Cr and Pt coatings, but this is strongly dependent on the type of sputtering device employed. In FE-SEM images there is almost no difference in contrast and particle size between the Au/Pd layer and the Pt layers of a similar thickness. The nuclei of Au/Pd are rather small with almost no growth to the sides or in height, making Au/Pd coatings a good alternative to chromium and platinum for FE-SEM of biological tissues because of its higher yield of secondary electrons.

This paper describes cuticular structures on the abdomen of the Oriental hornet (Vespa orientalis, Vespinae, Hymenoptera) in the region of the yellow stripes. A cross section in this region reveals the cuticle to resemble a notebook with more than 30 pages, the topmost pages (analogous to layers) being the thicker (up to 5 mu m or more in thickness) while the ones underneath are gradually thinner. The exterior of the cuticle displays pores which are dispersed at distances of 10-50 mu m apart. These pores are actually cuticular depressions which frequently possess eaves on their cephalic side whose internal diameter is 1-3 mu m near the surface but further down, in the region of the yellow pigment and the hypocuticle, this internal diameter broadens to about 20-30 mu m. These pores thus extend from the exocuticle down to the hypocuticle. In the spaces between the pores in the endocuticular region there are sinuses. Within these sinuses granules of yellow pigment are located. As for the pores, each of them represents the external outlet of a canal that is perpendicular to the cuticle. The canal walls are composed of the same layers making up the cuticle. Structurally, the canal encasing the pore resembles an upside down arrow. This arrow extends to about 35-40 mu m in depth and is narrow and hollow in its upper part-the shaft-for about 1-3 mu m, broadens into the shape of an onion in its lower head part (some 20-30 mu m in diameter). Down to its tip it becomes slightly sharpened to close at the hypocuticle by forming a concentric, dome shape structure that terminates in a nipple-shaped protuberance. Permeating through all layers of the cuticle are hemolymph, nerve fibers and tracheae. The parallel lamellae associated in the cuticle give the impression of an electrical capacitor. The present article discusses the structure of the cuticle as a photovoltaic system in which the endocuticle-lamellar layer and yellow pigment serves as a solar cell linked to an electrical capacitor. The manner whereby light energy and heat are collected, converted into electric energy, accumulated, transformed and used by the hornet is discussed. We assume that this source of energy is used for their thermoregulation by thermoelectric circuits.

The stereociliar structures of the guinea-pig cochlear organ of Corti were studied at low-voltage (1-5 kV) with field-emission scanning electron microscope (SEM) using various pre- and post-fixation methods, such as OTOTO (OsO4/thiocarbohydrazide/OsO4/thiocarbohydrazide/OsO4) and TAO (tannic acid/arginine/OsO4), and different dissection procedures of the cochlea. A perfusion and immersion pre-fixation with glutaraldehyde, in combination with removal of the bony wall and stria vascularis from the cochlea, followed by the TAO non-coating treatment, gave the best result at 2 kV acceleration voltage. Due to these new techniques, several interesting delicate structures of the stereocilia, in particular fine surface structures, were detected for the first time using SEM. These findings include the different types of cross-links and tip links, i.e., the fine surface morphology of the stereocilia and their attachments and imprints in the tectorial membrane (TM). One of the most interesting findings in this study is a network of long filamentous structures, which has been identified mainly at the top of the longest stereocilia and the undersurface of the TM and which may represent the glycocalyx. These findings and their possible implications in the process of mechanoelectrical transduction will be discussed.

A UV-hardening lacquer material based on polyurethane, used in Philips compact disc lens systems, was tested as suitable material for low-cost intraocular lenses (IOLs). A slightly changed composition (code number I-0.5A) came out as the best and was subsequenly tested, with reference to poly(methyl methacrylate) (PMMA), as a 'lobster-claw' lens in Hollander rabbit eyes for a period of 24 wk. No carcinogenic effects were found, while only minor vascularization of the cornea at the incision area occurred. The in vitro biocompatability tests of the material were positive. Scanning electron microscopy images of the IOLs at 2, 6, 12 and 24 wk postoperative showed portions of an acellular membrane, multinuclear giant cells which hyalinize and stay as pigmented plaques, microphages and fibroblasts with fibrillar strands, long pseudopodia, and broad microplicea. The chosen UV lacquer showed a foreign body reaction and properties comparable with PMMA, and is suitable for manufacturing low-cost IOLs.

The Morgagnian cataract lenses - pre-fixed with GA for SEM & TEM and post-fixed with tannic-acid-arginine-OsO4 for SEM and OsO4/K4Fe(CN)6 for TEM after staining with Ur-acetate/Pb-citrate - showed areas in the cortex with radial in stead of concentric running lensfibres, degeneration of lensfibres with vacuoles and defected ''ball & socket'' connections. The presence of oval/spherical bodies of 0.5-20 mum was acknowledged, the largest of them having a golfball appearance and originating from the cytoplasm of the degenerating lensfibres; the smallest of them with an approximate size of 0.2-0.5 mum seemed to be formed by budding off from the microvilli of the furrowed lensepithelium. The Brunescens cataract lenses showed at low magnification no difference between lensfibres from the cortical area and the nucleus. The desintegration process of the lensfibres was observable as degradation of the ball & socket system and the existance of holes in the lensfibre body and emerging of spherical bodies from the cytoplasm. The globular structures seemed to be covered with a thin coating and were partly filled with a low density membranous-like material. In TEM-sections of the cataractous lensmaterial vacuoles were visible consisting of a large number of smaller globules with a contents of low contrast low density membranous-like material, comparable with the globular structures seen in SEM.

Globular secondary cataract material, removed from 24 patients with ECCE after ophthalmic cleaning of the anterior capsule, were investigated with SEM and TEM. Besides spherical, somewhat oval shaped bodies of various shape and size comparable with those found in cataractous lenses, (an)aerobic bacteria and yeastcells were found in approximately 70% of the cases, all of them in eyes without intra-ocular inflammation. Probably these bacteria have been transferred from the conjunctiva during IOL.-implantation and were encapsulated without starting an inflammation.

The degeneration of the capsule epithelium of cataractous lenses has been studied with LM, SEM on TEM with emphases on TEM. The observed degeneration of the epithelial cells can be described as follows: The cell nucleus becomes picnotic and desintegrates as result of change of the chromatin. Degeneration of the cytoplasm starts with swelling of the mitochondria, coming into existence of filamentous networks and balloon-like bulges of the nucleus. Repelling of the cell nucleus due to a porosity of the plasm membrane. Collapse of the cell due to degeneration of the cell and loss of the cytoplasmic contents, leaving finally only a swollen framework of cell walls.

Secondary retention refers to the cessation of eruption of a tooth after emergence. This may be the result of pathological changes in the periodontal ligament. The aim of this study was to describe the morphological and histological aspects of the radicular surface of secondarily retained permanent molars. The roots of 12 secondarily retained molars and two control molars, were examined by means of scanning electron microscopy (SEM) and light microscopy (LM) in order to analyse the occurrence and localisation of ankylosis. With SEM it was observed that the root surface of retained molars showed local areas covered with bonelike tissue. LM of these areas showed that this tissue was bone in direct contact with the root surface (ankylosis). In 11 cases, the areas of ankylosis were observed in the bifurcation area and at the interradicular root surface. In the remaining case, ankylosis was located at the outer root surface. The results of this study endorse the assumption that focal ankylosis is an important factor in secondary retention. Treatment recommendations must be based on this fundamental principle, because orthodontic movement of ankylotic molars is not possible.

In this paper, data are presented on the in situ demineralization of human enamel as a function of the demineralization period. To quantify the mineral loss parameters versus time, it is important to obtain information on the kinetics, and thus on the mechanism of dental caries. The results show that for in situ enamel demineralization, the lesion depth as well as the mineral loss parameter both vary linearly with the demineralization time. This is in contrast to in vitro lesion formation where the third power, or the square power of the lesion depth is linearly related to the demineralization time. In in situ demineralization, the rate-determining step of the demineralization process is the inhibitor-controlled dissolution process at the enamel crystallite surfaces, while the inhibitor content (F-, proteins etc.) in the lesion originating from the plaque, saliva and enamel is high. Furthermore, the study indicates that in in situ demineralization, interprismatic mineral loss is very important.

Lens tissue from a Brunescens cataract was prepared for SEM study by prefixation with glutaraldehyde and postfixation with the tannic acid/arginine/OsO4 combination; for TEM study the material was prefixed with glutaraldehyde, postfixed with OsO4/K4Fe(CN), and poststained with uranyl acetate/lead citrate. At low magnification, in contrast to the Morgagni cataract, no difference could be seen between the lens fibres in the cortical and nuclear areas. Morphologically, the destruction of the ball and socket system and the development of holes and spherical structures was striking. The latter appeared to have a thin coating and, after fracture, were either empty or showed remnants of material resembling membranes. In sections of the cataractous material, larger vacuoles containing smaller spheres were indistinctly visible.

Lens tissue from a Morgagni cataract was examined by SEM and TEM. For SEM, after prefixation with glutaraldehyde and postfixation with the tannic acid/arginine/OsO4 non-coating (TAO) technique, and for TEM, after prefixation with glutaraldehyde, postfixation with OsO4/K4Fe(CN)6 and poststaining with uranyl acetate/lead citrate. The TAO technique seems to be a particularly suitable postfixation method for the SEM investigation of cararact tissue because of the presence of the protein structures present. The cortical region showed areas of radially, instead of concentrically, arranged lens fibres, degenerated lens fibres with holes (vacuoles), broken ball and socket connections between the lens fibres, and oval or spherical structures varying in size from 0.5-20 mum, the largest resembling a golfball, arising from the cytoplasm of degenerating lens fibres. The smallest, 0.2-0.5 mum, appear to have been expelled from the furrowed lens epithelium.

A new and simple technique for glaucoma filtering surgery is presented. It is based on the introduction of a stainless steel microspiral or gonioseton into a goniopuncture by way of an ab interno procedure, minimising surgical trauma to the filtration area. Preliminary results are presented.