Non-coating fixation techniques or redundancy of conductive coating, low kV FE-SEM operation and combined SEM/TEM of biological tissues.

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 non-coating 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 non-coating 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 pre-fixation mixture, which causes disrupted stereocilia to cling onto the bottom side of the tectorial membrane.

Micromorphology of the dorsal ocelli of the Oriental hornet.

The development of vision in animals throughout evolution has been reviewed by Sir Stewart Duke-Elder, whose survey of the sense of sight ranges from lowly Crustaceans to mammals and man. According to Duke-Elder each ocellus is formed by the "fusion of two or more ocelli, each with its own retina and pigment cup". This process of 'ocellation' probably occurred independently in a number of phyla. So far as Hymenoptera are concerned, at least in Bombus, only the median ocellus has retained any evidence of its 'dual' origin. In hornets, there are three ocelli which are organized on the dorsal part of the head and receive their innervation from the optical lobes that are located in the protocerebrum. Proceeding from the exterior to the interior, the ocelli are generally composed of a cornea, followed by corneogenic cell layers and then by a layer of sensory cells from which emerges the ocellar nerve. Thus, in wasps and hornets the ocelli consist of a group of visual cells beneath a common lens and they possibly accentuate the response to light stimuli that are perceived by the compound eyes. Recently, the ocelli have been ascribed roles in orientation and navigation. In honeybees the sensitivity of the ocelli to light at various wavelengths was compared with that of the compound eyes, and was found to be different, to wit: in the ocellus there were two peaks--one of UV light at a wavelength of 335-340 nm and the other of green light at 490 nm, whereas in compound eye the peaks were at 350 nm and 540 nm. From all the foregoing, it would seem that the role of the ocelli in insects in general and in hornets in particular is not yet sufficiently clear. The present study was undertaken to elucidate the structure of the ocelli in the Oriental hornet and possibly also their function.

Are ciliary hair cells and photoreceptors components of a gravitic system of the hornet Vespa orientalis?

Social wasps, including the sub-family Vespinae, are social insects that build combs beneath the ground which are directed towards the gravitic pull of the earth, and this in dim light or complete darkness. On the inner side of the frons plate in social wasps there is a gravity sensing apparatus composed of static and dynamic nerve fibers, some of which connect between the frons plate and the brain. It is highly probable that the interaction between the fibers and the various structures in the head is responsible for the proprioceptive ability of hornets, including gravity detection. Ishay et al called the apparatus involved, the "Ishay Organ", and also reported the presence of (stereo) cilia on the inner side of the frons plate. The frons plate which bears the gravity organs functions as a photovoltaic system. Additionally there are throughout the cuticle, at intervals of several micrometers apart, distinct pores which are the outlets of peripheral photoreceptors. In the past various investigators have studied the ciliar structures of different insects mainly by TEM, but we have now studied the hair cells in hornets by FE-SEM, to obtain a complete 3-dimensional image of the various hair cell structures involved.

A comparative study of thin coatings of Au/Pd, Pt and Cr produced by magnetron sputtering for FE-SEM.

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 1.5­3.0 nm, deposited by planar magnetron sputtering devices. The coating units were used under standard conditions for source­substrate 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.

Demonstration of the fine structure of stereocilia in the organ of Corti of the guinea pig by field emission scanning electron microscopy.

A combined perfusion- and immersion prefixation with glutaraldehyde followed by a tannic acid/arginine/osmium tetroxide (TAO) treatment of the guinea pig cochlea is described for field-emission gun scanning electron microscopy (FEG-SEM) observation of the fine structure of the stereocilia of the organ of Corti. Conventional osmium tetroxide postfixation methods in combination with a thin conductive coating failed to show the fine structure of the glycocalyx of the epithelial lining in the endolymphatic compartment of the cochlea, in particular, on the stereocilia surface. The antennulae-like glycocalyx covering of the stereocilia surface of the more pronounced rows of outer hair cells has been demonstrated only in ultrathin sections by means of cationic markers. The side- and tip-links connecting the stereocilia have been demonstrated both in scanning and transmission electron microscopy, although at that time these structures often were considered as artificial. However, they can be visualized with FEG-SEM at low accelerating voltage (2-3 kV), and at appropriate working distance and probe current, in combination with a glutaraldehyde perfusion/immersion prefixation and TAO postfixation. Stereo images enhance considerably the three-dimensional appreciation of the stereocilia with glycocalyx lining and side- and tip-links, proving that these connections are a structural part of the hair cell.

A micro-Raman spectroscopic study of hydrazine-treated human dental calculus.

Hydrazine has been used to remove organic components and to isolate the mineral(s) from human calculus. Micro-Raman measurements were performed on the mineral phase. After the hydrazine-treatment, not only a large reduction in fluorescence but also an increase in Raman signal was observed. The treatment was essential in minimizing thermally-induced chemical changes which could otherwise occur to the original calculus mineral due to the intense laser light. The Raman spectral features of the mineral were nearly all identical among the Raman spectra obtained at many randomly-selected sites by the micro-Raman microbe with a lateral resolution of approximately 1 micron, and were consistent with those of impure hydroxyapatite containing CO3(2-) and HPO4(2-). The spectra contained typical hydroxyapatite bands including PO4(3-)bands of the v1, v2, v3 and v4 modes and one OH- stretch band. Other minor bands due to the CO3(2-) v1 and v3 modes and bands possibly due to the HPO4(2-) v1, v2 and v4 modes were observable by the technique despite the hydrazine-treatment that could in principle remove the HPO4 and CO3 ions from the mineral. In comparison with pure synthetic hydroxyapatite, the intensity of the OH- stretch band relative to that of the PO4(3-) v1 band was approximately 70% weaker, and the bandwidth of the phosphate v1 band was 200% broader, reflecting various crystal imperfections presumably present in the calculus mineral.

Low-voltage field-emission scanning electron microscopy of non-coated guinea-pig hair cell stereocilia.

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/thiocrbohydrazide/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 technique, 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.

New material for low-cost intraocular lenses.

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 subsequently 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 biocompatibility 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.

Closing of dentinal tubules by glutardialdehyde treatment, a scanning electron microscopy study.

The properties of dentin are strongly influenced by the so-called smear layer. This layer is always present on the dentin surface after cutting, drilling, sawing, etc. The smear layer can be removed by various chemical treatments, such as those of acid etching or ethylenediaminetetraacetic acid (EDTA). These treatments remove the smear layer and open the tubules. In this paper, the effect on the smear layer of human dentin of treatment with a 2% glutardialdehyde (GDA) solution at pH 3.5 for 2 min and a 0.5-M EDTA solution at pH 7.4 for 4 min was investigated by scanning electron microscope (SEM). The dentin samples were dried by air or critical-point drying before SEM photography was employed. The number of open dentin tubules was quantified on micrographs of EDTA- and GDA+EDTA-treated dentin. The results show that the GDA treatment fixed part of the smear layer and the superficial dentin surface in such a way that at least 50% of the tubules remained closed after EDTA treatment. By closing the dentinal tubules, the GDA-fixed layer might have a positive effect on dentin hypersensitivity, root caries, and bonding of composite to dentin.

Natural and induced radiation caries: A SEM study.

Radiation caries is a well-known indirect side effect of head and neck radiotherapy. The initiation and progression of radiation caries were studied using an in situ model. The morphology of induced carious lesions was compared with that of natural radiation caries. Both natural and induced radiation caries showed the same patterns of decay; widespread areas with porosity of enamel, crater formation with exposure of subsurface enamel, preferential dissolution of prisms with hollowing out of prism cores, loss of large parts of surface enamel and loss of full enamel coverage exposing the underlying dentin. In irradiated patients most enamel slabs were severely demineralized within 6 weeks whereas in control subject the slabs showed no significant demineralization after 12 weeks. The model may offer an excellent opportunity to study preventive fluoride regimens because of the rapid induction of radiation caries and its similarity with natural lesions.

Healing of microvenous PTFE prostheses implanted into the rat femoral vein.

44 PTFE prostheses (Gore-Tex; ID 1 mm) were implanted into rats' femoral veins by means of the sleeve anastomotic technique and were evaluated at regular intervals from 1 h up till 24 weeks after implantation by means of light and electron microscopy to study in detail their healing process. All prostheses, except one at 1 week and one at 24 weeks after implantation, were patent at the time of removal. Upon implantation, the luminal surface of the prostheses became covered with a thin clot layer. From 1 week onwards, endothelial cells originating from the anastomotic sides grew in across the anastomoses. In addition, small capillary-like orifices were present at the anastomotic sites, from which endothelial cells also seemed to originate. At 2 weeks, in several areas in the mid-region of the prostheses, the fronts of regenerating endothelial cells had reached each other, and about 80% of the luminal surface was covered by endothelium and at 3 weeks, the prostheses were completely covered by an endothelial layer. These results demonstrate that PTFE microvenous prostheses heal exclusively by means of rapid ingrowth of endothelial cells originating from both sides at the anastomoses.

Microorganisms found in secondary cataract material of ECCE patients, a study with SEM and TEM.

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 yeast cells 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 Morgagnian and Brunescens cataract morphology studied with with SEM and TEM.

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 instead of concentric running lens fibres, degeneration of lens fibres with vacuoles and defected "ball & socket" connections. The presence of oval/spherical bodies of 0.5-20 microns was acknowledged, the largest of them having a golf-ball appearance and originating from the cytoplasm of the degenerating lens fibres; the smallest of them with an approximate size of 0.2-0.5 micron seemed to be formed by budding off from the microvilli of the furrowed lens epithelium. The Brunescens cataract lenses showed at low magnification no difference between lens fibres from the cortical area and the nucleus. The disintegration process of the lens fibres was observable as degradation of the ball & socket system and the existence of holes in the lens fibre 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 lens aterial 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.

Human capsule epithelial cell degeneration, A LM, SEM and TEM investigation.

The degeneration of the capsule epithelium of cataractous lenses has been studied with LM, SEM en TEM with emphasis on TEM. The observed degeneration of the epithelial cells can be described as follows: The cell nucleus becomes picnotic and disintegrates 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 plasma 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.

Lensfibre degeneration at cataract lenses. A LM, SEM and TEM investigation.

The degeneration process of lensfibres in a cataractous lens, described as the biochemical changes of a part of the lensproteins, can be characterised morphologically as follows: Emulsification of a part of the lensfibre-mass and the development of open spaces between the lensfibres with the formation of vacuoles (spherical structures) with a granular or a compact contents with high contrast, which is squeezed into the intercellular space. Degeneration of the ball & socket system interdigitation and microvilli domain system and the formation of almost empty rectangular structures in somewhat radial array. Balloon-like bulging of the cytoplasm of the lenscell, degeneration of the cytoplasm membrane and visualisation of a micro-filamentous network with enclosed cell organelles.

Combined Raman and SEM study on CaF2 formed on/in enamel by APF treatments.

Raman spectra containing the distinct band at 322 cm-1 due to CaF2 or CaF2-like material formed in/on fluoridated bovine enamel were recorded using a micro-Raman spectrograph. Due to increasing levels of background fluorescence with increasing thickness of enamel, the Raman measurements were carried out on thin regions of wedged enamel sections. The distribution of the CaF2 or CaF2-like material was estimated using a simple model. The results indicate that 1/3 of the total CaF2 was concentrated within the narrow depth < 2 microns with high CaF2 concentrations (> 10 wt%), and that the majority of the CaF2 was distributed over the depths up to 26 microns (1 wt% CaF2). SEM observations on fractured fluoridated enamel confirmed that morphological changes were present in the depth range comparable to that of the high CaF2 concentration region expected from the Raman analysis. In deeper regions where lower concentration (< 10%) but a large amount of CaF2 was still expected, the SEM images failed to distinguish between the normal and fluoridated enamel. After KOH treatment, the Raman spectra did not show evidence of the CaF2 peak and the SEM micrographs also confirmed the removal of globules. The peak position of the Raman band of the CaF2 formed by the fluoridation was identical to that of pure CaF2. However, the linewidth was 23 cm-1 (FWHM) and a factor of 2 broader than that of pure CaF2 (12 cm-1). This implies that the lattice dynamics of the CaF2 formed by fluoridation is different from of pure CaF2, and that the material formed is 'CaF2-like' or 'disordered CaF2'.

[Xerostomia-related dental caries]. / Xerostomie-gerelateerde cariës.

The initiation and prevention of xerostomia-related dental caries were studied using an in situ model. The morphology of induced carious lesions was compared with that of natural xerostomia-related dental caries. Both natural and induced xerostomia-related dental caries showed the same patterns of decay. Using the in situ model three preventive procedures were tested: neutral F gel applied every second day or weekly, and a daily rinse with a fluoride mouthwash for a period of six weeks. Application of fluoride gel or the use of the fluoride mouthrinse resulted in significant inhibition of the demineralization process. Of the procedures evaluated fluoride gel applied every second day was the most effective in preventing the onset of xerostomia-related dental caries.

Secondary retention of permanent molars: an assessment of ankylosis by scanning electron and light microscopy.

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.

(An)aerobic bacteria found in secondary-cataract material. A SEM/TEM study.

Twenty four patients, who had marked reduction of vision due to secondary-cataract developed after an ECCE, were treated by surgical cleaning of the posterior lens capsule. During this procedure globular secondary-cataract material was removed and collected for morphological examination by SEM and TEM. Fragments of various sizes and shapes, including some with a 'golf ball' structure, were seen; these closely resembled particles frequently found in cataractous lenses. In addition, in 18 patients micro-organisms were found: rod-shaped bacteria, cocci, and in 2 cases yeasts. These findings were the more remarkable because these were clinically quiet eyes with no signs of intra-ocular inflammation and cultures have been persistently negative. We imagine that these bacteria must have entered the eye during the cataract extraction and have settled there without causing an infection.

Study of the substructure of the Morgagni and Brunescens cataract with the TAO non-coating technique. Part 1: Morgagni cataract.

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 cataract 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 microns, the largest resembling a golfball, arising from the cytoplasm of degenerating lens fibres. The smallest, 0.2-0.5 microns, appear to have been expelled from the furrowed lens epithelium.