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Identification of mRNA-rich keratinocytes in the basal/suprabasal layers of psoriatic skin.

In this study, we examined the relative amounts of mRNA expressed in normal versus psoriatic epidermis, using in situ hybridization with a biotinylated oligonucleotide poly d(T) probe. The hybridization image was analyzed by Laser Scanning Confocal Microscopy (LSCM). In normal human skin, hybridization signals were detected homogeneously throughout the epidermis, mostly in nucleus. These signals disappeared following RNase T2 or RNase A treatment, indicating that the target for this probe is RNA; by implication, mRNA. In psoriatic lesions, the overall signal intensity was significantly elevated, especially in the basal/suprabasal layers. Moreover, those signals were most prominent in the cytoplasm, not in the nucleus. In contrast, the signals of uninvolved epidermis adjacent to the psoriatic lesions were indistinguishable from those of normal skin in both signal intensity and hybridization profile. Our data are consistent with the notion that one of the characteristic features of psoriasis is an elevated (or uncontrolled) synthesis of mRNA and proteins.

Biopsy, Needle↗

Near-field scanning optical microscopy in cell biology and cytogenetics.

Light microscopy has proven to be one of the most versatile analytical tools in cell biology and cytogenetics. The growing spectrum of scientific knowledge demands a continuous improvement of the optical resolution of the instruments. In far-field light microscopy, the attainable resolution is dictated by the limit of diffraction, which, in practice, is about 250 nm for high-numerical-aperture objective lenses. Near-field scanning optical microscopy (NSOM) was the first technique that has overcome this limit up to about one order of magnitude. Typically, the resolution range below 100 nm is accessed for biological applications. Using appropriately designed scanning probes allows for obtaining an extremely small near-field light excitation volume (some tens of nanometers in diameter). Because of the reduction of background illumination, high contrast imaging becomes feasible for light transmission and fluorescence microscopy. The height of the scanning probe is controlled by atomic force interactions between the specimen surface and the probe tip. The control signal can be used for the production of a topographic (nonoptical) image that can be acquired simultaneously. In this chapter, the principle of NSOM is described with respect to biological applications. A brief overview of some requirements in biology and applications described in the literature are given. Practical advice is focused on instruments with aperture-type illumination probes. Preparation protocols focussing on NSOM of cell surfaces and chromosomes are presented.

Animals↗

Electron microscopic identification of hemopoietic progenitor cells by exploiting their sugar-recognizing receptors using a newly developed minibead technique.

To identify hemopoietic progenitor cells in electron microscopic preparations we exploited the presence of "homing receptors" with galactosyl, mannosyl, and fucosyl specificities on their membrane surfaces. Amide-modified latex minibeads were covalently linked to the para-aminophenyl derivative of galactose, mannose, and fucose in pyranose form. Incubation of these probes with murine bone marrow cells led to specific binding of probes to a small proportion of marrow cells, comprising 0.5%, 0.6%, and 0.4% of the mononuclear marrow cell population, respectively, for galactosyl, mannosyl, and fucosyl probes. Specificity of the binding was demonstrated by inhibition in the presence of excess soluble sugars. The cells binding the probes were small, 4-5 microns, with characteristics similar to those described for progenitor cells. The implications of this technique for purification of hemopoietic progenitor cells are discussed.

Animals↗

Silicon-containing particles in synovial fluid: scanning electron microscopy coupled with analytical techniques allows an easy identification and differentiation from pathologically relevant crystals.

Silicon-containing particles were observed by scanning electron microscopy (SEM) in synovial fluid samples from patients with crystal-induced or inflammatory synovitis, or both. This material was an artefact produced by the technical procedures, but these particles could be easily differentiated from naturally occurring compounds by their morphology and their composition determined by analytical spectrometry.

Electron Probe Microanalysis↗

Connexin45, a major connexin of the rabbit sinoatrial node, is co-expressed with connexin43 in a restricted zone at the nodal-crista terminalis border.

The pacemaker of the heart, the sinoatrial (SA) node, is characterized by unique electrical coupling properties. To investigate the contribution of gap junction organization and composition to these properties, the spatial pattern of expression of three gap junctional proteins, connexin45 (Cx45), connexin40 (Cx40), and connexin43 (Cx43), was investigated by immunocytochemistry combined with confocal microscopy. The SA nodal regions of rabbits were dissected and rapidly frozen. Serial cryosections were double labeled for Cx45 and Cx43 and for Cx40 and Cx43, using pairs of antibody probes raised in different species. Dual-channel scanning confocal microscopy was applied to allow simultaneous visualization of the different connexins. Cx45 and Cx40, but not Cx43, were expressed in the central SA node. The major part of the SA nodal-crista terminalis border revealed a sharply demarcated boundary between Cx43-expressing myocytes of the crista terminalis and Cx45/Cx40-expressing myocytes of the node. On the endocardial side, however, a transitional zone between the crista terminalis and the periphery of the node was detected in which Cx43 and Cx45 expression merged. These distinct patterns of connexin compartmentation and merger identified suggest a morphological basis for minimization of contact between the tissues, thereby restricting the hyperpolarizing influence of the atrial muscle on the SA node while maintaining a communication route for directed exit of the impulse into the crista terminalis.

Animals↗

Preparation of aqueous standards for low temperature X-ray microanalysis.

A technique, using Nuclepore polycarbonate membrane filters as a containing medium for very small volumes of ionic standard solutions, to produce homogeneous ice standards is described. The standards are suitable for use in a scanning electron microscope. The relationship between elemental X-ray counts and ionic concentration is found to be linear. The method is rapid and simple. Minimum detectable concentrations are given.

Electron Probe Microanalysis↗

Preparation and characterisation of titania/hydroxyapatite composite coatings obtained by sol-gel process.

In the present work a titania network encapsulating a hydroxyapatite particulate phase is proposed as a bioceramic composite coating. The coating on a titanium substrate was produced starting from a sol containing a mixture of titania colloidal particles and hydroxyapatite submicron particles using the dip-coating technique. The microstructure, the morphology and the surface chemical composition of the coating were characterised using X-ray diffraction (XRD), scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS), respectively. Adhesion tests were also performed. These analyses showed that the obtained coating was chemically clean, homogeneous, rough, porous, with a low thickness and well-defined phase composition as well as a good adhesion to the substrate.

Biocompatible Materials↗

A simple method for correlative light, scanning electron microscopic and X-ray microanalytical examination of the same section.

Thin paraffin sections, mounted on scanning specimen holders previously coated with polyester film tape (Minnesota Mining and MFG Co., Scotch film tape No. 850 gold), were processed for light microscopy (LM) in the conventional way, then covered with celloxin shellac and examined in the LM by using the upper illuminating source. After removal of the shellac from the surface of the sample by immersion in acetone, the sections were air-dried, coated with a copper layer in a vacuum evaporator and examined in a scanning electron microscope (SEM). The method allows: (i) high-quality LM possibilities for establishment of the diagnosis in pathological cases; (ii) SEM examination of the same area as observed in LM; and (iii) EPMA measurements of insoluble precipitates embedded in the tissue. The usefulness of the proposed method is obvious in cases where the composition of a precipitate on LM scale is to be compared with the LM appearance of the surrounding tissue.

Animals↗

The inorganic content of pleromin in tooth plates of the living holocephalan, Chimaera phantasma, consists of a crystalline calcium phosphate known as beta-Ca3(PO4)2 (whitlockite).

The tooth plates in the living Holocephalan, Chimaera phantasma were studied by various techniques. They consisted of osteodentin and hypermineralized pleromin (kosmin). The degree of mineralization on the latter was as much as in the enameloid of Chondrichthyes and Osteichthyes. Scanning electron microscope observation indicated that the pleromin consisted of large and granular crystals. X-ray powder diffractometry, electron microprobe analysis and analytical electron microscopy revealed that the inorganic constituent of pleromin included, as an essential element, a beta-Ca3(PO4)2 (whitlockite) structure containing a small amount of Mg.

Animals↗

Nonlinear resistance versus length in single-walled carbon nanotubes.

In this work fundamental properties of the electrical transport of single-walled carbon nanotubes as a function of their length are investigated. For this purpose, we have developed a new technique that allows us to characterize electronic transport properties of single-walled carbon nanotubes by probing them at different spots. This technique uses scanning force microscopy to make mechanical and electrical nanocontacts at any selected spot of a given image. We have applied this technique to molecules with high intrinsic resistance. The results show a nonlinear resistance vs distance behavior as the nanotube is probed along its length. This is an indication of elastic electronic transport in one-dimensional systems.

Journal Article↗

Solid solubility of tin in silver-mercury gamma 1 in dental amalgams.

The solid solubility of Sn in gamma 1 has been studied by examining a series of gamma 1 samples containing 0.25-2.0 wt% Sn by scanning electron microscopy and energy-dispersive X-ray analysis. It has been observed that the addition of Sn to gamma 1 leads to the formation of mainly an intergranular Sn-Hg compound. On the basis of this observation it has been concluded that the solubility of Sn in gamma 1 is virtually nil.

Chemical Phenomena↗

Internalization of iron-transferrin complex by murine L1210 leukemia cells and rat reticulocytes demonstrated by a minibead probe.

To determine if the cellular uptake of iron is associated with internalization of iron-transferrin (TF) complex by the cell, we synthesized a visual probe in which TF is covalently bound to amide-modified latex minibead, submicrometer in size (0.345 micron). Incubation of the probe with L1210 leukemia cells and rat reticulocytes led to the binding of the probe to the cell surface visualized and semiquantified by scanning and transmission electron microscopy. The binding was inhibited by preincubation with nonderivatized iron-TF complex. Internalization of the probe occurred through clathrin-coated pits and vesicles. Minibeads derivatized by nontransport proteins or glycine as well as nonderivatized minibeads did not appreciably bind to the cells and were not internalized. Ethylamine, an inhibitor of receptor-mediated endocytosis abolished the internalization but not the binding of the probe which, then, accumulated on the cell surface. These findings provide direct evidence for internalization of TF during the iron uptake.

Animals↗

Characterization of microorganisms isolated from lignite excavated from the Záhorie coal mine (southwestern Slovakia).

Microorganisms were isolated from lignite freshly excavated in the Záhorie coal mine (southwestern Slovakia) under conditions excluding contamination with either soil or air-borne microorganisms. The isolates represented both Prokarya and Eukarya (fungi). All were able to grow on standard media, although some microorganisms were unstable and became extinct during storage of coal samples. Bacteria belonged to the genera Bacillus, Staphylococcus, and Rhodococcus, according to both morphological criteria and ITS sequences. Several bacterial isolates were resistant to antibiotics. The presence of anaerobic bacteria was also documented, although they have not yet been identified. Fungal isolates were typified by using their ITS sequences. They belonged to the genera Trichoderma (Hypocrea), Penicillium, Epicoccum, Metarhizium (Cordyceps), and Cladosporium. Several fungi produced compounds with antibiotic action against standard bacterial strains. The evidence for the presence of microorganisms in native lignite was obtained by means of fluorescence microscopy, scanning electron microscopy, and electron microprobe analysis. Results demonstrated that microorganisms were able to survive in the low-rank coal over a long time period.

Anti-Bacterial Agents↗

Analytical characterization of collagen- and/or hydroxyapatite-modified polypyrrole films electrosynthesized on Ti-substrates for the development of new bioactive surfaces.

The design and development of new bioactive surfaces on titanium-based materials employed in orthopedic implants is described. The new biosurfaces consist of thin polypyrrole films, directly grown on implant materials and modified by the inclusion of hydroxyapatite and/or collagen during the polymer electrodeposition step. The experimental procedure has been optimized in terms of loading and distribution of the bioactive components. X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) investigations have been performed in order to control the effectiveness of film modifications. In particular, XPS has been used to check the presence of biocompounds in the surface and sub-surface region of the polymer film, which is a critical requisite for a positive interface interaction between the biomaterial and the surrounding tissue.

Biocompatible Materials↗

[Potassium urate in urinary tract calculi].

In a study of renal calculi, performed with scanning electronic microscope, Edax, element mapping and semiquantitative analysis with a Nova 2 computer, we have found, among the urates, ratios K/Na from 0.016, in the almost pure monosodium urate stones, to 1.856 in other areas. The presence of monopotassium urate has, to our knowledge, no previous reference in the literature on human lithiasis.

Electron Probe Microanalysis↗

Characterization of AC mode scanning ion-conductance microscopy.

A scanning ion-conductance microscope (SICM) with a vibrating probe has been recently developed (vSICM). In this system, the amplitude of the AC ionic current is detected by using a lock-in amplifier locked to the vibration frequency of the probe. Such a scheme allows for a better control of the tip position because the AC ionic current is more sensitive to the probe-surface distance than the DC ionic current used previously. In this paper, we demonstrate the utility of this technique to the imaging of topographically rough specimens and high-resolution imaging over selected small areas. We also show that it is possible to record the DC ionic current simultaneously during the scan, which can reveal additional information not apparent in the images obtained with the AC ionic current.

Electric Conductivity↗

Tissue responses to natural aragonite (Margaritifera shell) implants in vivo.

The purpose of this study was to access tissue reactions to the outer prismatic (prism) and the inner nacreous (nacre) layers of the fresh water Margaritifera shell. The materials, in granule form, were implanted into the back muscles and femurs of rats for 1, 2, 4, 8 and 16 weeks. In the back muscles, a foreign body reaction was observed around the implants, starting from one week after implantation and reaching maximal proportions at two weeks. After four weeks, a thin layer of fibrous tissue encapsulated the implanted particles. The external surface of the material stained strongly with acid fuchsin, indicating degradation of implant. At femoral sites, newly formed bone was directly applied to the implant surfaces. The outer-most parts of the organic sheets in prisms were not degraded until 16 weeks after implantation and were embedded in the newly formed bone. The interface between bone and the implants showed close fusion by scanning electron microscopy (SEM). Energy dispersive X-ray analysis (EDAX) demonstrated a phosphorous-rich zone in the interface between bone and the implants, and no electron-dense layer in the interface was found by transmission electron microscopy (TEM). We conclude that Margaritifera shells are biocompatible, biodegradable and osteoconductive materials. Bonding between this natural aragonite and bone seems to occur via a phosphorous-rich intermediate layer.

Animals↗