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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↗

Electron spectroscopy and atomic force microscopy studies of DNA adsorption on mica.

Various methods for the deposition of deoxyribonucleic acid (DNA) molecules on mica are investigated to determine their reproducibility, and to quantify their ability to bind DNA. The use of these deposition methods for sample preparation for biological scanning tunneling microscopy (STM) and atomic force microscopy (AFM) studies is discussed. Auger electron spectroscopy (AES) and electron spectroscopy for chemical analysis (ESCA) were used to investigate the quantity of DNA adsorbed. AFM images of DNA deposited using the methods investigated are presented. The combination of AFM results with AES and ESCA results provides a basic understanding of the deposition techniques studied and illustrates that electron spectroscopy can be a useful addition to studies of this nature.

Adsorption↗

Cerebral astrocyte response to micromachined silicon implants.

The treatment of neurologic disorders and the restoration of lost function due to trauma by neuroprosthetic devices has been pursued for over 20 years. The methodology for fabricating miniature devices with sophisticated electronic functions to interface with nervous system tissue is available, having been well established by the integrated circuit industry. Unfortunately, the effectiveness of these devices is severely limited by the tissue reaction to the insertion and continuous presence of the implant, a foreign object. This study was designed to document the response of reactive astrocytes in the hope that this information will be useful in specifying new fabrication technologies and devices capable of prolonged functioning in the brain. Model probes fabricated from single crystal silicon wafers were implanted into the cerebral cortices of rats. The probes had a 1 x 1-mm tab, for handling, and a 2-mm-long shaft with a trapezoidal cross-section (200-microm base, 60microm width at the top, and 130 microm height). The tissue response was studied by light and scanning electron microscopy at postinsertion times ranging from 2 to 12 weeks. A continuous sheath of cells was found to surround the insertion site in all tissue studied and was well developed but loosely organized at 2 weeks. By 6 and 12 weeks, the sheath was highly compacted and continuous, isolating the probe from the brain. At 2 and 4 weeks, the sheath was disrupted when the probe was removed from the fixed tissue, indicating that cells attached more strongly to the surface of the probe than to the nearby tissue. The later times showed much less disruption. Scanning electron microscopy of the probes showed adherent cells or cell fragments at all time points. Thus, as the sheath became compact, the cells on the probe and the cells in the sheath had decreased adhesion to each other. Immunocytochemistry demonstrated that the sheath was labeled with antibodies to glial fibrillary acidic protein (GFAP), an indicator for reactive gliosis. The tissue surrounding the insertion site showed an increased number of GFAP-positive cells which tended to return to control levels as a function of time after probe insertion. It was concluded that reactive gliosis is an important part of the process forming the cellular sheath. Further, the continuous presence of the probe appears to result in a sustained response that produces and maintains a compact sheath, at least partially composed of reactive glia, which isolates the probe from the brain.

Animals↗

Diagnostic applications of scanning electron microscopy and microanalysis in pathology.

Microanalytical technology developed within the last decade provides important information in diagnostic pathology. Scanning electron microscopy, including backscattered electron imaging and energy dispersive X-ray analysis should become at least as valuable as polarized light microscopy, histochemistry and conventional transmission electron microscopy. Other as yet less available techniques such as the ion microprobe and laser Raman microprobe are also valuable. The pathologist should consider the use of microanalytic techniques in any disease process in which endogenous or exogenous materials may be present in the tissues, in the same manner in which one would perform stains for microorganisms. Cases are presented illustrating the tissue preparation and results of scanning electron microscopy and energy dispersive X-ray analysis in diagnosis.

Electron Probe Microanalysis↗

Observation of impurities in ice.

This paper presents a new method for determining the microstructural location of impurities in polycrystalline ice, which involves allowing the ice to sublimate under vacuum and then identifying the concentrated impurities using energy dispersive X-ray microanalysis in a low-vacuum scanning electron microscope. The method allows the presence of impurities in both the grain boundaries and the lattice of natural polycrystalline ice to be observed. Using this technique on natural ice, filaments consisting chiefly of NaCl were observed in some grain boundaries, small amounts of sulfur and chlorine were found in the grain interiors of the ice, and large concentrations of sulfur were observed in inclusions.

Electron Probe Microanalysis↗

Elemental mapping using the Ga 3d and In 4d transitions in the epsilon2 absorption spectra derived from EELS.

It is proposed that by using the valence-band states in electron energy loss spectroscopy, high-spatial resolution maps of quantitative elemental composition may be acquired with high acquisition rates. Further, it is shown that by using the epsilon(2) spectrum instead of single scattering data, the noise in the observed transitions and associated maps is significantly reduced. The epsilon(2) spectra are derived through a Kramers-Kronig transformation from electron energy loss spectra obtained in a scanning transmission electron microscope. Using transitions that occur in the epsilon(2) absorption spectrum (<40eV), quantitative elemental maps for III-V device structures have been produced. An example is provided using the Ga 3d transition to map a GaInNAs/GaAs laser structure. Weaker transitions such as In 4d have also been used to verify the Ga elemental distribution.

Electron Probe Microanalysis↗

Genuine myotubular myopathy.

Two patients, a father and his 14-year-old son, were suffering from a facioperoneal syndrome, and muscle biopsy findings were consistent with a myotubular myopathy. The father exhibited central nuclei in most muscle fibers, but his son had typical changes exclusively in hypotrophic type I fibers. The cytochemical and ultrastructural analysis revealed a spectrum of pathological changes typical of myotubular myopathy. Energy-dispersive electron probe x-ray microanalysis was performed on 6- to 12-microns thick freeze-dried cryosections visualized in the scanning or scanning transmission mode of electron microscopy. We found a high intracellular sodium and chlorine concentration and a low potassium concentration in comparison with control muscles. These changes pointed in the direction similar to results from human fetal muscle. The changes in the intracellular elemental composition may indicate a membrane pump dysfunction, which might be caused by a partial arrest in muscle fiber maturation.

Adolescent↗

Cell analysis with the new Leipzig high-energy ion nanoprobe.

The high-energy ion nanoprobe LIPSION at the University of Leipzig has been in operation since 1998. The ultrastable, 3.5 MV SINLETRON accelerator supplies the H+ or He+ ion beam. A magnetic scanning system moves the focused beam across the sample. At present, a resolution of 41 +/- 4 nm in the low current mode and 300 nm at 5 pA can be achieved. The experimental chamber is equipped with electron-, energy dispersive X-ray-, and particle detectors. They can be used simultaneously to analyse the sample by means of PIXE (particle induced X-ray emission), RBS (Rutherford backscattering), and in the case of thin sections or monolayer samples STIM (scanning transmission ion microscopy). A goniometer allows the application of channeling measurements in single crystals in combination with these methods. In contrast to previous publication describing microbeam facility at LIPSION, the current biomedical research has concentrated on microscopy and tomography on chondrocytes in pig cartilages and fixed single endothelial cells (HUVEC). For the irradiation of single living cells, an external beam facility with irradiation platform, fast beamgate and mini-Petri dishes is under construction.

Animals↗

Investigations of phagosomes, mitochondria, and acidic granules in human neutrophils using fluorescent probes.

The oxidative burst is frequently evaluated by the conversion of dihydrorhodamine 123 (DHR) to rhodamine 123 (R123) and hydroethidium (HE) to ethidium with the use of flow cytometry (FCM). Added R123 accumulates in mitochondria, but during phagocytosis R123 originating from DHR has been observed in neutrophil granules. The present study was designed to identify the site of reactive oxygen species (ROS) formation and the intracellular traffic of R123 in neutrophils by using mitochondrial membrane potential probes and the lysosomotropic probe LysoTracker Red, which have not previously been applied to neutrophils. Quiescent and phagocytosing human peripheral blood neutrophils were incubated with DHR, HE, R123, MitoTracker Green (MTG), MitoTracker Red (CMX-Ros), and LysoTracker Red alone and in all combinations of red and green probes, and studied by FCM and confocal laser scanning microscopy (CLSM). Phagosomes were filled with R123 originating from DHR. Phagocytosis also triggered the oxidative burst in oxidative response granules that differed from acidic granules. All the neutrophils stained with mitochondrial and lysosomotropic dyes. Added R123 and MTG selectively accumulated in mitochondria. Added R123, MTG, and DHR increased the fluorescence of CMX-Ros and LysoTracker Red. This is the first FCM and CLSM demonstration of ROS formation in phagosomes. A distinct subpopulation of neutrophil granules, termed oxidative response granules, also was identified. Neutrophil mitochondrial membrane potential may be evaluated by incubating the cells with R123 and MTG, but results with CMX-Ros should be interpreted with caution. HE and DHR seem to measure a common pathway in the oxidative burst. The simultaneous application of several probes for investigations of organelles carries the risk of probe interference.

Acids↗

Calcium content and concretions of pineal glands of young and old rats. A scanning and X-ray microanalytical study.

Calcium content and pineal concretions were studied in young (2-3 months) and old (28 months) Wistar rats. Samples, deep-frozen by liquid propane isopentane and freeze-dried were analysed by means of X-ray microanalysis in a scanning electron microscope. Total semi-quantitative measurements revealed that pineals of old rats showed a marked increase of calcium compared with the pineals of young rats. It is thus suggested that a calcium-rich environment is responsible for the growth of pineal concretions, which only appear in old rats. Pineal calcifications in rats could thus be an indicator of aging and/or of a degenerating state.

Aging↗

Study of the biological films formed during the pitting of aluminium in human plasma.

Film formation during the pitting corrosion of aluminium in human plasma is studied using conventional electrochemical techniques complemented with scanning electron microscopy as well as energy-dispersive x-ray, electron microprobe and immunoelectrophoresis analysis. The anodic polarization curve of aluminium in human plasma at 37 degrees C shows a passive region related to a low corrosion rate that extends up to -0.66 V (s.c.e.). At more positive potential values than -0.66 V, the breakdown of the oxide protective film occurs due to the chloride anions, leading to an intense localized dissolution of the metal (pitting corrosion). Scanning electron microscopy of the metal surface reveals a thick non-adherent precipitate covering pits of a crystallographic feature. Energy-dispersive x-ray and the microprobe analysis show the presence of aluminium, chloride and carbon signals. Immunoelectrophoresis of the precipitate resuspended in physiological solution shows a strong protein denaturization. Results suggest that the corrosion products are formed during pitting by the interaction of the saturated AlCl3 salt present within the pits and the adsorbed plasma proteins.

Aluminum↗

Anammox enrichment from different conventional sludges.

Three sets of sequencing batch reactor (SBR) were used for Anammox enrichment from conventional sludges including upflow anaerobic sludge blanket, activated sludge, and anaerobic digestion sludge. After four months of operation, the Anammox activity occurred in all reactors allowing continuous removal of ammonium and nitrite. The morphology of the cultivated Anammox sludge was observed using scanning electron microscope. The photographs showed that the obtained culture was mostly spherical in shape, presumably Anammox culture. There were also filamentous-like bacteria co-existing in the system. Fluorescence in situ hybridization (FISH) analysis using 16S rRNA targeting oligonucleotide probes PLA46 and Amx820 showed that the dominant population developed in all SBRs was hybridized with both PLA46 and Amx820 gene probes. It means that the cultivated biomass in all SBRs was classified in the group of Planctomycetales bacteria with respect to the anaerobic ammonium-oxidizing bacteria, Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis. Numerous time sequences were tested in this experiment. The shortest workable reaction time was found in the range from 5 to 7 h. Good quiescence of sludge was obtained at 30 min of settle period followed by a discharge period of 15 min. A long-term performance showed a near perfect removal of nitrite based on the influent NO2(-)-N concentration of 50-70 mg l(-1). The maximum ammonia removal efficiency was 80% with the influent NH4(+)-N concentration of 40-60 mg l(-1). It is, therefore, concluded that Anammox cultivation from conventional sludges was highly possible under control environment within four months.

Ammonia↗

Scanning electron microscopy and fluorescent in situ hybridization of experimental Brachyspira (Serpulina) pilosicoli infection in growing pigs.

Two groups of six 8-week-old pigs were challenged with 1x10(9) cfu Brachyspira (Serpulina) pilosicoli or Serpulina intermedia daily for 3 consecutive days to study the pathology of porcine colonic spirochetosis by scanning electron microscopy (SEM) and fluorescent in situ hybridization (FISH) with oligonucleotide probes targeting ribosomal RNA specific for B. pilosicoli and the genus Brachyspira/Serpulina. Six pigs served as noninoculated controls. The animals were euthanatized successively between postinoculation days 14 and 24. B. pilosicoli was reisolated in feces from all of the inoculated pigs; however, only two pigs developed transient watery diarrhea. S. intermedia was reisolated from four of the inoculated pigs, but clinical signs were not observed. Gross examination of the B. pilosicoli-infected pigs revealed dilated large intestines with a hyperemic mucosa, whereas the large intestines of the S. intermedia-inoculated pigs and the control pigs appeared normal. SEM examination of B. pilosicoli-infected pigs revealed degenerated epithelial cells and spirochetal colonization of the colonic mucosa in four pigs. By FISH, B. pilosicoli cells were found colonizing and invading the surface epithelium and the crypts in all the pigs. Spirochetal crypt colonization markedly exceeded the occurrence of spirochetes on the mucosal surface. SEM examination of S. intermedia-inoculated pigs revealed no abnormalities, and Serpulina cells were detected only sporadically in the otherwise normal-appearing mucosa of four pigs by FISH. The results provide further evidence that B. pilosicoli is associated with colitis in pigs, although the gross lesions are mild. The spirochete is capable of colonizing the large intestine, inducing mucosal damage, invasion of the crypt and surface epithelium, and focal infiltration of the lamina propria. In addition, the study shows the applicability of FISH for specific identification of B. pilosicoli in formalin-fixed tissue.

Animals↗

Synthesis, surface properties and performance of thiosulphate-substituted plasticized poly(vinyl chloride).

Plasticized poly(vinyl chloride) (PVC) was surface modified by nucleophilic substitution of the chlorine atoms of PVC by thiosulphate in aqueous media in the presence of a phase-transfer catalyst. The properties of the modified surface were evaluated by contact angle measurements, attenuated total reflection Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and scanning electron microscopy. Migration of the plasticizer di-(2-ethylhexyl) phthalate from control as well as modified PVC was examined in hexane, in cottonseed oil and in poly(ethylene glycol)-400 (PEG-400). While the modified PVC was found to be highly migration resistant in hexane, cottonseed oil and PEG-400 extracted the plasticizer. The modified PVC was found to cause haemolysis and was cytotoxic. A possible explanation for the difference in performance of the modified material in different extraction media and its toxicity is presented from a mechanistic view of the substitution process.

Animals↗

Scanning electron microscopy and energy dispersive analysis of machined denture base surfaces.

To relate the characteristics of rotary instruments to the surfaces they produce, acrylic resin, Molloplast B, and Novus were investigated with energy dispersive analysis and scanning electron microscopy (secondary and backscatter images) before and after machining. The chemical composition of cutting instruments, material surfaces, and residues was identified. Machined debris embedded in Molloplast B after machining with the Molloplast stone was found to contain a mean lead content of 45%. High concentrations of barium sulphate were discovered on the arbor band-machined surface of Novus. These differences were related to clinically appropriate instrumentation, and, therefore, biocompatibility studies that intimately relate to the in vivo situation should be considered for new materials.

Dental Polishing↗

The use of extraction replicas in scanning electron microscopy for the elemental analysis of marginal seals.

Elemental analysis of marginal seal material on 20 bulk samples of occlusally restored teeth have shown that, generally, more elements were detected in the seal material analyzed on the bulk restoration or fractured tooth surfaces than when such material was extracted using a nitrocellulose replica prior to analysis. No mercury was detected in any of the extracted seal material and only three of the replica specimens contained silver. This implies that the technique of using extraction replicas to remove material from the amalgam-tooth interface for subsequent x-ray microanalysis can provide an accurate elemental composition. The details of fabrication and the advantages of using high resolution nitrocellulose replicas for extracting marginal seal material from teeth for subsequent x-ray microanalysis are described.

Dental Amalgam↗