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Light microscopy, scanning electron microscopy, and microprobe analysis of bone response to zinc and nonzinc amalgam implants.

Freshly mixed, unset zinc-free and zinc-containing analgam was implanted in the right tibia of 32 rats. Half of the specimens were examined by the light microscope and the other half by the scanning electron microscope and x-ray microprobe analysis. It was found that amalgam is well tolerated by the rat osseous tissue, and there were no histologic reaction differences between zinc and zinc free amalgam. The surfaces of the implants were covered by an organic film at 3 weeks and with bone at later intervals. Very little corrosion products containing sulfur were observed on the amalgam surface at all intervals. Bone adjacent to the amalgam contained tin and sulfur irrespective of the presence of zinc in the alloy, indicating outward migration of specific components of the alloy.

Animals↗

A morphological and genetic analysis of conidiophore development in Neurospora crassa.

The filamentous fungus Neurospora crassa responds to nutrient deprivation and dessication by producing asexual spores, or conidia. These conidia are derived from differentiated aerial structures called conidiophores. The process of conidiation was analyzed in wild-type and morphological mutants using scanning electron microscopy (SEM) and specific fluorescent probes. The first discernible morphological step of conidiation is the transition from growth by hyphal tip elongation to growth by repeated apical budding, resulting in the formation of chains of proconidia that resemble beads on a string. The initial proconidial chains are morphologically distinct from those that form later and are capable of reverting to hyphal growth, whereas the later chains are committed to conidiation. As the proconidial chains are formed, nuclei migrate into the conidiophore, and cross-walls arise between adjoining proconidia in a series of steps that have been defined by staining with Calcofluor, a fluorescent chitin-binding probe. The chains ultimately disarticulate in several discrete stages into free, morphologically mature conidia. Different conidiation-defective mutants were shown to be blocked at distinct stages in conidiation. Our observations permit us to derive a developmental timeline of conidiation relating the occurrence of morphological changes and the stage blocked in specific mutants.

Gene Expression Regulation↗

Redistribution of parasite and host cell membrane components during Toxoplasma gondii invasion.

The initial association of tachyzoites of Toxoplasma gondii with a host cell induces an endocytic process which leads to the formation of a vacuole known as the parasitophorous vacuole (PV). We analyzed the parasite-host cell interaction process using either parasites or host cells whose membrane was previously labeled with probes specific for proteins, sialoglycoconjugates and lipids, and then allowed to interact for periods varying from 5 minutes to 24 hours. The fate of the fluorescents probes was followed by confocal laser scanning microscopy. In host cells previously labeled with PKH26, FITC-Thiosemicarbazide or DTAF, which label membrane proteins, siloglycoconjugates and lipids, respectively, a uniform labeling of the cell surface was observed before interaction. When allowed to interact with T. gondii, labeling for PKH26 and DTAF, but not for FITC-Thiosemicarbazide, was observed initially at the region of contact between the two cells and subsequently on the membrane lining the PV and the intravacuolar parasites. These observations show that some, but not all, membrane components contribute to the formation of the PV membrane. Previously labeled parasites attach to the host cell surface but lose the fluorescent probes during the invasion process so that no labeled parasites were seen within the PV. These observations point to the existence of a dynamic process of membrane-associated components of the parasite and host cell during the interaction process.

Animals↗

Redistribution of plasma-membrane surface molecules during formation of the Leishmania amazonensis-containing parasitophorous vacuole.

Leishmania amazonensis presents two developmental stages that gain access to the host macrophage through phagocytosis. The protozoan resides in a membrane-bound compartment, the parasitophorous vacuole (PV), which can fuse with the endocytic system. For evaluation of the parasite/host-cell interaction process and of PV biogenesis, the two parasite forms or host-cell membrane whose surface had previously been labeled with specific probes for lipids, proteins, and sialoglycoconjugates were allowed to interact for periods varying from 5 to 15 min for adhesion and from 30 to 60 min for PV formation. The fate of fluorescent probes was followed by confocal laser scanning microscopy. In host cells previously labeled with PKH26, DTAF and FITC-thiosemicarbazide, which label membrane lipids, proteins, and sialoglycoconjugates, respectively, interaction with both protozoan forms revealed that adhesion to the macrophage was sufficient for labeling of the parasite surface. In addition, recently formed PVs displayed strongly labeled intravacuolar parasites, except for amastigote-macrophage interaction in a DTAF-labeled macrophage that displayed slight labeling of intravacuolar parasites, with the membrane lining the PV evidently being stained. Therefore, the vacuole modulation presents some particularities such that different host-cell membrane components may be selected, depending on the protozoan form involved. Thereafter, amastigotes labeled with the probes mentioned above displayed a diffuse labeling pattern after interaction with unlabeled macrophages, suggesting the spreading of Leishmania surface molecules during the initial parasite-invasion stages. In particular, intravacuolar DTAF-labeled amastigotes showed a delineating halo around the PV, with the intravacuolar parasite being partially labeled. Promastigotes could not be labeled with 5-(4,6-dichlorotriazinyl)aminofluorescein (DTAF) or with fluorescein-5-thiosemicarbazide, but promastigotes labeled with PKH26 lost the fluorescent probe during the invasion process such that slightly labeled promastigotes were seen inside the PV. These observations indicate the existence of a dynamic process of exchange of membrane-associated glycoproteins and lipids between the parasite and the host cell.

Animals↗

Atomic force microscopy of macromolecular interactions.

The introduction of functional imaging tools and techniques that operate at molecular-length scales has provided investigators with unique approaches to characterizing biomolecular structure and function relationships. Recent advances in the field of scanning probe techniques and, in particular, atomic force microscopy have yielded tantalizing insights into the dynamics of protein self-assembly and the mechanics of protein unfolding.

Crystallography, X-Ray↗

Comparison of probes for microbial contamination following use in periodontal pockets of various depths.

BACKGROUND: It has been speculated that periodontal probes can transmit periodontal pathogens from site to site. The purpose of this study was to evaluate the potential for bacterial transmission as a function of periodontal probe design. METHODS: Four different periodontal probes were used to measure probing depths ranging from 0 to 3 mm and > or = 4 mm. Following measurement of each pocket, probes were transported to a laboratory and cultured on blood agar, and colony forming units (CFUs) were determined for total aerobic, anaerobic, and dark-pigmented colonies (DPCs). Eight randomly selected probe tips, representing each probe type and probing depth category, were selected for examination by scanning electron microscopy (SEM) to determine distribution and morphotypes of adhering microbes. RESULTS: Results showed no statistically significant main effect for probes with respect to CFUs. However, there was a statistically significant increase in CFUs for deeper pockets when compared to pockets 0 to 3 mm. SEM observations were consistent among groups, regardless of probe design or probing depth. All probes exhibited a rough surface texture. Microbes were observed as single organisms or in aggregates and were adherent along the entire length of the probe up to the maximum probing depth. CONCLUSIONS: There was no difference in CFUs obtained as a function of periodontal probes. The rough surfaces of the probes used in this study likely promoted bacterial adherence through two different mechanisms: through scraping of the pocket walls by the roughened surfaces of the probe and by the high surface-free energy of the metal probes, facilitating bacterial adherence.

Alloys↗

rRNA sequence-based scanning electron microscopic detection of bacteria.

A new scanning electron microscopic method was developed for gaining both phylogenetic and morphological information about target microbes using in situ hybridization with rRNA-targeted oligonucleotide probes (SEM-ISH). Target cells were hybridized with oligonucleotide probes after gold labeling. Gold enhancement was used for amplification of probe signals from hybridized cells. The hybridized cells released a strong backscatter electron signal due to accumulation of gold atoms inside cells. SEM-ISH was applied to analyze bacterial community composition in freshwater samples, and bacterial cell counts determined by SEM-ISH with rRNA-targeted probes for major phyla within the domain Bacteria were highly correlated to those by fluorescent in situ hybridization (FISH). The bacterial composition on surface of river sediment particles before and after cell dispersion treatment by sonication was successfully revealed by SEM-ISH. Direct enumeration of bacterial cells on the surface of sonicated sediment particles by SEM-ISH demonstrated that members of Cytophaga-Flavobacterium existed tightly on the surface of particles. SEM-ISH allows defining the number and distribution of phylogenetically defined cells adherent to material surfaces, which is difficult in FISH, and it gives new insight into electron microscopic studies of microorganisms in their natural environment.

Bacteria↗

Qualitative electron probe analysis of secretory ameloblasts and odontoblasts in the rat incisor.

Rapidly frozen growing rat incisors were freeze fractured and freeze dried in preparation for energy dispersive X-ray emission microanalysis in a scanning electron microscope. Ca levels were found to be elevated in the distal cell body of odontoblasts, whereas Ca was uniformly low over all parts of the cell body of secretory ameloblasts. The results suggest fundamental differences in the mechanisms by which these two cell types process Ca, and that Ca possibly diffuses through the secretory ameloblast layer on its way to the enamel.

Ameloblasts↗

Silica accumulation in Triticum aestivum L. and Dactylis glomerata L.

The silica accumulation in orchard grass (Dactylis glomerata L.) and wheat (Triticum aestivum L.) has been studied in plant samples grown under defined conditions in a pot trial. The plant habit and the quantity of biomineralised silica within the selected Gramineae depend to a remarkable extent on the soil. The plants grew with different soil pH values and silica additives. By means of atomic absorption spectrometry, the silicon enrichment in different plant parts was determined. In dried plant parts the silica bodies can be well distinguished by variable pressure scanning electron microscopy in the back scattering mode. They are located in silica cells below the epidermis and in epidermal appendices (bristles, prickle hairs). The silica bodies showed a defined morphology, structure and composition which was elucidated by the combined performance of scanning electron microscopy in combination with X-ray spectroscopy, solid-state nuclear resonance, X-ray diffraction and Raman spectroscopy. The composition was near to stoichiometric SiO(2) (41 weight% silicon, 56 weight % oxygen), and the SiO(4/2)tetrahedra were arranged preferentially in three-dimensional networks; a smaller proportion was in chains and layers. The silica bodies with an overall amorphous structure contained crystalline precipitates, which could be indexed by alpha-quartz.

Dactylis↗

The role of proteins in the nucleation and formation of calcium-containing deposits on biomaterial surfaces.

In experiments in vivo using diffusion chambers, the morphology and composition of calcium-containing deposits on natural and artificial biomaterials that had no direct contact with cells were studied using scanning electron microscopy with energy dispersion X-ray microanalysis. It was revealed that the formation of a protein layer containing protein-calcium complexes is the key event in biomaterial calcification. A mechanism of formation of a calcium-containing protein matrix that creates the conditions for supersaturation of the crystal-forming medium over critical value has been proposed. The formation of nuclei of insoluble calcium phosphate starts predominantly deep in an adsorbed protein layer enriched by calcium ions.

Biocompatible Materials↗

Fluorescence in situ hybridization on human metaphase chromosomes detected by near-field scanning optical microscopy.

Fluorescence in situ hybridization on human metaphase chromosomes is detected by near-field scanning optical microscopy. This combination of cytochemical and scanning probe techniques enables the localization and identification of several fluorescently labelled genomic DNA fragments on a single chromosome with an unprecedented resolution. Three nucleic acid probes are used: pUC1.77, p1-79 and the plasmid probe alpha-spectrin. The hybridization signals are very well resolved in the near-field fluorescence images, while the exact location of the probes can be correlated accurately with the chromosome topography as afforded by the shear force image.

Biotin↗

Preparation and characterization of copper-doped cobalt oxide electrodes.

Cobalt oxide (Co3O4) and copper-doped cobalt oxide (CuxCo(3-x)O4) films have been prepared onto titanium support by the thermal decomposition method. The electrodes have been characterized by different techniques such as cyclic voltammetry, scanning electron microscopy, X-ray diffraction, and X-ray photoelectron spectroscopy (XPS). The effect on the electrochemical and crystallographic properties and surface morphology of the amount of copper in the oxide layer has been analyzed. The XPS spectra correspond to a characteristic monophasic Cu-Co spinel oxides when x is below 1. However, when the copper content exceeds that for the stoichiometric CuCo2O4 spinel, a new CuO phase segregates at the surface. The analysis of the surface cation distribution indicates that Cu(II) has preference for octahedral sites.

Cations, Divalent↗

Morphological evaluation of osteoblasts cultured on different calcium phosphate ceramics.

The objective of these investigations was to develop an in vitro test system for evaluating novel rapidly resorbable calcium phosphate ceramics of varying composition. Rat bone marrow cells were cultured on the disc-shaped test substrates for 14 days. Five calcium phosphates were examined: R1 CaNaPO4; R1/M2, composed of CaNaPO4 and MgNaPO4; R1/2, composed of CaNaPO4 and Mg2SiO4; R1 + 9% SiO2 consisting of CaNaPO4 and 9% SiO2 (wt%) and R17, Ca2KNa(PO4)2. Two studies were performed. In study I cultures were re-fed every two to three days. In study II the medium was changed daily, and calcium and phosphate concentrations of the medium were determined daily. Specimens were prepared for light microscopy and morphometric evaluation of the cell-covered substrate area, scanning electron microscopy and energy-dispersive X-ray analysis. With all materials tested except for R1/2, an increase of cellular growth was observed after changing the medium daily. Of the different calcium phosphate ceramics tested, R17 and R1/M2 facilitated osteoblast growth and elaboration of extracellular matrix to the highest degree. The inhibition of cell growth encountered with R1 in study I and R1/2 in both studies seemed to be related to a high phosphate-ion release from these materials.

Absorption↗

Scanning electron microscopic study of the effect of citrate and pyrophosphate on calcium oxalate crystal morphology.

The effects of citrate and pyrophosphate on the morphology of calcium oxalate monohydrate crystals formed in a supersaturated calcium oxalate solution were examined by scanning electron microscopy. Within the normal range of urinary concentrations, citrate and pyrophosphate affected the morphology of the crystals. The length/width ratio of the crystals decreased at higher concentrations of citrate and pyrophosphate. Citrate seems to have a dual mechanism of inhibition of crystal growth (binding to the crystal surfaces) and of complexation (with calcium in the solution). Pyrophosphate is believed to bind preferentially to calcium in the solid phase rather than in the solution phase, as it affected crystal morphology at very low concentrations and was detected on the crystals by X-ray microanalysis. Pyrophosphate seems to bind to the monohydrate but not to the dihydrate.

Calcium Oxalate↗

Modified drug release from inert matrix tablets prepared from formulations of identical composition but different organisations.

This paper develops for the first time a concept to modify the release rate of a fixed formulation by changing only the organisation of the mix used to prepare the tablets (ordered mixing). To estimate the influence of the organisation of binary mixes, several mixes of ethylcellulose and niflumic acid of the same composition but different organisation were compacted. The tablet surfaces were examined by energy dispersive X-ray microanalysis before the release experiments. Finally, the cross-sections of the remaining matrix were examined by scanning electronic microscopy. Excipient-excipient and excipient-drug interactions are the major factors influencing the drug release rate from the tablets. In the case of interacting materials, the initial release behaviour depends on the tablet surface presented to the dissolution media. The dissolution properties of the tablets are governed by the percolating material. When the inert excipient is percolating, the release rate increases linearly with the excipient/drug size ratio, whereas when the drug is the only material percolating through the system, its release rate is independent of the size ratio. When both materials are percolating through the system, the release rate is independent of the component particle sizes.

Cellulose↗

The application of scanning transmission electron microscopy (STEM) to the study of thin anodic films on nickel and nickel-molybdenum alloys.

Anodized thin-film samples of nickel, molybdenum and a nickel-13 w/o molybdenum alloy have been analysed by scanning transmission electron microscopy, using selected area diffraction, microdiffraction and X-ray micro-analysis. Thin-film samples were obtained by ion thinning with argon and by electropolishing in acetic acid-perchloric acid. Electropolishing is the preferred technique for these studies, producing a surface with roughness on a scale of 1 nm. The anodized films on nickel and nickel 13 w/o molybdenum alloy exhibit a rugosity with a 'particle' size of 3 nm. The crystal structure of the films is similar to f.c.c. nickel oxide. Molybdenum enrichment was detected in the anodized alloy surfaces but there is no electron diffraction evidence for a crystalline molybdenum oxide. The electron diffraction pattern from anodized pure molybdenum suggests that the surface film is amorphous.

Alloys↗