A scanning electron microscopic investigation of in vitro osteogenesis.
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Animals↗
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Results for “Microscopy, Scanning Probe”
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Fluorescence lifetime imaging with near-field scanning optical microscopy.
Near-field scanning optical microscopy (NSOM) is a high-resolution scanning probe technique capable of obtaining simultaneous optical and topographic images with spatial resolution of tens of nanometers. We have integrated time-correlated single-photon counting and NSOM to obtain images of fluorescence lifetimes with high spatial resolution. The technique can be used to measure either full fluorescence lifetime decays at individual spots with a spatial resolution of <100 nm or NSOM fluorescence images using fluorescence lifetime as a contrast mechanism. For imaging, a pulsed Ti:sapphire laser was used for sample excitation and fluorescent photons were time correlated and sorted into two time delay bins. The intensity in these bins can be used to estimate the fluorescence lifetime at each pixel in the image. The technique is demonstrated on thin films of poly(9,9'-dioctylfluorene) (PDOF). The fluorescence of PDOF is the results of both inter- and intrapolymer emitting species that can be easily distinguished in the time domain. Fluorescence lifetime imaging with near-field scanning optical microscopy demonstrates how photochemical degradation of the polymer leads to a quenching of short-delay intrachain emission and an increase in the long-delay photons associated with interpolymer emitting species. The images also show how intra- and interpolymer species are uniformly distributed in the films.
Journal Article↗
The determination of absolute concentrations of elements in thin (0.1 micrometer) sections of biological tissue by x-ray microanalysis in the scanning electron microscope and calibration with monoenergetic alpha particle scattering techniques.
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Alpha Particles↗
[Mechanisms of the development of anemia in stomach cancer].
Structural and metabolic statuses of erythrocytes were evaluated using cytophoto- and interferometry, fine-layer chromatography, scanning and transmission electron microscopy, electrophoresis and fluorescent probing in 48 stomach cancer patients. Structural and metabolic disorders were detected in mature erythrocytes in patients with and without anemia. Changes in the structure and metabolism of peripheral blood erythrocytes taking place prior to quantitative changes were identified. Anemia development pathways were dependent on enhanced hemolysis of circulating erythrocytes and influx of immature cells from the bone marrow. The changing statuses of the membranes and metabolism appeared to play quite a role in erythrons being damaged by tumor process. Dysfunctional erythrocytes of stomach cancer patients need to be corrected by membrane-stabilizing and -repairing drugs.
Adult↗
Laser confocal scanning microscopy of the surface membrane/T-tubular system and the sarcoplasmic reticulum in insect striated muscle stained with DilC18(3).
The structure of the surface membrane/transverse tubular (T-tubular) system and of the sarcoplasmic reticulum (SR) of the labial adductor muscle of the honey bee (Apis mellifera) was examined by laser confocal scanning microscopy, after staining with the fluorescent membrane probe DiIC18(3). The following components of the surface membrane/T-tubular system were visualized: transverse tubular networks that are located in the A-band close to the A-I junction and form dyads with the SR, longitudinal tubules that link the T-tubular networks within the between sarcomeres, and surface invaginations of larger diameter that contain tracheoles. The well developed SR forms a dense network of branching and anastomosing tubules in the A-band. A few tubular elements in the interfibrillar space in the I-band link the SR of adjacent sarcomeres. This study demonstrates the advantages of the laser confocal microscope and lipophilic fluorescent dyes for studying the 3-D structure of cellular membrane systems.
Animals↗
Use of a new fluorescent probe, seminaphthofluorescein-calcein, for determination of intracellular pH by simultaneous dual-emission imaging laser scanning confocal microscopy.
A new pH indicator, seminaphthofluorescein (SNAFL)-calcein acetoxymethyl ester, was used for intracellular pH (pHi) measurement in living MDCK cells with a laser scanning confocal microscope (LSCM) equipped with an Argon/Krypton laser and dual-excitation and dual-emission (FITC/Texas Red) filter set. SNAFL-calcein excitation maxima are approximately 492/540 nm (acid/base) and emission maxima are approximately 535/625 nm (acid/base) with a pKa value at approximately 7.0. The absorption/emission spectra of SNAFL-calcein indicate that the ratio of emission intensities of its basic/acidic forms is pH dependent. With an Argon/Krypton LSCM, we were able to monitor the acidic and basic forms of this dye simultaneously using dual-excitation (488/568 nm) and dual-emission (525-614 nm/> or = 615 nm) wavelengths (lambda s). The simultaneous dual-excitation/emission LSCM system allows for efficient recording of pHi dynamics (time resolution approximately 1 sec) in living cells. We have analyzed emission stability of the dye at different temperatures (22 degrees C and 37 degrees C) and constant pH, and at the same temperature (22 degrees C) but various pHs (6.6, 7.0, and 7.4). Bleaching rate is slightly higher at 37 degree C than that at 22 degrees C. The basic form of the dye (lambda Em approximately 625 nm) has a slightly higher bleaching rate than the acidic form (lambda Em approximately 535 nm) in standard culture medium (pH 7.3) at either 22 degree C or 37 degrees C. The pHi in MDCK cells calculated from ratio images (535 nm/625 nm) was 7.19 +/- 0.03 (mean +/- SEM, n = 20). Calibration experiments show that the useful pH range of SNAFL-calcein appears to be between 6.2 and 7.8, as the dye is difficult to calibrate outside this pH range.
Animals↗
[The effect of magnesite emission into the environment on the respiratory tract in rabbits studies with scanning electron microscopy with additional equipment].
Changes induced by magnesite dusts on the mucosa relief of the airways of rabbits are described. X-ray microanalysis and examination by raster electron microscopy were used to determine and verify the changes.
Air Pollutants↗
Mineralized dermal layer of the Brazilian tree-frog Corythomantis greeningi.
Some species of anuran amphibians possess a calcified dermal layer (the Eberth-Kastschenko layer) located between the "stratum spongiosum" and the "stratum compactum." This layer consists of calcium phosphate deposits, proteoglycans, and glycosaminoglycans. Although regarded as a protective layer against desiccation, a calcium reservoir, or possibly a remnant of a dermal skeleton present in anuran ancestors, very little is known about its origin, structure, and function. Thus, we studied the structure and composition of the mineralized dermal layer of Corythomantis greeningi, a peculiar hylid from the Brazilian semiarid region (caatinga), using conventional and cryosubstitution methods combined with transmission, scanning, and analytical electron microscopy. Results show that the dermal layer consists of dense, closely juxtaposed, globular structures. Although the electron opacity of the globules was variable, depending on the type of preparation, crystal-like inclusions were present in all of them, as confirmed by dark field microscopy. Electron probe X-ray microanalysis showed calcium, phosphorus, and oxygen, and electron diffraction revealed a crystalline structure comparable to that of a hydroxyapatite.
Animals↗
An optical submicrometer calcium sensor with conductance sensing capability.
The identification of chemical species and the measurement of their concentrations with high (submicrometer) spatial resolution are of considerable importance in cell biology. In this article we report the first successful development of a > or = 0.1-micron Ca2+ sensor based on a pulled micropipet, filled with a conducting porous sol-gel glass which was doped with the fluorescent calcium green 1 Ca2+ indicator. Such sensors are potentially capable of measuring Ca2+ concentrations as low as 10(-8) M, in confined volumes, with a three-dimensional resolution which exceeds approximately 0.1 micron. A major advantage of the sensor is its capability to be integrated into a multifunctional probe which will measure chemical analyte concentrations and ion conductance.
Animals↗
Application of a pH-sensitive fluoroprobe (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms.
An important feature of microbial biofilms is the development of four-dimensional physical and chemical gradients in space and time. There is need for novel approaches to probe these so-called microenvironments to determine their effect on biofilm-specific processes. In this study, we describe the use of seminaphthorhodafluor-4F 5-(and-6) carboxylic acid (C-SNARF-4) for pH microenvironment analysis in Pseudomonas aeruginosa biofilms. C-SNARF-4 is a fluorescent ratiometric probe that allows pH quantification independent of probe concentration and/or laser intensity. By confocal scanning laser microscopy, C-SNARF-4 revealed pH heterogeneity throughout the biofilm in both the x,y and x,z planes, with values ranging from pH 5.6 (within the biofilm) to pH 7.0 (bulk fluid). pH values were typically remarkably different than those just a few micrometers away. Although this probe has been successfully used in a number of eukaryotic systems, problems have been reported which describe spectral emission changes as a result of macromolecular interactions with the fluorophore. To assess how the biofilm environment may influence fluorescent properties of the dye, fluorescence of C-SNARF-4 was quantified via spectrofluorometry while the probe was suspended in various concentrations of representative biofilm matrix components (i.e., proteins, polysaccharides, and bacterial cells) and growth medium. Surprisingly, our data demonstrate that few changes in emission spectra occur as a result of matrix interactions below pH 7. These studies suggest that C-SNARF-4 can be used as a reliable indicator of pH microenvironments, which may help elucidate their influence on the medical and geobiological roles of natural biofilms.
Benzopyrans↗
[A case of Morquio mucopolysaccharidosis (scanning microscopy and x-ray microanalysis studies].
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Child, Preschool↗
Scanning-force techniques to monitor time-dependent changes in topography and adhesion force of proteins on surfaces.
Scanning-force microscopy (SFM) investigations were conducted to probe the influences of the interactions of proteins with surfaces relevant in medicine. These interactions are an important feature in the area of biofilm formation. The adsorption of proteins leads to changes in topography, which was monitored for the build up of protein layers of hen egg-white lysozyme and bovine serum albumin (BSA) on mica in real time in phosphate-buffered aqueous solution over a time period of 10 min. Phase imaging was additionally applied to compare material contrasts and to evaluate this method for further application in this field. The adhesion forces that develop on a time scale below 20 s between a protein-modified SFM tip and titanium surfaces (TiO(2), TiAl6V4 and TiAl6Nb7) were investigated. The influences of the parameters loading force and interaction time between the protein and the surface were monitored as well as the influence of protein structure. The interaction time dependency of the adhesion force could be described with a kinetic model of two consecutive first-order reactions. For the maximal adhesion force a correlation to the ratio of the amino acids cysteine, proline and glycine has been proposed.
Adhesiveness↗
In vivo contribution of Class III alcohol dehydrogenase (ADH3) to alcohol metabolism through activation by cytoplasmic solution hydrophobicity.
Alcohol metabolism in vivo cannot be explained solely by the action of the classical alcohol dehydrogenase, Class I ADH (ADH1). Over the past three decades, attempts to identify the metabolizing enzymes responsible for the ADH1-independent pathway have focused on the microsomal ethanol oxidizing system (MEOS) and catalase, but have failed to clarify their roles in systemic alcohol metabolism. In this study, we used Adh3-null mutant mice to demonstrate that Class III ADH (ADH3), a ubiquitous enzyme of ancient origin, contributes to alcohol metabolism in vivo dose-dependently resulting in a diminution of acute alcohol intoxication. Although the ethanol oxidation activity of ADH3 in vitro is low due to its very high Km, it was found to exhibit a markedly enhanced catalytic efficiency (kcat/Km) toward ethanol when the solution hydrophobicity of the reaction medium was increased with a hydrophobic substance. Confocal laser scanning microscopy with Nile red as a hydrophobic probe revealed a cytoplasmic solution of mouse liver cells to be much more hydrophobic than the buffer solution used for in vitro experiments. So, the in vivo contribution of high-Km ADH3 to alcohol metabolism is likely to involve activation in a hydrophobic solution. Thus, the present study demonstrated that ADH3 plays an important role in systemic ethanol metabolism at higher levels of blood ethanol through activation by cytoplasmic solution hydrophobicity.
Alcohol Dehydrogenase↗
Vulnerability to ROS-induced cell death in ageing articular cartilage: the role of antioxidant enzyme activity.
OBJECTIVES: To test the hypothesis that age-related loss of chondrocytes in cartilage is associated with impaired reactive oxygen species (ROS) homeostasis resulting from reduced antioxidant defence. METHODS: Cell numbers: The total number of chondrocytes in the articular cartilage of the femoral head of young, mature and old rats was estimated using an unbiased stereological method. ROS quantification: Fluorescence intensity in chondrocytes was quantified using the oxygen free radical sensing probe dihydrorhodamine 123 (DHR 123), confocal laser scanning microscopy and densitometric image analysis. In order to delineate the reactive species, explants were pre-treated with N-acetylcysteine (NAC) or N(G)-nitro-l-arginine methyl ester (l-NAME) prior to ROS quantification. Induction of intracellular ROS: Explants were incubated in the redox-cycling drug menadione after which they underwent ROS quantification and cell-viability assay. Antioxidant enzyme activity: The activity of catalase, superoxide dismutase (SOD) and glutathione peroxidase (GPX) was measured. RESULTS: Chondrocyte numbers: A significant and progressive loss of chondrocytes was observed with ageing. Cellular ROS levels: A significant age-related increase in cellular ROS-induced fluorescence was demonstrated. NAC significantly reduced ROS levels in old chondrocytes only. Induction of intracellular ROS: Menadione increased cellular ROS levels dose-dependently in young and old chondrocytes, with a greater effect in the latter. Old chondrocytes were more vulnerable to menadione-induced cytotoxicity. Antioxidant enzymes: Catalase activity declined significantly in aged cartilage whilst SOD and GPX activities were unaltered. CONCLUSIONS: Substantial loss of chondrocytes occurs in rat articular cartilage which may result from increased vulnerability to elevated intracellular ROS levels, consequent upon a decline in antioxidant defence.
Animals↗
Exposure of the rat small intestine to raw kidney beans results in reorganization of absorptive cell microvilli.
BACKGROUND/AIMS: A single exposure to raw kidney beans (RKB) results in vesiculation, shortening, and then regrowth of microvilli in the rat small intestine. This study investigated changes that occur in the structure of microvilli 2-10 hours after RKB exposure. METHODS: Circumferences of microvilli from absorptive cells obtained sequentially after challenge with RKB or chow were assigned to one of three groups: small, intermediate, or large. The distribution and concentration of actin in intact mucosae or isolated epithelial sheets were determined by confocal laser scanning microscopy, immunocytochemistry, and immunoblot analysis with specific probes. RESULTS: Six hours after exposure to RKB, most microvilli were large, abnormal in shape, and contained significantly more actin filaments than large microvilli from control rats. In addition, the fluorescence intensity of F-actin increased within injured microvilli without changes in the total intracellular actin concentration. By 8-10 hours after challenge with RKB, some microvilli remained larger than those of control rats but had resumed their normal shape and contained fewer actin filaments than at 6 hours. CONCLUSIONS: Exposure of the rat small intestine to RKB results in enlargement of absorptive cell microvilli and reorganization of membrane and core actin filaments without changes in intracellular actin concentration. Enlarged microvilli are rapidly repaired.
Actins↗
Recent advances in buccal drug delivery and absorption--in vitro and in vivo studies.
In the first part of this study, the aim was to characterize transport of fluorescein isothiocyanate (FITC)-labelled dextrans of different molecular weights as model compounds for peptides and proteins through buccal mucosa. The penetration of these dextrans through porcine buccal mucosa (a nonkeratinized epithelium, comparable to human buccal mucosa) was investigated by measuring transbuccal fluxes and by analyzing the distribution of the fluorescent probe in the epithelium, using confocal laser scanning microscopy for visualizing permeation pathways. The results revealed that passage of hydrophilic compounds such as the FITC-dextrans through porcine buccal epithelium is restricted to permeants with a molecular weight lower than 20 kDa. The permeabilities of buccal mucosa for the 4 and 10 kDa FITC-dextran (of the order of 10(-8) cm/s) were not significantly different from each other or from the much smaller compound FITC. The confocal images of the distribution pattern of FITC-dextrans showed that the paracellular route is the major pathway through buccal epithelium. In the in vivo part of this study, buccal delivery of FITC-labelled dextran 4400 (FD4) and the peptide drug buserelin was investigated in vivo, in pigs. The delivery device consisted of an application chamber with a solution of FD4 or buserelin, and was attached to the buccal mucosa for 4 h using an adhesive patch. A randomized cross-over study including intravenous administration and buccal delivery without and with 10 mM sodium glycodeoxycholate (GDC) as an absorption enhancer was performed in pigs. After buccal administration, steady-state plasma levels were rapidly achieved. Co-administration of 10 mM GDC increased the absolute bioavailability from 1.8+/-0.5 to 12.7+/-2.0% for FD4. From the present studies, it is concluded that buccal administration is a suitable route for the delivery for macromolecules and hydrophilic compounds such as peptide drugs.
Animals↗
Characterization of lipid in mature enamel using confocal laser scanning microscopy.
OBJECTIVE: To characterize the lipid components of organic matrix in mature human enamel using Confocal Laser Scanning Microscopy (CLSM) coupled with a hydrophobic fluorescent probe. METHODS: Twenty-four longitudinal sections of human enamel were fixed with 3.7% paraformaldehyde (PFA), partially decalcified with 0.5 M of EDTA, and labelled with a fluorescent probe (Nile red) before CLSM characterization. Based on the fluorescence spectra of Nile red in ethanol (1 microgram/ml), each enamel section was evaluated with 543 nm light source and with 590 nm long pass filter. Spectrophotometric analysis was carried out to characterize the autofluorescence and Nile red lipid fluorescence in mature enamel. Special optical parameters of the microscope were chosen to rule out the intrinsic fluorescence of the samples, and that induced by PFA. RESULTS: The intensity of autofluorescence and PFA-induced fluorescence were negligible above 565 nm; whereas the fluorescence of Nile red peaked at around 600 nm. Lipid material was identified in the cross-striations, the lines of Retzius, the Hunter-Schreger bands, inter-prismatic spaces, and inter-prismatic spaces in the mature human enamel. CONCLUSIONS: This technique successfully revealed the distribution of lipid components of organic matrix in mature human enamel and may be promising in assessing the changes of enamel organic elements in the developmental, pathological or experimental conditions.
Dental Enamel↗
Hypoglycaemic effect of a novel insulin buccal formulation on rabbits.
Transmucosal delivery is a suitable route for insulin non-injection administration. In this study, the hypoglycaemic effect of INSULIN BUCCAL SPRAY (IBS), a formulation with soybean lecithin and propanediol combined as absorption enhancer for insulin on diabetic rabbits and rats, were investigated. The hypoglycaemic rate was calculated and the pharmacodynamics and pharmacokinetics of the formulation in rabbits were studied. The results show that when the diabetic rabbits were administrated with IBS in dosages of 0.5, 1.5 and 4.5Ukg(-1), the blood glucose level decreased significantly compared with that of the control group and the hypoglycaemic effect lasted over 5h. The blood glucose decreasing rates are 22.4, 48.1 and 53.5%, respectively. The average bioavailability of IBS by buccal delivery versus subcutaneous injection is 29.2%. Meanwhile, the diabetic rats were administrated with IBS in dosages of 1.0, 3.0 and 9.0Ukg(-1), the blood glucose level decreased significantly compared with that of the control group and the hypoglycaemic effect lasted over 4h. The blood glucose decreasing rates are 24.6, 47.5 and 59.6%, respectively. Furthermore, the penetration of fluorescein isothiocyanate (FITC)-labelled insulin through rabbit buccal mucosa was investigated by scanning the distribution of the fluorescent probe in the epithelium using confocal laser scanning microscopy. The results revealed that FITC-insulin can pass through the buccal mucosa promoted by the enhancer and the passage of insulin across the epithelium includes both intracellular and paracellular routes. From the rabbit and rat experimental results showed that IBS is an effective buccal delivery system, which is promising for clinical trial and the future clinical application.
Administration, Buccal↗