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Probing the building blocks of eumelanins using scanning electron microscopy.

Scanning electron microscopy (SEM) is used to examine the structure of natural and synthetic melanins. Eumelanin from Sepia officinalis and synthetic eumelanin are found to be structurally dissimilar. The natural sample has a significant structural order with subunits that have a lateral dimension of approximately 15 nm. The synthetic samples, on the other hand, appear to be amorphous solids. These results lend support for the existence of fundamental structural units proposed from the analyses of wide-angle X-ray diffraction measurements and previous mass-spectrometry results. These findings also provide insight into the disparate photophysical behavior of Sepia and synthetic eumelanin.

Animals↗

HAADF-STEM imaging with sub-angstrom probes: a full Bloch wave analysis.

A full coherent Bloch wave calculation is presented to investigate high-angle annular dark-field image formation for sub-angstrom probes in scanning transmission electron microscopy (STEM). With increasing illumination angle, the contribution of the 1s bound state increases to a maximum at an optimum probe angle, after which we find increasing contributions from high-angle plane wave states around the periphery of the objective aperture. Examination of image contributions from different depths within a crystal shows an oscillatory behavior due to the beating between 1s and non-1s states. The oscillation period reduces with decreasing probe size, while the relative contribution from a specific depth increases. This signifies a changeover from a projection mode of imaging to a depth-slicing mode of imaging. This new mode appears capable of resolving three-dimensional atomic structures in future generation aberration-corrected STEM.

Algorithms↗

Structural conformation of in vitro and in vivo aged orthodontic elastomeric modules.

The objectives of this study were to investigate the structural characteristics of open and closed elastomeric modules in the as-received condition, and following (a) 24-hour in vitro loading determined as a 50 per cent extension relative to their original length; (b) 24-hour intra-oral exposure; (c) 3-week intra-oral exposure. Ten specimens of each type (open and closed) of the three brands of elastic chains were included in the study (total of 60). All specimens were subjected to (a) optical transmission microscopy utilizing phase contrast and polarized light modes, (b) micro MIR FT-IR spectroscopy, and (c) scanning electron microscopy and electron probe X-ray microanalysis. Stretched elastomers demonstrated a honeycomb pattern of filament detachment corresponding to strained areas. The chains possessing a well-differentiated inter-modular link presented higher strain concentration gradients located in the link. Phase contrast imaging revealed that the stress adsorption mechanism of these materials involved macromolecular chain orientation and elongation co-axially to the applied load. The specimens retrieved after 24 hours had acquired a proteinaceous film rich in alcohol groups with minimal evidence of phosphate mineralization, while the 3-week retrieved specimens presented precipitation of Ca- and P-forming calcium phosphates. The high protein content of the biofilm organized on the surface of these materials, as well as the calcification pattern found, were similar to a non-specific mechanism of film adsorption of biomaterials exposed to body fluids. The results have clinical implications for aspects of retraction control through sliding mechanics with the use of elastomeric ligatures, and the potential detrimental effects on dental and periodontal tissues such as decalcification and gingival inflammation, respectively. In addition, the experimental protocols adopted for the in vitro study of the force decay of elastomerics should be modified to include parameters pertinent to adsorption phenomena and calcification processes documented to occur in vivo.

Biofilms↗

Fluorescent in situ hybridisation on tissue sections: a quantitative approach with confocal laser scanning microscopy.

The use of fluorescent detection methods in association with digital microscopy technologies is an innovative approach for tissue localisation of messenger RNA. The success of such methods relies on the tissue preservation, local availability of the probe and on the existence of high resolution tridimensional analysis systems. Cryostatic sections, mild denaturation, short oligonucleotide probes (20mer) and confocal laser scanning microscopy allow the fulfillment of all these conditions avoiding photobleaching and tissue autofluorescence. In this paper, we describe in detail a method for in situ hybridisation set up with digoxigenin-coupled oligonucleotide complementary to beta-actin mRNA as a probe and an anti-hapten fluorescent antibody as second step for detecting specific hybridisation. Fluorescence was analysed by means of a confocal laser scanning microscope (CLSM) that provides images with low out-of-focus blurring also with relatively low numerical aperture (NA) objectives. We propose also an easy method to perform semi-quantitative thresholding analysis which allows to discriminate between background and specific signal.

Humans↗

Optimizing the environment for sub-0.2 nm scanning transmission electron microscopy.

Sub-0.2 nm probes can now be readily obtained on Schottky field-emission microscopes. However, environmental instabilities are proving to be the limiting factors for atomic resolution spectroscopy and distortion-free annular-dark field imaging. This is a result of the long acquisition times and the serial nature of the scanning system where instabilities result in image distortions rather than reductions in contrast. Troubleshooting the most common environmental problems is discussed here. In addition to the expected sensitivity to mechanical vibration, electromagnetic interference and temperature variations, air-pressure fluctuations are found to have a significant impact on microscopes with side-entry goiniometers.

Journal Article↗

Hexahedrally based crystals in human tooth enamel.

Mg-containing calcium phosphate crystals including pseudocuboidal, rhombohedral shapes and groupings of quadrangular blades cubically arranged were found in human tooth enamel by scanning electron microscopy and by electron probe microanalysis. In caries-free old enamel, these hexahedrally based crystals measuring 0.5-2.5 microns in length were observed in some crevices of tufts and lamellae. The crystals were rarely seen in the inner crevices of caries-free exfoliated deciduous enamel and none could be seen in sound young enamel. In brown-coloured old enamel possessing arrested caries with lamellae, some of the lamellae contained crystals measuring 0.1-1.5 mu in length adjacent to half-dissolved prisms. These crystals, identified as Mg-containing whitlockite, will grow during a long period after eruption of the tooth or during the enamel caries process.

Aged↗

Exceptional case of bone resorption in an osteo-odonto-keratoprosthesis. A scanning electron microscopy and X-ray microanalysis study.

This article reports the findings of investigations on an osteo-odonto-keratoprosthesis in an eye that was enucleated owing to severe complications 12 years after implantation. Scanning electron microscopy and electron probe X-ray microanalysis showed extensive resorption of the bone that was used as a supporting element in the kind of transcorneal prosthesis developed by Strampelli. The destructive process, in addition to surgical trauma, has been associated with the early and recurrent bacterial infections relating to the presence of Staphylococcus epidermidis. The need to control the occurrence of primary bacterial infections in traumatized tissues during operations as well as further infectious situations, given the enhanced antibiotic-resistence of bacteria, is emphasized.

Bone Resorption↗

The effect of dietary xylitol on recalcifying and newly formed cortical long bone in rats.

Thirty-six 3-week-old male Wistar rats were labeled with a single intraperitoneal tetracycline injection. Twenty-four of them were then fed a Ca-deficient basal diet for 3 weeks, while the control group received the basal diet supplemented with CaCO3 (12 g/kg). The tetracycline labeling was then repeated and six animals in each group were decapitated. The diet of the remaining formerly Ca-deficient animals was returned to normal, and half the test rats also received xylitol supplementation (50 g/kg). After 4 weeks of rehabilitation the labeling was repeated and the animals were decapitated and their tibias were prepared. The tibias were measured in terms of weight and density and cross sections were prepared for the examination of mineral content. Bone element analysis was performed by scanning electron microscopy with electron-probe microanalysis, examining separately the bone areas formed during the various dietary periods. Areas of the former Ca-deficient and newly formed cortical bone were identified by tetracycline fluorescence under ultraviolet light, and the amount of cortical bone in each group was measured. The mineralization-promoting effect of dietary xylitol as compared with CaCO3 supplementation alone was seen more clearly in the newly formed periosteal bone than in remineralization of the formerly Ca-deficient bone, the concentrations of Ca and P being significantly elevated (P < 0.05), as also was the total mineral content (P < 0.01). The cortical bone volume was similar following the CaCO3 and CaCO3 + xylitol supplementations, suggesting unaltered formation of the organic matrix. The results show that the effect of xylitol on bone during dietary Ca rehabilitation particularly concerns newly formed bone mineral.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

Standards for quantification of elements in the otolithic membrane by electron probe X-ray microanalysis: calibration curves and electron beam sensitivity.

An absolute quantitative standardization technique has been developed to measure Ca and K weight fractions (WF) in the otolithic membrane of the saccule and utricle by scanning electron microscopy and electron probe X-ray analysis using the peak-to-background (P/B) ratio method. Microcrystalline salt standards were used to calibrate Ca and K K alpha P/B or Y = (P/B).Z2/A (Z = atomic number; A = atomic weight) against WF at 10, 15, 20 and 25 kV accelerating voltage. The effect of voltage on the calibration, plotting the coefficient of correlation (r) as a function of voltage, was not dependent on the voltage in the range 10-25 kV for Ca standards. K standards were also independent when P/B was corrected for Z2/A. Background counts in the otoconia (Bo) were obtained at 5, 25, 50, 100, 200 and 500 s and used to test the electron beam sensitivity of saccular and utricular otoconia. Bo was not dependent on the spectra acquisition time, with the exception of Bo under K alpha K peak in the saccule at 10 kV. Ca and K WF were determined at 10, 15, 20 and 25 kV in the saccule and utricle, showing similar values regardless of the voltage used. This method of calibration offers several advantages, such as stability, homogeneity, known composition of the standards, high reproducibility at different voltages even without Z2/A correction and the similarity between the otoconia and crystal standards. We recommend the application of this method for other elements and biomineral systems.

Animals↗

New dimension in nano-imaging: breaking through the diffraction limit with scanning near-field optical microscopy.

In recent years scanning near-field optical microscopy (SNOM) has developed into a powerful surface analytical technique for observing specimens with lateral resolution equal to or even better than 100 nm. A large number of applications, from material science to biology, have been reported. In this paper, two different kinds of near-field optical microscopy, aperture and scattering-type SNOM, are reviewed together with recent studies in surface analysis and biology. Here, near-field optical techniques are discussed in comparison with related methods, such as scanning probe and standard optical microscopy, with respect to their specific advantages and fields of application.

Microscopy↗

Different methods of tissue preparation for X-ray microanalysis combined with scanning electron microscopy based on zebra fish, Brachydanio rerio muscle fibres.

Four preparation methods for electron probe X-ray microanalysis (EPXMA) of muscle fibres were compared: (1) dissection, freezing in LN2 (liquid nitrogen), cutting in cryostat, freeze-drying and analysis; (2) dissection, immersing in Tissue-Tek, freezing in LN2, cutting in cryostat, the following steps as in method 1; (3) dissection, freezing in LN2, freeze-drying, separation of fibres into groups; (4) dissection, cutting into 2 mm thick slices by razor blade, freezing in LN2, following steps as in method 1. The contents of Na, Cl, and K as well as K/Na, Cl/K, ratios were taken as criteria of good preservation of muscle fibres. The best results were obtained by method 1. Good morphological preservation can be routinely observed in sections prepared by methods 3 and 4. However, the diffusible elements and K/Na, Cl/K ratios were not retained at relatively constant level among the individual samples. Fibres prepared by methods 1 and 3 showed, despite of freezing artefacts, high K/Na, and low Cl/K ratio. After method 1, high level of the elemental contents was retained, and it did not differ significantly among the samples, showing relatively low standard deviation values.

Animals↗

Probing the structure of the SARS coronavirus using scanning electron microscopy.

A novel coronavirus, SARS-CoV, has been confirmed to be the aetiological agent of SARS. Transmission electron microscope (TEM) images played an important role in initial identification of the pathogen. In order to obtain greater morphological detail of SARS-CoV than could be obtained by TEM, we used ultra-high resolution scanning electron microscopy (SEM) to image the virus particles. We show here the three-dimensional appearance of SARS-CoV. Enhanced detail of the ultrastructure reveals the trimeric structure of the 10-20 nm spikes on the virion surface. These results contribute to characterization of the SARS agent and development of new antiviral strategies.

Humans↗

[Development of calcium fluorescent probes and their application in life sciences].

It is well known that calcium is the key second intracellular messenger. Techniques using calcium fluorescent probes have been rapidly developed during the last decade, and have become one of the most important methods to measure intracellular free Ca2+ concentrations. This trend was even more accelerated after the development of a new generation of calcium fluorescent probes and the laser scanning confocal microscopy. In this article we will review the development of calcium fluorescent probes, the mechanisms and methods of measuring intracellular free Ca2+ concentrations, the properties of new calcium fluorescent probes, and their applications in life sciences.

Aminoquinolines↗

Balance of hydrophobic and electrostatic forces in the pH response of weak polyelectrolyte capsules.

A detailed study of the role of solution pH and ionic strength on the swelling behavior of capsules composed of the weak polyelectrolytes poly(4-vinylpyridine) (P4VP) and poly(methacrylic acid) (PMA) with different numbers of layers was carried out. The polyelectrolyte layers were assembled onto silicon oxide particles and multilayer formation was followed by zeta-potential measurements. Hollow capsules were investigated by scanning electron microscopy and atomic force microscopy. The pH-dependent behavior of P4VP/PMA capsules was probed in aqueous media using confocal laser scanning microscopy. All systems exhibited a pronounced swelling at the edges of stability, at pHs of 2 and 8.1. The swelling degree increased when more polymer material was adsorbed. The swollen state can be attributed to uncompensated positive and negative charges within the multilayers, and it is stabilized by counteracting hydrophobic interactions. The swelling was related to the electrostatic interactions by infrared spectroscopy and zeta-potential measurements. The stability of the capsules as well as the swelling degree at a given pH could be tuned, when the ionic strength of the medium was altered.

Biophysics↗