Structure and mineralization of the spearing mantis shrimp (Stomatopoda; Lysiosquillina maculata) body and spike cuticles.

Stomatopoda is a crustacean order including sophisticated predators called spearing and smashing mantis shrimps that are separated from the well-studied Eumalacotraca since the Devonian. The spearing mantis shrimp has developed a spiky dactyl capable of impaling fishes or crustaceans in a fraction of second. In this high velocity hunting technique, the spikes undergo an intense mechanical constraint to which their exoskeleton (or cuticle) has to be adapted. To better understand the spike cuticle internal architecture and composition, electron microscopy, X-ray microanalysis and Raman spectroscopy were used on the spikes of 7 individuals (collected in French Polynesia and Indonesia), but also on parts of the body cuticle that have less mechanical stress to bear. In the body cuticle, several specificities linked to the group were found, allowing to determine the basic structure from which the spike cuticle has evolved. Results also highlighted that the body cuticle of mantis shrimps could be a model close to the ancestral arthropod cuticle by the aspect of its biological layers (epi- and procuticle including exo- and endocuticle) as well as by the Ca-carbonate/phosphate mineral content of these layers. In contrast, the spike cuticle exhibits a deeply modified organization in four functional regions overprinted on the biological layers. Each of them has specific fibre arrangement or mineral content (fluorapatite, ACP or phosphate-rich Ca-carbonate) and is thought to assume specific mechanical roles, conferring appropriate properties on the entire spike. These results agree with an evolution of smashing mantis shrimps from primitive stabbing/spearing shrimps, and thus also allowed a better understanding of the structural modifications described in previous studies on the dactyl club of smashing mantis shrimps.

A scoping study of component-specific toxicity of mercury in urban road dusts from three international locations.

This scoping study presents an investigation of the total and bioaccessible mercury concentrations in road dust (RD) from three international urban sites, where a one-off sampling campaign was conducted at each. This was done to address the hypothesis that the matrix in which mercury is found influences its ability to become accessible to the body once inhaled. For that purpose, the samples were analysed for total and pulmonary bioaccessible mercury and the data compared to the chemical structure of individual particles by SEM. The results obtained from this study suggest that a high mercury content does not necessarily equate to high bioaccessibility, a phenomenon which could be ascribed to the chemical character of the individual particles. It was found that the Manchester samples contained more pulmonary soluble mercury species (as determined by elemental associations of Hg and Cl) in comparison to the other two samples, Curitiba, Brazil, and Johannesburg, South Africa. This finding ultimately underlines the necessity to conduct a site-specific in-depth analysis of RD, to determine the concentration, chemical structure and molecular speciation of the materials within the complex matrix of RD. Therefore, rather than simply assuming that higher bulk concentrations equate to more significant potential human health concerns, the leaching potential of the metal/element in its specific form (for example as a mineral) should be ascertained. The importance of individual particle behaviour in the determination of human health risk is therefore highlighted.

Quantitative imaging of Candida utilis and its organelles by soft X-ray Nano-CT.

Soft X-ray microscopy has excellent characteristics for imaging cells and subcellular structures. In this paper, the yeast strain, Candida utilis, was imaged by soft X-ray microscopy and three-dimensional volumes were reconstructed with the SART-TV method. We performed segmentation on the reconstruction in three dimensions and identified several types of subcellular architecture within the specimen cells based on their linear absorption coefficient (LAC) values. Organelles can be identified by the correlation between the soft X-ray LAC values and the subcellular architectures. Quantitative analyses of the volume ratio of organelles to whole cell in different phases were also carried out according to the three-dimensional datasets. With such excellent features, soft X-ray imaging has a great influence in the field of biological cellular and subcellular research.

Quantitative x-ray microanalysis of model biological samples in the SEM using remote standards and the XPP analytical model.

It is shown that accurate x-ray microanalysis of frozen-hydrated and dry organic compounds, such as model biological samples, is possible with a silicon drift detector in combination with XPP (exponential model of Pouchou and Pichoir matrix correction) software using 'remote standards'. This type of analysis is also referred to as 'standardless analysis'. Analyses from selected areas or elemental images (maps) were identical. Improvements in x-ray microanalytical hardware and software, together with developments in cryotechniques, have made the quantitative analysis of cryoplaned frozen-hydrated biological samples in the scanning electron microscope a much simpler procedure. The increased effectiveness of pulse pile-up rejection renders the analysis of Na, with ultrathin window detectors, in the presence of very high concentrations of O, from ice, more accurate. The accurate analysis of Ca (2 mmol kg-1 ) in the presence of high concentrations of K is possible. Careful sublimation of surface frost from frozen-hydrated samples resulted in a small increase in analysed elemental concentrations. A more prolonged sublimation from the same resurfaced sample and other similar samples resulted in higher element concentrations.

Elemental mapping of Neuromelanin organelles of human Substantia Nigra: correlative ultrastructural and chemical analysis by analytical transmission electron microscopy and nano-secondary ion mass spectrometry.

Neuromelanin (NM) is a compound which highly accumulates mainly in catecholamine neurons of the substantia nigra (SN), and is contained in organelles (NM-containing organelles) with lipid bodies and proteins. These neurons selectively degenerate in Parkinson's disease and NM can play either a protective or toxic role. NM-containing organelles of SN were investigated by Analytical Electron Microscopy (AEM) and Nano-Secondary Ion Mass Spectrometry (NanoSIMS) within human tissue sections with respect to ultrastructure and elemental composition. Within the NM-containing organelle, the single NM granules and lipid bodies had sizes of about 200-600 nm. Energy-Dispersive X-ray microanalysis spectra of the NM granules and lipid bodies were acquired with 100 nm beam diameter in AEM, NanoSIMS yielded elemental maps with a lateral resolution of about 150 nm. AEM yielded the quantitative elemental composition of NM granules and bound metals, e.g., iron with a mole fraction of about 0.15 atomic percent. Chemical analyses by AEM and NanoSIMS were consistent at the subcellular level so that nanoSIMS measurements have been quantitated. In NM granules of SN from healthy subjects, a significant amount of S, Fe, and Cu was found. In lipid bodies an amount of P consistent with the presence of phospholipids was measured. The improved detection limits of nanoSIMS offer new possibilities for chemical mapping, high-sensitivity trace element detection, and reduced acquisition times. Variations between individual NM granules can now be investigated effectively and quantitatively by NanoSIMS mapping Cu and Fe. This should yield new insight into the changes in chemical composition of NM pigments during healthy aging and disease. Neuromelanin-containing organelles of dopamine neurons in normal human substantia nigra were investigated by analytical electron mircoscopy and secondary ion mass spectroscopy (NanoSIMS) yielding the ultrastructure and elemental composition. In neuromelanin granules a significant amount of S, Fe and Cu was found. In lipid bodies an amount of P consistent with the presence of phospholipids was measured. The improved sensitivity of NanoSIMS shows differences in chemical composition between individual neuromelanin granules and allows to study chemical changes of neuromelanin organelles during aging and disease.

Development and comparison of the methods for quantitative electron probe X-ray microanalysis analysis of thin specimens and their application to biological material.

In recent years, there has been a return to the use of electron probe X-ray microanalysis for biological studies but this has occurred at a time when the Hall programme which acted as the mainstay for biological microanalysis is no longer easily available. Commercial quantitative routines rely on the Cliff-Lorimer method that was originally developed for materials science applications. Here, the development of these two main routines for obtaining quantitative data from thin specimens is outlined and the limitations that are likely to be met when the Cliff-Lorimer routine is applied to biological specimens is discussed. The effects of specimen preparation on element content is briefly summarized and the problems encountered when using quantitative analysis on resin-embedded materials emphasized.

Cells on biomaterials--some aspects of elemental analysis by means of electron probes.

Electron probe X-ray microanalysis enables concomitant observation of specimens and analysis of their elemental composition. The method is attractive for engineers developing tissue-compatible biomaterials. Either changes in element composition of cells or biomaterial can be defined according to well-established preparation and quantification procedures. However, the qualitative and quantitative elemental analysis appears more complicated when cells or thin tissue sections are deposited on biomaterials. X-ray spectra generated at the cell/tissue-biomaterial interface are modelled using a Monte Carlo simulation of a cell deposited on borosilicate glass. Enhanced electron backscattering from borosilicate glass was noted until the thickness of the biological layer deposited on the substrate reached 1.25 µm. It resulted in significant increase in X-ray intensities typical for the elements present in the cellular part. In this case, the mean atomic number value of the biomaterial determines the strength of this effect. When elements are present in the cells only, the positive linear relationship appears between X-ray intensities and cell thickness. Then, spatial dimensions of X-ray emission for the particular elements are exclusively in the range of the biological part and the intensities of X-rays become constant. When the elements are present in both the cell and the biomaterial, X-ray intensities are registered for the biological part and the substrate simultaneously leading to a negative linear relationship of X-ray intensities in the function of cell thickness. In the case of the analysis of an element typical for the biomaterial, strong decrease in X-ray emission is observed in the function of cell thickness as the effect of X-ray absorption and the limited excitation range to biological part rather than to the substrate. Correction procedures for calculations of element concentrations in thin films and coatings deposited on substrates are well established in materials science, but little is known about factors that have to be taken into account to accurately quantify bioelements in thin and semi-thick biological samples. Thus thorough tests of currently available quantification procedures are required to verify their applicability to cells or tissues deposited on the biomaterials.

Fiber analysis vignettes: Electron microscopy to the rescue!

There has been considerable interest in the exposure doses that contribute to the various asbestos-associated diseases. Epidemiological studies have shown important differences in the contributions of the various fiber types to asbestos-related diseases, with the amphiboles showing a greater degree of potency as compared to chrysotile. However, epidemiological studies have occasionally provided misleading results. Over the past several decades, there have been several examples where fiber analysis using electron microscopy produced unexpected results which were important to our understanding of disease-exposure relationships. It is the purpose of this article to summarize these fiber analysis vignettes.

Approaches to single-nanoparticle catalysis.

Nanoparticles are among the most important industrial catalysts, with applications ranging from chemical manufacturing to energy conversion and storage. Heterogeneity is a general feature among these nanoparticles, with their individual differences in size, shape, and surface sites leading to variable, particle-specific catalytic activity. Assessing the activity of individual nanoparticles, preferably with subparticle resolution, is thus desired and vital to the development of efficient catalysts. It is challenging to measure the activity of single-nanoparticle catalysts, however. Several experimental approaches have been developed to monitor catalysis on single nanoparticles, including electrochemical methods, single-molecule fluorescence microscopy, surface plasmon resonance spectroscopy, X-ray microscopy, and surface-enhanced Raman spectroscopy. This review focuses on these experimental approaches, the associated methods and strategies, and selected applications in studying single-nanoparticle catalysis with chemical selectivity, sensitivity, or subparticle spatial resolution.

[Reparative Osteogenesis and Angiogenesis in Low Intensity Electromagnetic Radiation of Ultra-High Frequency].

BACKGROUND: Non-drug correction of reparative bone tissue regeneration in different pathological states - one of the most actual problems of modern medicine. OBJECTIVE: Our aim was to conduct morphological analysis of the influence of electromagnetic radiation of ultra-high frequency and low intensity on reparative osteogenesis and angiogenesis in fracture treatment under transosseous osteosynthesis. METHODS: A controlled nonrandomized study was carried out. In the experiment conducted on rats we modeled tibial fracture with reposition and fixation of the bone fragments both in control and experimental groups. In the animals of the experimental group the fracture zone was exposed to low intensity electromagnetic radiation of ultra-high frequency. Exposure simulation was performed in the control group. The operated bones were examined using radiography, light and electronic microscopy, X-ray electronic probe microanalysis. RESULTS: It has been established that electromagnetic radiation of ultra-high frequency sessions in fracture treatment stimulate secretory activity and degranulation of mast cells, produce microcirculatory bed vascular permeability increase, endotheliocyte migration phenotype expression, provide endovascular endothelial outgrowth formation, activate reparative osteogenesis and angiogenesis while fracture reparation becomes the one of the primary type. The full periosteal, intermediary and intraosteal bone union was defined in 28 days. CONCLUSION: Among the therapeutic benefits of electromagnetic radiation of ultra-high frequency in fracture treatment we can detect mast cell secretorv activity stimulation and endovascular anziozenesis activation.

[Chemical microanalysis of mineral deposits on explanted hydrophilic acrylic intraocular lenses].

AIM: to perform chemical microanalysis of mineral deposits on the surface of explanted hydrophilic acrylic intraocular lenses (IOL). MATERIAL AND METHODS: Two soft IOLs made of hydrophilic acryl (one, however, hydrophobic surface coated) and explanted 3 and 6 years after implantation were examined by scanning electron microscopy (EVO LS10, "Karl Zeiss", Germany). Chemical composition of the lens surface was studied using an energy-dispersive spectrometer (EDS X-Max50, Oxford, Great Britain). RESULTS: Chemical microanalysis allowed identification of the deposits, which turned out to be non-stoichiometric hydroxylapatite (also, hydroxyapatite (HA)) crystals with zinc impurity (up to 1.4%weight). CONCLUSION: The two samples represent two stages of a single process. The early stage is associated with newly formed HA crystals that are unable to cause any significant changes to the lens surface. However, as spherocrystals grow, they exert a crystallization effort that moves their growth centers apart with subsequent lens rupture and deformation. Crystal morphology undergoes dynamic changes: while primary (newly formed) crystals are sheaf-like, mature are spheres. A growing HA is non-stoichiometric. Zinc abundance accounts for appearance of its separate mineral phase. Hydrophilic properties of acrylic polymer determine its high affinity for HA crystals. Hydrophobic coating (sample no.1) does not completely prevent lens opacification due to mineral deposits on its surface.

Fabrication of a sealed electrochemical microcell for in situ soft X-ray microspectroscopy and testing with in situ co-polypyrrole composite electrodeposition for Pt-free oxygen electrocatalysis.

In this paper we report on the fabrication and testing of a novel concept of sealed electrochemical microcell for in situ soft X-ray microspectroscopy in transmission, dedicated for nonvacuum compatible electrolytes. The microcell, fabricated using ultraviolet lithography, at variance with previous versions of electrochemical wet cells, that featured an optical window glued on top of the electrode system and a very limited electrolyte volume, the device presented here is a single solid block based around a microfabricated channel with fixed optical windows and apt for microfluidic work. Moreover, this cell allows to employ an advanced electrodic geometry developed in our group - so far used only in open electrochemical cells for work with vacuum-compatible electrolytes - also with low-vapor pressure liquids, possibly saturated with the required gases. The cell optimal electrode design allows three-electrode electrochemical control typical of traditional electrochemical experiments. The first electrochemical experiments with this new cell explore the electrochemical growth of a Co-polypyrrole, a composite electrocatalyst material with promising performance to replace the expensive Pt catalyst in fuel-cell oxygen electrodes. Morphological and chemical-state distributions of Co codeposited with polypyrrole has been followed as a function of time and position, yielding unprecedented information on the processes relevant to the synthesis of this catalyst.

Scanning ion-selective electrode technique and X-ray microanalysis provide direct evidence of contrasting Na+ transport ability from root to shoot in salt-sensitive cucumber and salt-tolerant pumpkin under NaCl stress.

Grafting onto salt-tolerant pumpkin rootstock can increase cucumber salt tolerance. Previous studies have suggested that this can be attributed to pumpkin roots with higher capacity to limit the transport of Na(+) to the shoot than cucumber roots. However, the mechanism remains unclear. This study investigated the transport of Na(+) in salt-tolerant pumpkin and salt-sensitive cucumber plants under high (200 mM) or moderate (90 mM) NaCl stress. Scanning ion-selective electrode technique showed that pumpkin roots exhibited a higher capacity to extrude Na(+), and a correspondingly increased H(+) influx under 200 or 90 mM NaCl stress. The 200 mM NaCl induced Na(+)/H(+) exchange in the root was inhibited by amiloride (a Na(+)/H(+) antiporter inhibitor) or vanadate [a plasma membrane (PM) H(+) -ATPase inhibitor], indicating that Na(+) exclusion in salt stressed pumpkin and cucumber roots was the result of an active Na(+)/H(+) antiporter across the PM, and the Na(+)/H(+) antiporter system in salt stressed pumpkin roots was sufficient to exclude Na(+) X-ray microanalysis showed higher Na(+) in the cortex, but lower Na(+) in the stele of pumpkin roots than that in cucumber roots under 90 mM NaCl stress, suggesting that the highly vacuolated root cortical cells of pumpkin roots could sequester more Na(+), limit the radial transport of Na(+) to the stele and thus restrict the transport of Na(+) to the shoot. These results provide direct evidence for pumpkin roots with higher capacity to limit the transport of Na(+) to the shoot than cucumber roots.

Evaluation of X-ray microanalysis for characterization of dental enamel.

Elemental analysis of dental hard tissues is of importance. The aim of this study is to evaluate X-ray microanalysis (XRMA) of bovine enamel in a scanning electron microscope (SEM) with different coatings. The buccal surface of bovine incisors was polished flat, one-third was coated with carbon, one-third with gold, leaving one-third uncoated for XRMA in an SEM equipped with an energy-dispersive microanalysis system. The elements oxygen, sodium, magnesium, phosphorous, chlorine, potassium, and calcium were analyzed using their respective characteristic K X-ray series. Comparisons were made with analyses of glass produced by fusion of the bovine enamel, showing that oxygen analyses using the K X-ray series are reliable and preferable to calculating oxygen by stoichiometry for natural enamel. For the gold-coated and uncoated analyses, carbon was also measured using the K X-ray series. Small area Analyses in small areas (80 × 80 µm) in variable pressure-SEM mode with low vacuum (20 Pa), without any coating, midway between 40 µm wide gold lines 140 µm apart to avoid build-up of electrostatic charge is the preferred method, especially if carbon is included in the analysis. The analyses of bovine enamel are sufficiently reproducible to be regarded as quantitative for all elements except carbon.

Comparative study of the measurement of enamel demineralization and remineralization using transverse microradiography and electron probe microanalysis.

Transverse microradiography (TMR) and electron probe microanalysis (EPMA) are commonly used for characterizing dental tissues. TMR utilizes an approximately monochromatic X-ray beam to determine the mass attenuation of the sample, which is converted to volume percent mineral (vol%min). An EPMA stimulates the emission of characteristic X-rays from a variable volume of sample (dependent on density) to provide compositional information. The aim of this study was to compare the assessment of sound, demineralized, and remineralized enamel using both techniques. Human enamel samples were demineralized and a part of each was subsequently remineralized. The same line profile through each demineralized lesion was analyzed using TMR and EPMA to determine vol%min and wt% elemental composition and atomic concentration ratio information, respectively. The vol%min and wt% values determined by each technique were significantly correlated but the absolute values were not similar. This was attributable to the complex ultrastructural composition, the variable density of the samples analyzed, and the nonlinear interaction of the EPMA-generated X-rays. EPMA remains an important technique for obtaining atomic ratio information, but its limitations in determining absolute mineral content indicate that it should not be used in place of TMR for determining the mineral density of dental hard tissues.

Hydroxyapatite coatings on nanotubular titanium dioxide thin films prepared by radio frequency magnetron sputtering.

In this study, hydroxyapatite (HA) was coated on anodized titanium (Ti) surfaces through radio frequency magnetron sputtering in order to improve biological response of the titanium surface. All the samples were blasted with resorbable blasting media (RBM). RBM-blasted Ti surface, anodized Ti surface, as-sputtered HA coating on the anodized Ti surface, and heat-treated HA coating on the anodized Ti surface were prepared. The samples were characterized using scanning electron microscopy and X-ray photoemission spectroscopy, and biologic responses were evaluated. The top of the TiO2 nanotubes was not closed by HA particles when the coating time is less than 15 minutes. It was demonstrated that the heat-treated HA was well-crystallized and this enhanced the cell attachment of the anodized Ti surface.

Improved algorithm for elemental analysis by laser-induced breakdown spectroscopy.

We propose a calibration-free algorithm for retrieval of elemental concentrations using laser-induced breakdown spectroscopy. This is a simple and improved version of other ratio-based algorithms as it needs only one trial parameter to estimate all other concentrations. The present algorithm has been used to estimate the composition of a brass sample, and the results agree within 1% with electron probe microanalyzer measurements.

Laboratory-based cryogenic soft x-ray tomography with correlative cryo-light and electron microscopy.

Here we present a novel laboratory-based cryogenic soft X-ray microscope for whole cell tomography of frozen hydrated samples. We demonstrate the capabilities of this compact cryogenic microscope by visualizing internal subcellular structures of Saccharomyces cerevisiae cells. The microscope is shown to achieve better than 50 nm half-pitch spatial resolution with a Siemens star test sample. For whole biological cells, the microscope can image specimens up to 5 µm thick. Structures as small as 90 nm can be detected in tomographic reconstructions following a low cumulative radiation dose of only 7.2 MGy. Furthermore, the design of the specimen chamber utilizes a standard sample support that permits multimodal correlative imaging of the exact same unstained yeast cell via cryo-fluorescence light microscopy, cryo-soft X-ray microscopy, and cryo-transmission electron microscopy. This completely laboratory-based cryogenic soft X-ray microscope will enable greater access to three-dimensional ultrastructure determination of biological whole cells without chemical fixation or physical sectioning.

Analysis of biologically-derived small particles--searching for geometry correction factors using Monte Carlo simulation.

A Monte Carlo simulation was used to determine geometry correction factors that increase accuracy of quantitative X-ray microanalysis of laterally semithick biological materials. A model composed of cellulose with homogeneously distributed biological elements and lateral dimensions between 0.5-25 µm was chosen. The specimen was exposed to 5, 10, and 15 keV electrons, the net intensities of characteristic X-rays registered for the elements, and presented as a function of the lateral dimensions of the model. This showed the double decay exponential function fitted the distribution of X-ray intensities in relation to the model size. The applicability of the function as a correction method was successfully tested for 30 specimens with varying composition and dimensions. The value of relative error decreased from ±60% to ±5% when the correction was applied. Moreover, the minimal lateral size of the material was defined, below which the correction is not required. The simulation also revealed that the difference of the weighted sum of Z²/A between the unknown and the standard could reach 25% without significant influence on the quantitative results. The correction method could be helpful for accurate assessment of elemental composition in biological or organic matrices, when their lateral dimensions are smaller than the distribution range.

Rapid and accurate analysis of an X-ray fluorescence microscopy data set through gaussian mixture-based soft clustering methods.

X-ray fluorescence (XRF) microscopy is an important tool for studying trace metals in biology, enabling simultaneous detection of multiple elements of interest and allowing quantification of metals in organelles without the need for subcellular fractionation. Currently, analysis of XRF images is often done using manually defined regions of interest (ROIs). However, since advances in synchrotron instrumentation have enabled the collection of very large data sets encompassing hundreds of cells, manual approaches are becoming increasingly impractical. We describe here the use of soft clustering to identify cell ROIs based on elemental contents, using data collected over a sample of the malaria parasite Plasmodium falciparum as a test case. Soft clustering was able to successfully classify regions in infected erythrocytes as "parasite," "food vacuole," "host," or "background." In contrast, hard clustering using the k-means algorithm was found to have difficulty in distinguishing cells from background.While initial tests showed convergence on two or three distinct solutions in 60% of the cells studied, subsequent modifications to the clustering routine improved results to yield 100% consistency in image segmentation. Data extracted using soft cluster ROIs were found to be as accurate as data extracted using manually defined ROIs, and analysis time was considerably improved.