Studies on cultural metal artifacts can benefit greatly from microscopy techniques. The examination of microstructural features can provide relevant information about ancient manufacturing techniques, as well as about corrosion/degradation processes. In the present work, advantages of the use of multifocus imaging techniques in optical microscopy for the study of archaeological metals are presented. An archaeometallurgical study of a large collection of bronzes demonstrates the possibility of a microstructural study with no need for sample removal, which is a great advantage in the study of cultural objects. In addition, the study of mounted samples illustrates the advantages of the multifocus technique in the examination of particular corrosion features, with the possibility of three-dimensional reconstructions.

Salicornia ramosissima J. Woods (Chenopodiaceae), included in the species aggregate S. europaea agg., is an annual succulent halophyte and one of the most salt tolerant plants, broadly distributed in the salt marshes and salt pans of Ria de Aveiro and in many others of the Iberian Peninsula. Salicornia L. has been used not only as a seasoned vegetable, salad and fermented food in coastal areas of Europe and Asia, but also as folk medicine for disorders such as constipation, obesity and diabetes. To corroborate this, the literature reports immunomodulatory, antioxidative, anti-inflammatory, anti-hyperlipidemic and antidiabetic effects. Moreover, some bioactive compounds from its aerial parts were recently isolated and identified, exhibiting also antioxidant and cytotoxic activities.

Factors such as the proper botanical identification, season and harvest site, extraction and purification methods, characterisation of the active constituents and the extract effects in a dose-and time-dependent manner are crucial to assess the therapeutic potential of herbal extracts. The aim of this study was to investigate the possible hepatic and renal effects of an ethanolic extract of S. ramosissima on mice.

Aerial portions of S. ramosissima were collected from a salt pan in Ria de Aveiro (Portugal). The ethanolic extract (50 mg/kg b.w.) was orally administered, during 3 weeks, to male ICR-CD1 mice, purchased from Harlan (Spain). Control group was also considered. Liver and kidney were collected and prepared for histology. Animal procedures were followed according to guidelines for ethics and animal care.

Central and portal vein congestion and severe hydropic changes of the hepatocytes were noted in the liver of exposed group, compared with controls (Figs. 1A, 1B). Renal profile of control group evidenced normal features (Figs.1C, 1E). However, significant histological alterations were found in the exposed group: cortical interstitial haemorrhages (Fig. 1D), inflammatory cell infiltration and tubular cell degeneration within medulla and cortico-medullary junction (Fig. 1F).

In conclusion, this pilot study has demonstrated considerable effects in the mouse metabolism of S. ramosissima ethanolic extract, with significant hepatic and renal lesions. Further studies are needed to correlate these data with isolated active constituents for a more reliable evaluation of the potential of this species.

Acknowledgements

Grants were provided by CICECO, QOPNA and CESAM.

Biosynthetic calcite samples were investigated using combined synchrotron X-ray microspectroscopy mapping. These samples were prepared with bacteria isolated from the Large cave of Arcy-sur-Cure in which prehistoric figures are masked by an opaque calcite layer. The biotic or abiotic origin of this layer is the issue of the present work. As previously known, a large community of bacteria may be involved in the CaCO3 formation in caves. A mixture of calcite/vaterite was obtained from bacteria isolated from the cave. Therefore, we can offer conclusions on their calcifying capability. The rare presence of vaterite in cave environments may be treated as a marker of biotic carbonate formations. Moreover, an amorphous calcium phosphate phase was present in the form of a calcite/vaterite mixture in the biotic model samples. This mixture of phases could be used as a tracer of the biotic process of CaCO3 formation. These biotic tracer phases were not identified using the applied analytical methods in the natural samples taken from the opaque calcite layers that covered the prehistoric figures of the Large cave. In this case, based on the obtained results, the biotic calcite formation process is likely to be considered as an undetectable effect at minimum.

Most blood vessels contain elastin that provides the vessels with the resilience and flexibility necessary to control hemodynamics. Pathophysiological hemodynamic changes affect the remodeling of elastic components, but little is known about their structural properties. The present study was designed to elucidate, in detail, the three-dimensional (3D) architecture of delicate elastic fibers in small vessels, and to reveal their architectural pattern in a rat model. The fine vascular elastic components were observed by a newly developed scanning electron microscopy technique using a formic acid digestion with vascular casts. This method successfully visualized the 3D architecture of elastic fibers in small blood vessels, even arterioles and venules. The subendothelial elastic fibers in such small vessels assemble into a sheet of meshwork running longitudinally, while larger vessels have a higher density of mesh and thicker mesh fibers. The quantitative analysis revealed that arterioles had a wider range of mesh density than venules; the ratio of density to vessel size was higher than that in venules. The new method was useful for evaluating the subendothelial elastic fibers of small vessels and for demonstrating differences in the architecture of different types of vessels.

Atomic-scale tomography (AST) is defined and its place in microscopy is considered. Arguments are made that AST, as defined, would be the ultimate microscopy. The available pathways for achieving AST are examined and we conclude that atom probe tomography (APT) may be a viable basis for AST on its own and that APT in conjunction with transmission electron microscopy is a likely path as well. Some possible configurations of instrumentation for achieving AST are described. The concept of metaimages is introduced where data from multiple techniques are melded to create synergies in a multidimensional data structure. When coupled with integrated computational materials engineering, structure–properties microscopy is envisioned. The implications of AST for science and technology are explored.

Sponsored by the Microscopy Society of America (MSA) and the Microanalysis Society (MAS)

The use of microreactors in (bio)chemical processes has been gaining relevance in the last decade. The low consumption of reagents, the possibility of continuous operation and the faster translation from lab- to production scale are some of the several advantages of these devices. The whole results in cost reductions in process development. Enzyme catalyzed reactions have proved to be an excellent alternative to the use of chemicals due to its ability to catalyze the most complex chemical processes under benign experimental and environmental conditions. In this way, enzymes may be crucial to the implementation of a much more sustainable chemical industry.

The present work is within such scope, using as model system the immobilization of invertase in glass (silica) microchannels, for the production of fructose syrups through sucrose hydrolysis. The immobilization of the enzyme was achieved through treatment of the substrate with a sequence of coatings (Figure 1). Activation of the inner surface of the microchannels with 3-aminopropyltriethoxysilane (APTES) was followed by the introduction of a spacer, glutaraldehyde. Lastly the enzyme solution was introduced in the presence of sodium cyanoborohydride, in order to enhance the stability of the support-enzyme binding.

The characterization of the coatings at each stage of the immobilization protocol was carried out to confirm the change of the microchannel surface. Such studies were performed using Atomic Force Microscopy (AFM) and Scanning Electron Microscopy (SEM) analysis (Figure 1).

The results obtained, namely the shifts on the surface roughness corroborate that the several coatings were successfully applied and the enzyme immobilized. Moreover, the immobilization approach used proved to be highly effective, resulting on successful continuous use of the microreactor for a period of 30 days with roughly constant full conversion of a sucrose solution of 50g/l, at a flow rate of 7µl/minute (Figure 2).

Future work will involve a more extensive characterization of the several coatings by Quartz Crystal Microbalance which will be decisive to achieve a better comprehension of the coating phenomena and hence optimize the immobilization process.

The authors would like to thank Fundação para a Ciência e a Tecnologia, Portugal, for financial support through contracts under the program Ciência 2007 awarded to P. Fernandes, for the doctoral grant SFRH/BD/74818/2010 awarded to F. Carvalho and for the doctoral grant SFRH/BD/71990 /2010 awarded to Patrizia Paradiso.

In this study, we replaced the expensive blade material with an aluminum–bronze alloy that has excellent corrosion resistance and cavitation characteristics and developed the corrosion protection method to improve durability using an electrochemical method. The objective of this study was to identify the electrochemical corrosion protection conditions to minimize cavitation damage due to generating hydrogen gas (2H2O + 2e− → 2OH− + H2) by means of hydrogen overvoltage before the impact pressure of the cavity is transferred to the surface. In the constant potential experiment under the cavitation environment, the energy was reflected or cancelled out by collision of the cavities with the hydrogen gas generated by the hydrogen overvoltage. As a result, the optimal corrosion prevention potential in the dynamic state is assumed to be the range of −1.4 to −1.7 V, which is the range at which active polarization took place.

Studies of human bloodstains on nonbiological materials have been previously carried out using a high-vacuum scanning electron microscope (HV-SEM) in secondary-electron mode without any sample treatment. To assess whether biological substrates can affect the morphology of human erythrocytes in bloodstains, three fragments of different biological material (bone, shell, and wood) were smeared with peripheral human blood. Afterward, the bloodstains were directly examined in secondary-electron mode by an HV-SEM following a procedure initially standardized to be used in uncoated human bloodstains on stone. The obtained results suggest that HV-SEM is suitable for examining untreated bloodstains on biological substrate and that the morphology of erythrocytes in human bloodstains is not affected by the biological nature of the substrate. A cautionary issue regarding bloodstains on nondehydrated biological substrates is that the waiting time required for initiating the HV-SEM examination is by far higher than when using inorganic bloodstain substrates.

Fluorescence resonance energy transfer (FRET) measurement based on partial acceptor photobleaching (PbFRET) is easy to implement without external references. However, the current PbFRET methods are inapplicable to the construct with multiple acceptors, which largely increase the Förster distance. Here, we proposed a linear theory for the dependence of the acceptor photobleaching probability of construct with multiple acceptors on the photobleaching degree (x) and developed a multiple acceptors PbFRET method (Ma-PbFRET) to measure the FRET efficiency of construct with multiple acceptors (n) by measuring the fluorescence intensities of both donor and acceptor channels before and after acceptor photobleaching. The Ma-PbFRET method was validated by measuring the FRET efficiency of construct with two or three acceptors under different x in living cells. Our experimental results demonstrate that the Ma-PbFRET method is capable of exactly quantifying the FRET efficiency of construct with multiple acceptors, providing a simple and powerful tool to investigate the assembly/disassembly of biomolecular complexes with larger distance in living cells.

Pollen grain morphology has been widely used in the classification of the Acanthaceae, where external pollen wall features have proved useful in determining relationships between taxa. Although detailed information has been accumulated using light microscopy, transmission electron microscopy and scanning electron microscopy (SEM) techniques, internal pollen wall features lack investigation and the techniques are cumbersome. A new technique involving precise cross sectioning or slicing of pollen grains at a selected position for examining wall ultrastructure, using a focused ion beam-scanning electron microscope (FIB-SEM), has been explored and promising results have been obtained. The FIB-SEM offers a good technique for reliable, high resolution, three-dimensional (3D) viewing of the internal structure of the pollen grain wall.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

A new approach for the simulation of dynamic electron backscatter diffraction (EBSD) patterns is introduced. The computational approach merges deterministic dynamic electron-scattering computations based on Bloch waves with a stochastic Monte Carlo (MC) simulation of the energy, depth, and directional distributions of the backscattered electrons (BSEs). An efficient numerical scheme is introduced, based on a modified Lambert projection, for the computation of the scintillator electron count as a function of the position and orientation of the EBSD detector; the approach allows for the rapid computation of an individual EBSD pattern by bi-linear interpolation of a master EBSD pattern. The master pattern stores the BSE yield as a function of the electron exit direction and exit energy and is used along with weight factors extracted from the MC simulation to obtain energy-weighted simulated EBSD patterns. Example simulations for nickel yield realistic patterns and energy-dependent trends in pattern blurring versus filter window energies are in agreement with experimental energy-filtered EBSD observations reported in the literature.

Welcome to the second special section of Microscopy and Microanalysis focused on electron backscatter diffraction (EBSD), which follows the June 2011 issue. The content of the previous special section was provided by participants at EBSD 2010, the second Microanalysis Society (MAS) topical conference dedicated to EBSD in the United States. The present 2013 special section includes work from participants at both EBSD 2012, the third of such topical conferences (held June 19–21, 2012 at Carnegie Mellon University, Pittsburgh, PA), and EMAS 2012, the European Microanalysis Society's 10th Regional Workshop that included three EBSD sessions (held June 17–20 at the Institute for Geosciences and Earth Resources, Padua, Italy).

Nanostructure evolution during low temperature aging of three binary Fe-Cr alloys has been investigated by atom probe tomography. A new method based on radial distribution function (RDF) analysis to quantify the composition wavelength and amplitude of spinodal decomposition is proposed. Wavelengths estimated from RDF have a power-law type evolution and are in reasonable agreement with wavelengths estimated using other more conventional methods. The main advantages of the proposed method are the following: (1) Selecting a box size to generate the frequency diagram, which is known to generate bias in the evaluation of amplitude, is avoided. (2) The determination of amplitude is systematic and utilizes the wavelength evaluated first to subsequently evaluate the amplitude. (3) The RDF is capable of representing very subtle decomposition, which is not possible using frequency diagrams, and thus a proposed theoretical treatment of the experimental RDF creates the possibility to determine amplitude at very early stages of spinodal decomposition.

Electron backscatter diffraction (EBSD) was used in Conservation Science for characterization of ancient materials collected from works of art. The results demonstrate the feasibility of EBSD analysis on heterogeneous matrices as very small samples of paint layers collected from paintings. Two reference pigments were selected from those used by artists to investigate the relationship existing between EBSD pattern quality and properties of the investigated material (i.e., average atomic number, density, and Mohs hardness). The technique was also tested to investigate the pigment phases on two real samples collected from Romanino's Santa Giustina altarpiece, an oil on wood painting dated 1514 (Civic Museum, Padova, Italy). Results show for the first time the acquisition of EBSD patterns from painting samples mounted in resin, i.e., painting cross sections, opening a new powerful tool to elucidate the pigment phases avoiding large sampling on works of arts and to further study the complex mechanisms of pigment deterioration.

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

Podosomes are cellular adhesion structures involved in matrix degradation and invasion that comprise an actin core and a ring of cytoskeletal adaptor proteins. They are most often identified by staining with phalloidin, which binds F-actin and therefore visualizes the core. However, not only podosomes, but also many other cytoskeletal structures contain actin, which makes podosome segmentation by automated image processing difficult. Here, we have developed a quantitative image analysis algorithm that is optimized to identify podosome cores within a typical sample stained with phalloidin. By sequential local and global thresholding, our analysis identifies up to 76% of podosome cores excluding other F-actin-based structures. Based on the overlap in podosome identifications and quantification of podosome numbers, our algorithm performs equally well compared to three experts. Using our algorithm we show effects of actin polymerization and myosin II inhibition on the actin intensity in both podosome core and associated actin network. Furthermore, by expanding the core segmentations, we reveal a previously unappreciated differential distribution of cytoskeletal adaptor proteins within the podosome ring. These applications illustrate that our algorithm is a valuable tool for rapid and accurate large-scale analysis of podosomes to increase our understanding of these characteristic adhesion structures.

A very rare subset of Epidermolysis bullosa simplex (EBS), termed “EBS superficialis” (EBSS), has been described in two families. EBS superficialis is a rare autosomal dominant form of suprabasal EBS in which blistering arises just below the stratum corneum.

The original report of EBSS was subsequently questioned by genetic analysis of one of the families that identified a mutation in a collagen gene responsible for dystrophic epidermolysis bullosa (DEB), thus implying that this entity is in fact a variant of DEB. Nonetheless, the existence of some form of EBSS can not be ruled out.

We performed a transmission electron microscopy study of a skin biopsy from a 36 year old woman suspect of having EBSS. A skin biopsy was sequentially fixed in 3% glutaraldehyde in sodium cacodylate buffer 0.1M pH 7.3, osmium tetroxide in the same buffer and uranyl acetate in acetate-acetic acid buffer 0.1M pH 5. Dehydration was performed in ethanol and embedding in an Epon-Araldite mixture. Thin sections contrasted with uranyl acetate and lead cytrate were observed and photographed in a JEOL 100SX electron microscope.

She had a few intact acral bullae in the neonatal period and superficial erosions and crusts in friction areas since then. Her one year-old boy, her older sister and the latter’s adolescent son had the same clinical picture. The patient does not display any symptoms expected for DEB.

We found profound alterations of the structure of the outer layers of the epidermis. Degenerative changes were seen in the cells of the upper layers of the stratum spinosum. Nuclei are lost and flattened cells made mostly of compressed tonofilaments lay below a cleft that develops between the compressed cells and the granular cell layer (Fig. 1). The granular cell layer is very reduced and represented by a few damaged cells that stay attached to the stratum corneum (Fig. 2).

The alterations do not seem to be related to collagen defects, and in the reported case the cleft develops in the altered upper region of the prickle-cell layer, just below the granular layer.