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

This paper aims at identifying the provenance of Goryeo celadons by understanding its microstructural characteristics, such as particles, blisters, forms and amount of pores, and the presence of crystal formation, bodies, and glazes and its boundary, using an optical microscope and scanning electron microscopy (SEM). The analysis of the reproduced samples shows that the glazed layer of the sherd fired at higher temperatures has lower viscosity and therefore it encourages the blisters to be combined together and the layer to become more transparent. In addition, the result showed that the vitrification and melting process of clay minerals such as feldspars and quartzs on the bodies was accelerated for those samples. To factor such characteristics of the microstructure and apply it to the sherds, the samples could be divided into six categories based on status, such as small particles with many small pores or mainly large and small circular pores in the bodies, only a limited number of varied sized blisters in the glazes, and a few blisters and needle-shaped crystals on the boundary surface. In conclusion, the analysis of the microstructural characteristics using an optical microscope and SEM have proven to be useful as a categorizing reference factor in a provenance study on Goryeo celadons.

A fully automatic approach to locate polyomavirus particles in images of transmission electronic microscopy is presented, that can localize intact particles, many damaged capsids and an acceptable percentage of superposed ones; the performance is quantified in 25 electron micrographs containing nearly 390 particles, compared to the interpretation of two independent electron microscopy experts. As these particles often does not have a well-defined edge, in highly textured backgrounds (Figure 1) the initial guess of candidate points from local entropy proportion measures retains a large set of points, in order to avoid false negatives.

Based in human criteria - focused on the pattern generated by the capsomeres typical of polyomavirus surface, this approach uses indicators calculated from the local co-occurrence matrix of grey levels to assess the textured pattern and prune the initial set of candidates; in some more complicated backgrounds about 2-10% of the elements will survive. A restricted set of the points accepted (Figure 2) is used to evaluate typical average and variance and reduce the set of survivors accordingly. These intermediate points are locally evaluated using i) a statistical index concerning the radiometric distribution of a circular neighborhood around the centroid of each candidate, and ii) a structural index resuming the expected morphological characteristics of eight radial intensity profiles encompassing the area of the possible particle. All the parameterization is based on the particle expected dimensions.

This hierarchical approach attains 95% efficiency in the detection of intact virus particles, tolerating a certain lack of differentiation in the borders and a certain amount of shape alterations. Only 5 to 7% (according to the reference used) of the intact particles are missed by the algorithm – cases of particles with abnormal radial structures, particles with different degrees of agglutination or particles showing empty (or with different degrees of permeation) capsids. Superposed particles are detected with a success rate of 71% to 77%. In the case of distorted and/or doubtful particles, a correct detection of 72% is attained; these particles are the main source of differences between experts (a difference of 48% between both interpretations).

This kind of algorithm is "tailored" for one virus family particle, in this case, the polyomavirus; it can be parameterized for different working magnifications, but cannot be applied to different virus, that has different texture, structure and typical radiometry characteristics. We believe that similar results can be obtained in other virus families with the necessary adaptations.

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

Disputes on the painting methods of Goguryeo murals can mainly be categorized into whether the murals adapted eastern secco or western fresco; however, the murals have their own unique methods as well. There are different viewpoints among experts on interpreting the painting methods. This study involved the creation of research samples to discover the painting methods under dispute and may help discover the methods based on scanning electron microscopy energy-dispersive X-ray spectroscopy (SEM-EDX) studies. Goguryeo murals introduced pseudo-fresco rather than buon fresco methods. Unlike fresco techniques in the West, Goguryeo painters mixed traditional soft binders and adapted typical secco painting techniques for paintings, borders, and corrections after drying. The disputed issues may be resolved by these techniques, and samples may be produced based on the analyzed data. Therefore, many questions can finally be answered through SEM-EDX elemental mapping.

Zinc is a trace metal that is regularly found at pollution sites, amongst other metals and pollutants, contributing to the toxicity of marine ecosystems. In this study zinc acute toxicity tests were conducted on the sea anemone Actinia equina. This sea anemone presents a wide geographic distribution on rocky shore marine habitats, is quite resistant to some degree of various forms of contaminants and plays an important ecological key role in the marine environments.

After a period of acclimatising on a flow through system, organisms of the same average size were exposed to different concentrations of zinc solution during a period of 96 hours (0 µg/l, 10 µg/l, 50 µg/l, 100 µg/l, and 200µg/l). The histological processing of the specimens was performed using standard procedures, slightly modified due to the peculiar nature of the biological material.

The general effects exhibited necrosis of tentacles, mesenterial filaments, and in the gastrodermal regions (Figures 1, 2 and 3). It were observed tentacular cysts, black particulate accumulations in the gastrodermis, hypertrophied cells in the lobes of the mesenteries filaments, and spirocyst and associated necrosis (Fig. 2-3). All anemones exhibited extensive areas of tissue necrosis, degeneration of the epidermis in various stages, with loss of mucous cells, vacuolation, necrosis and ptychocysts from the epidermis, necrosis of retractor muscles and ducts (Figures 2-3). Neoplasms of the gastrointestinal tract and gonads were observed in internal structures. In the former, modifications of the mesenteries such as intensive fragmentation, vacuolization and epithelial thinning and in the latter abnormal gonad development and necrosis were noted (Figures 2-3).

The data suggests that Actinia equina may be adversely affected by acute exposures to high levels of zinc as seen during laboratory exposures of other invertebrates and is also probably sensitive to acute exposures to metal’s toxic sediments in the marine environment. The loss of mucous secretory cells and ptychocysts from the epidermis suggest that energy reserves for cell replenishment were adversely affected. Mucus production in benthic cnidarians is necessary to prevent burial by sedimentation and to protect against toxins or invading microorganisms. The serious effects on the gastrodermis integrity indicate that sea water in the coelenteron is no longer circulated. The observed modifications could be considered as a general cnidarians response to stress.

Further investigations of bioaccumulation, physiology, and histopathology in this anemone and other anthozoans from polluted environments should prove to be valuable in pollution monitoring studies, as they have for other benthic invertebrates

Acknowledgments

This work was supported by the Portuguese Foundation for the Science and Technology – Portugal and FEDER funds, through the Projects: PTDC/MAR/464729/2006 and FCT/CNPq (Brazil), Project 6818, Programme 19/ 004.

Several problematic specimens, especially when composed of a complex three-dimensional architecture or very high or ultralow ranges in regional thickness and density, can be observed in improved clarity and precision when universal variable brightfield–darkfield contrast (UVBDC) is used. In this method, two different partial images are optically superimposed and interfere with each other, contributing to complementary visual information: a brightfield and a darkfield image. These images can be generated with concentric-peripheral, paraxial, or axial illuminating light. In all variants, variable transitions between bright- and darkfield are achievable. By use of a pancratic condenser (zoom system), the illuminating light can be universally adjusted and optimally adapted to each type of specimen and each type of objective (glass and mirror lenses). The concentric-peripheral variant is preferably carried out with normal glass lenses, the axial variant with mirror lenses. Glass lenses can also be used for UVBDC based on axial or paraxial light when combined with a special contrast tube, which is described in detail. Which technical variant of UVBDC might lead to the best result may be determined by the particular properties of the specimen, but all techniques described promise significant improvements in image quality and visual information.

Atom probe is a nanoscale technique for creating three-dimensional spatially and chemically resolved point datasets, primarily of metallic or semiconductor materials. While atom probe can achieve local high-level resolution, the spatial coherence of the technique is highly dependent upon the evaporative physics in the material and can often result in large geometric distortions in experimental results. The distortions originate from uncertainties in the projection function between the field evaporating specimen and the ion detector. Here we explore the possibility of continuum numerical approximations to the evaporative behavior during an atom probe experiment, and the subsequent propagation of ions to the detector, with particular emphasis placed on the solution of axisymmetric systems, such as isolated particles and multilayer systems. Ultimately, this method may prove critical in rapid modeling of tip shape evolution in atom probe tomography, which itself is a key factor in the rapid generation of spatially accurate reconstructions in atom probe datasets.

Engineered nanomaterials such as nanoparticles (NPs) are increasingly being used for commercial purposes in products within medicine, electronics, sporting goods, tires, textiles and cosmetics. They comprise diverse types of materials from metals, polymers, ceramic to biomaterials and have been defined as particles with at least one dimension in the order up to 100 nm1. The higher toxicological potential of NPs is mostly due to their small size, wide surface, increase of their chemical reactivity and biological activity and the capacity to generate free radicals. NPs also can have the ability to penetrate trough the biological barriers and to move easily through the biological systems. Therefore, is mandatory to assess the toxicity of these nanomaterials, since their industrial production and uses will also result in releases to the environment with unclear consequences.

The aim of the present work is to evaluate the toxicity of titanium dioxide (TiO2) NPs on gill gluthatione-S-transferase activity (GST), lipid peroxidation and on structure of the gills of two freshwater fish species (Carassius auratus and Danio rerio). Suspensions of TiO2 NPs, with an average size of 21 nm, were prepared using distillate water and then ultrasonicated (10 min, 35 KHz). The suspensions were added to 10L of tap water in exposure tanks, to obtain nominal concentrations (0.01; 0.1; 1, 10; 100 TiO2 mg/L). The test fish, C. auratus (N=144) and D. rerio (N=80), were randomly distributed by 6 exposure tanks and an additional tank with clean tap water was used as control. Fish were sampled after 7, 14, and 21 days. Six fish from both species were left for depuration in clean tap water during 14 days and then sacrificed. The GST activity was determined by following the procedure described by Habig et al. and lipid peroxidation was measured based on the Thiobarbituric Acid Reactive Species method. The tissues were processed essentially according to Martoja and Martoja for light microscopy (LM). For transmission electron microscopy (TEM) the samples were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite according to standard procedures. The histological and ultrastructural observations were performed using a Leica-ATC 2000 microscope and a JEOL 100-SX electron microscope respectively.

The results show a significant increase of GST in gills tissues for C.auratus exposed to 10 and 100 mg/L TiO2 NPs and a decrease following the depuration period. With respect to D. rerio a significant increase was observed in fish exposed to 1, 10 and 100 mg/L TiO2 NPs. Lipid peroxidation are in agreement with GST results but showing a significant increase for fish (C.auratus and D. rerio) exposed to concentrations of 0.1 TiO2 mg/L NPs and higher. Usually, the oxidative stress caused by exposure to TiO2 NPs is attributed to hydroxyl radicals (OH) generated by photochemical (UV/vis) processes but it may be also related to specific properties of TiO2 NPs such as size, surface area and solubility that can influence the degree of toxicity. The results from LM observations (Fig. 1) showed that exposure to TiO2 NPs affected gill tissues, with changes being detected in both fish species exposed to 0.1 TiO2 mg/L NPs and higher which is in accordance with biochemical results. Changes include different degrees of hyperplasia (from low to complete fusion of lamellae). The TEM analysis revealed that TiO2 NPs were internalized by gills epithelial cells accumulating in vacuoles inside these cells (Fig. 2). After the depuration period it was observed that the capability for gills to recover was not complete. The results show a strong response to oxidative stress caused by exposure to TiO2 NPs, possibly because they are in direct contact with the exposure medium and function as a first barrier against external aggression. However, the gills changes observed following exposure and a partial recover after depuration suggest that TiO2 NPs may cause deleterious effects in fish gills compromising fish homeostasis.

The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through grant PTDC/CTM/099446/2008 and through project no. PEst-C/EQB/LA0006/2011 granted to Requimte.

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

A new method for determining the vitrification rate of pottery depending on the firing temperature was devised using secondary electron images (SEI) of scanning electron microscope (SEM). Several tests were performed to establish the appropriate operating conditions of SEM and reproducibility as well as to examine the applicability of the method. The grayscale values converted from each pixel of SEI were used to determine the vitrification rate of pottery, which in our study were artificially fired specimens composed of three types of clay. A comparison between the vitrification rate value and appearance temperature of minerals shows that mullite formation starts at 1,100°C, during which the vitrification rate rapidly increases by over 10%. In consequence, the result presented here demonstrates that the new method can be applied to estimate the firing temperature of pottery.

White and black deposits have been frequently observed on the surface of Korean stone cultural heritages, and they are considered as damage factors in both conservation and esthetic points of view. In order to set up the appropriate conservation remedy, it is important to know their origins, characteristics, and compositions. In this study, optical and scanning electron microscopes (SEMs) equipped with an energy-dispersive spectrometer (EDS) and X-ray diffractometer were employed to determine the white and black deposits. It was found that both deposits consisted mainly of calcium carbonate (calcite) and calcium sulfate (gypsum). The calcite and gypsum were characterized by bladed, rhombohedral, tabular, and amorphous morphologies under a SEM. The black deposit was not only composed of calcite or gypsum, but also accompanied amorphous and irregular matrix. SEM-EDS analysis revealed an abundance of silicon, aluminum, iron, phosphorus, and carbon on the matrix, which were major elements of soil, atmospheric deposits, and organisms. The white deposit, on the other hand, barely contained those coloring substances. These salts and deposited substances were caused by chemical reaction and physical adhesion between rock-forming minerals, lime mortar, sulfur in polluted air environment, soil dust, and microorganisms.

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