Transmission electron microscopy (TEM), fluorescence microscopy, and confocal Raman microscopy can be used to characterize ultrastructural and compositional heterogeneity of plant cell walls. In this study, TEM observations revealed the ultrastructural characterization of Cornus alba L. fiber, vessel, axial parenchyma, ray parenchyma, and pit membrane between cells, notably with the ray parenchyma consisting of two well-defined layers. Fluorescence microscopy evidenced that cell corner middle lamella was more lignified than adjacent compound middle lamella and secondary wall with variation in lignification level from cell to cell. In situ Raman images showed that the inhomogeneity in cell wall components (cellulose and lignin) among different cells and within morphologically distinct cell wall layers. As the significant precursors of lignin biosynthesis, the pattern of coniferyl alcohol and aldehyde (joint abbreviation Lignin-CAA for both structures) distribution in fiber cell wall was also identified by Raman images, with higher concentration occurring in the fiber secondary wall where there was the highest cellulose concentration. Moreover, noteworthy was the observation that higher concentration of lignin and very minor amounts of cellulose were visualized in the pit membrane areas. These complementary microanalytical methods provide more accurate and complete information with regard to ultrastructural and compositional characterization of plant cell walls.

The interfacial layer between the Al2O3 layer and the Si3N4 layer formed after postdeposition annealing (PDA) of TaN/Al2O3/Si3N4/SiO2/Si (TANOS) stacks was investigated using transmission electron microscopy (TEM), scanning transmission electron microscopy, and electron energy loss spectroscopy (EELS). From the result of the TEM analysis, it was found that the 2-nm-thick interface layer between Al2O3 and Si3N4 layers was amorphous. The high-loss EELS analysis showed that the phases of the interfacial layer weakly bound together instead of the substoichiometric silicon oxide and amorphous Al2O3 near the bottom interface of the crystalline Al2O3. The low-loss EELS analysis showed that aluminum existed in metallic state at the interface. Therefore, we speculated that SiOxNy could be formed by oxidation of Si3N4 during PDA and that metallic aluminum could be formed by the decomposition of weakly bound amorphous Al2O3 during electron irradiation. These complicated reactions near the interface could induce oxygen deficiency in the Al2O3 layer and finally degrade the retention properties of TANOS stacks.

Electron energy-loss spectroscopy (EELS) attached to current transmission electron microscopes can probe not only element-selective chemical information, but also site-selective information that depends on the position that a specific element occupies in a crystal lattice. The latter information is exploited by utilizing the Bloch waves symmetry in the crystal, which changes with its orientation with respect to the incident electron wave (electron channeling). We demonstrate the orientation dependence of the cross-section of the electron energy-loss near-edge structure for particular crystalline sites of spinel ferrites, by quantitatively taking into account the dynamical diffraction effects with a large number of the diffracted beams. The theoretical results are consistent with a set of experiments in which the transition metal sites in spinel crystal structures are selectively excited. A new measurement scheme for site-selective EELS using a two-dimensional position-sensitive detector is proposed and validated by theoretical predictions and trial experiments.

Cr(III)-tris(picolinate), [Cr(pic)3], is a very common dietary supplement, recommended for humans, cattle and swine. Chromium is considered an essential trace element, when in oxidation state +3, with some of its compounds seeming to have a beneficial effect on blood sugar regulation mechanisms. However, the safety of the use of a particularly popular Cr(III) compound, ie [Cr(pic)3], remains debatable. Clastogenic, and mutagenic features have been reported by Stearns and co-workers, although surrounded by a controversial and contradictory multitude of publications on this subject. The present work aims to study the effects of [Cr(pic)3] on mice spermatogenesis.

Cr(III)-tris(picolinate) was synthesized and characterized according to the literature. Its composition as a mononuclear complex was tested by ESI-MS and by X-ray powder diffraction followed by single-crystal simulation calculations.

Male adult CDI mice from Harlan (Spain) were divided in groups and orally given 25 mg/kg and 50 mg/kg/body weigh/daily of [Cr(pic)3] for two weeks. Controls were also done. Behaviour and body weight were monitored throughout the experiments. After sacrifice, testis were collected, weighed, and fixed in Bouin´s solution. Organs were then prepared for histology using routine techniques. Animal experiments were conducted according to ethics procedures. Histological sections of control group evidenced normal regular features (Fig. 1a). However considerable damage was observed in both experimental groups in a dose dependent manner. In fact, seminiferous tubules showed degenerative changes within epithelium, namely vacuolation and sloughing of immature germ cells into the lumen in the group given the lowest dose (Fig.1 b,c). The high dosed group displayed more conspicuous injury within testis, namely strongly atrophic seminiferous tubules devoid of germs cells and strong vacuolation (Figs.1d-f).

The results of this study have shown an increased risk of adverse events in mice receiving 50 mg/kg/body weight of [Cr(pic)3]. However, little potential for adverse reproductive and developmental effects namely on progeny was recently described for male mice fed a diet containing 200 mg/kg/day [Cr(pic)3]. In conclusion, concerns about using dietary supplements based on [Cr(pic)3] remain to be elucidated in future work.

This work was financed by CICECO, Aveiro University, Portugal.

Mesua ferrea is traditionally used for treating bleeding piles, fever, and renal diseases. It has been reported to have antimircobial activity. In the present study, antibacterial efficacy of leaf and fruit extracts on the growth and morphology of Staphylococcus aureus is evaluated. Both extracts display good antibacterial activity against S. aureus with a minimum inhibition concentration of 0.048 mg/mL. Both extracts are bacteriostatic at a minimum bacteriostatic concentration of 0.39 mg/mL. The bacteriostatic activity lasts for 24 h, and then cells start to grow as normal as shown in time-kill analysis. Scanning electron microscopy study indicated potential detrimental effect of the extracts of leaf and fruits of M. ferrea on the morphology of S. aureus. The treatment with the extracts caused extensive lysis of the cells, leakage of intracellular constituents, and aggregation of cytoplasmic contents forming an open meshwork of the matrix.

The formation and morphological evolution of germanides formed in a ternary Ni/Ta-interlayer/Ge system were examined by ex situ and in situ annealing experiments. The Ni germanide film formed in the Ni/Ta-interlayer/Ge system maintained continuity up to 550°C, whereas agglomeration of the Ni germanide occurred in the Ni/Ge system without Ta-interlayer. Through microstructural and chemical analysis of the Ni/Ta-interlayer/Ge system during and after in situ annealing in a transmission electron microscope, it was confirmed that the Ta atoms remained uniformly on the top of the newly formed Ni germanide layer during the diffusion reaction. Consequently, the agglomeration of the Ni germanide film was retarded and the thermal stability was improved by the Ta incorporation.

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

We report the installation and performance evaluation of a probe aberration-corrected high-resolution JEOL JEM-ARM200F transmission electron microscope (TEM). We provide details on construction of the room that enables us to obtain scanning transmission electron microscope (STEM) data without any evident distortions/noise from the external environment. The microscope routinely delivers expected performance. We show that the highest STEM spatial resolution and energy resolution achieved with this microscope are 0.078 nm and 0.34 eV, respectively. We report a direct comparative evaluation of the performance of this microscope with a Schottky thermal field-emission gun versus a cold field-emission gun. Cold field-emission illumination improves spatial resolution of the high current probe for analytical spectroscopy, the TEM information limit, and the electron energy resolution compared to the Schottky thermal field-emission source.

Depth scan confocal Raman microscopy was employed to map water and air spatial distributions in immersed superhydrophobic films. Due to the lack of visible nanobubbles on flat surfaces, we have probed heterogeneous surfaces where solid–liquid, liquid–vapor, and vapor–solid coexist. Depth scan profiles show liquid exclusion (vapor) layers inside the fiber arrangement and water in contact only at the fiber apex.

We describe a novel method for in situ measurement of the local thickness of a freely suspended solid-state membrane after thinning with a focused helium ion beam. The technique utilizes a custom stage for the helium ion microscope that allows the secondary electron detector used for normal imaging to collect information from ions transmitted through the sample. We find that relative brightness in the transmission image scales directly with the membrane thickness as determined by atomic force microscopy measurements.

Duplex stainless steels (DSS) may be defined as a category of steels with a two-phase ferritic–austenitic microstructure, which combines good mechanical and corrosion properties. However, these steels can undergo significant microstructural modification as a consequence of either thermo-mechanical treatments (ferrite decomposition, which causes σ- and χ-phase formation and nitride precipitation) or plastic deformation at room temperature [austenite transformation into strain-induced martensite (SIM)]. These secondary phases noticeably affect the properties of DSS, and therefore are of huge industrial interest. In the present work, SIM formation was investigated in a 2101 lean DSS. The material was subjected to cold rolling at various degrees of deformation (from 10 to 80% thickness reduction) and the microstructure developed after plastic deformation was investigated by electron backscattered diffraction, X-ray diffraction measurements, and hardness and magnetic tests. It was observed that SIM formed as a consequence of deformations higher than ~20% and residual austenite was still observed at 80% of thickness reduction. Furthermore, a direct relationship was found between microstructure and magnetic properties.

Plasmodium falciparum causes the most severe/lethal form of malaria, a parasitic infection that affects 500 million people worldwide and leads to the death of nearly one million/year, about 91% being due to P. falciparum. The malaria pigment or hemozoin crystals (HZ) are formed in an enzyme-independent polymerization of heme released during haemoglobin digestion by the parasite during the intra-erythrocytic life cycle. The quinoline and artemisinin based antimalarials (the only available treatment options) appear to act by disrupting the formation of HZ. Therefore, the development of antimalarial agents based on the physicochemical process of heme crystallization appears a good option to drug design.

We have produced β-haematin following an assay adapted from the method described by Basilico et al, with modifications. Briefly, hemin (ferriprotoporphyrin IX chloride; Sigma-Aldrich) was dissolved NaOH and polymerized by the addition of acetic acid and incubation at 37°C. Resulting pellet washed with dimethyl sulfoxide (DMSO) to remove unpolymerized hemin and resuspended in H2O. To extract native HZ (nHZ) from P. falciparum (clone Dd2)-infected erythrocytes, when parasitemia reached >10%, parasites were harvested by saponin lysis, washed with phosphate-buffered saline (PBS) and sonicated in 2% sodium dodecyl sulfate (SDS), further washed in 2% SDS and pellet was resuspended (10 mM Tris-HCl (pH 8.0); 0.5% SDS; 2mg/ml of proteinase K) and incubated at 37°C overnight. The nHZ pellet was washed in 2% SDS and in distilled H2O, then resuspended in distilled H2O water and sonicated again prior to use to minimize aggregation and maintain the nHZ in suspension. β-haematin and native nHZ crystals were analysed by Scanning Electron Microscopy (JEOL 7001FSEM) (Fig.1; A, B.1 and C.1) and Transmission Electron Microscopy (Hitachi H8100) (Fig.1; B.2 and C.2).

Here we report the crystal morphology of nHZ produced by the Plasmodium falciparum clone Dd2 and of β-haematin produced with the above protocol. Chemically, HZ is a stable dimer of iron(III)(protoporphyrin[PP]-IX), also known as β-hematin and consists of heme units dimerized through reciprocal iron-carboxylate bonds. Even though the parasite nHZ and synthetic β-hematin are isostructural, there are large differences in the crystal morphologies of nHZ (Dd2) and β-hematin as evidenced by SEM and TEM images (Fig.1). nHZ crystals are remarkably uniform in size and shape, adopting a parallelipede morphology with high aspect ratio of about 800x200x200 nm (Fig. 1 C1 and C2). Different protocols to prepare β-haematin yield diverse material, which can be poorly crystalline and heterogeneous within the same sample. The present results show that β-haematin is rather homogeneous and constituted by needle-like particles of sizes similar to those presented by nHZ ones (compare B1 with C1). Higher resolution observations evidence differences between the β-haematin and nHZ materials, since the latter seems constituted by uniform flat layers and the former shows flaked ones (Fig. B2).

Morphology seems to be of biological relevance, since structure-function studies revealed that the size and shape of the synthetic crystals influence their ability to activate, for instance, the inflammatory responses in vitro and in vivo. Several protocols have been developed to measure drug inhibition of heme crystallization in vitro although it has been difficult to standardize these assays. In the present study, we report a new method for β-haematin preparation that renders homogenous crystals, both in shape and size, favourable for reproducible drug inhibition of heme crystallization assays.

This work was supported by CMDT through PEst-OE/SAU/LA0018/2011 grant and FCT through PTDC/SAU-FAR/114864/2009 and PEst-OE/CTM-UI0084/2011 grants.

A fast method for determination of the Co-valence state by electron energy loss spectroscopy in a transmission electron microscope is presented. We suggest the distance between the Co-L3 and Co-L2 white-lines as a reliable property for the determination of Co-valence states between 2+ and 3+. The determination of the Co-L2,3 white-line distance can be automated and is therefore well suited for the evaluation of large data sets that are collected for line scans and mappings. Data with a low signal-to-noise due to short acquisition times can be processed by applying principal component analysis. The new technique was applied to study the Co-valence state of Ba0.5Sr0.5Co0.8Fe0.2O3-d (BSCF), which is hampered by the superposition of the Ba-M4,5 white-lines on the Co-L2,3 white-lines. The Co-valence state of the cubic BSCF phase was determined to be 2.2+ (±0.2) after annealing for 100 h at 650°C, compared to an increased valence state of 2.8+ (±0.2) for the hexagonal phase. These results support models that correlate the instability of the cubic BSCF phase with an increased Co-valence state at temperatures below 840°C.

Solid particles in snow deposits, sampled in mining and Pb-processing area of Žerjav, Slovenia, have been investigated using scanning electron microscopy/energy-dispersive X-ray spectroscopy (SEM/EDS). Identified particles were classified as geogenic–anthropogenic, anthropogenic, and secondary weathering products. Geogenic–anthropogenic particles were represented by scarce Zn- and Pb-bearing ore minerals, originating from mine waste deposit. The most important anthropogenic metal-bearing particles in snow were Pb-, Sb- and Sn-bearing oxides and sulphides. The morphology of these particles showed that they formed at temperatures above their melting points. They were most abundant in snow sampled closest to the Pb-processing plant and least abundant in snow taken farthest from the plant, thus indicating that Pb processing was their predominant source between the last snowfall and the time of sampling. SEM/EDS analysis showed that Sb and Sn contents in these anthropogenic phases were higher and more variable than in natural Pb-bearing ore minerals. The most important secondary weathering products were Pb- and Zn-containing Fe-oxy-hydroxides whose elemental composition and morphology indicated that they mostly resulted from oxidation of metal-bearing sulphides emitted from the Pb-processing plant. This study demonstrated the importance of single particle analysis using SEM/EDS for differentiation between various sources of metals in the environment.

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

A new technique that allows direct three-dimensional (3D) investigations of mesopores in carbon materials and quantitative characterization of their physical properties is reported. Focused ion beam nanotomography (FIB-nt) is performed by a serial sectioning procedure with a dual beam FIB-scanning electron microscopy instrument. Mesoporous carbons (MPCs) with tailored mesopore size are produced by carbonization of resorcinol-formaldehyde gels in the presence of a cationic surfactant as a pore stabilizer. A visual 3D morphology representation of disordered porous carbon is shown. Pore size distribution of MPCs is determined by the FIB-nt technique and nitrogen sorption isotherm methods to compare both results. The obtained MPCs exhibit pore sizes of 4.7, 7.2, and 18.3 nm, and a specific surface area of ca. 560 m2/g.

Mass transport within collagen-based matrices is critical to tissue development, repair, and pathogenesis, as well as the design of next-generation tissue engineering strategies. This work shows how collagen precursors, specified by intermolecular cross-link composition, provide independent control of collagen matrix mechanical and transport properties. Collagen matrices were prepared from tissue-extracted monomers or oligomers. Viscoelastic behavior was measured in oscillatory shear and unconfined compression. Matrix permeability and diffusivity were measured using gravity-driven permeametry and integrated optical imaging, respectively. Both collagen types showed an increase in stiffness and permeability hindrance with increasing collagen concentration (fibril density); however, different physical property–concentration relationships were noted. Diffusivity was not affected by concentration for either collagen type over the range tested. In general, oligomer matrices exhibited a substantial increase in stiffness and only a modest decrease in transport properties when compared with monomer matrices prepared at the same concentration. The observed differences in viscoelastic and transport properties were largely attributed to increased levels of interfibril branching within oligomer matrices. The ability to relate physical properties to relevant microstructure parameters, including fibril density and interfibril branching, is expected to advance the understanding of cell–matrix signaling, as well as facilitate model-based prediction and design of matrix-based therapeutic strategies.

Electron backscatter diffraction (EBSD) and electron probe microanalysis (EPMA) measurements are combined to characterize an industrial produced dual-phase steel containing some bainite fraction. High-resolution carbon mappings acquired on a field emission electron microprobe are utilized to validate and improve the identification of the constituents (ferrite, martensite, and bainite) performed by EBSD using the image quality and kernel average misorientation. The combination eliminates the ambiguity between the identification of bainite and transformation-induced dislocation zones, encountered if only the kernel average misorientation is considered. The detection of carbon in high misorientation regions confirms the presence of bainite. These results are corroborated by secondary electron images after nital etching. Limitations of this combined method due to differences between the spatial resolution of EBSD and EPMA are assessed. Moreover, a quantification procedure adapted to carbon analysis is presented and used to measure the carbon concentration in martensite and bainite on a submicrometer scale. From measurements on reference materials, this method gives an accuracy of 0.02 wt% C and a precision better than 0.05 wt% C despite unavoidable effects of hydrocarbon contamination.

Nanoparticles (NPs), particles with at least one dimension less than 100 nm, are used in many industrial applications and to produce new types of materials with unique physicochemical properties. The aquatic environment is commonly the ultimate recipient for NPs and there is uncertainty of exposure as understanding and data regarding the potential detrimental effects of NPs on aquatic biota are missing. In this study, titanium dioxide (TiO2) was chosen for its potential use in technology and diverse industrial applications. The objective of this work is to evaluate the toxicity of TiO2 NPs on total liver glutathione-S-transferase (GST), lipid peroxidation and tissue structure of the livers of two freshwater fish species (Carassius auratus and Danio rerio).

Stock suspensions of TiO2 NPs, with an average size of 21 nm, were prepared using distilled 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. Immediately after sampling the fish were processed for enzymatic determination and histopathology. 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 ultra-structural observations were carried out using a Leica microscope (Leica-ATC 2000) and a JEOL 100-SX electron microscope respectively.

The results showed increased activities of the GST in livers with increasing TiO2 NP concentrations after 7 days of exposure, however after 14 days a trend to decrease was observed for both species. The GST results suggest that the increase of activity of these detoxification enzymes can be a response to oxidative stress caused by the generation of reactive oxygen species by the NP. On the other hand, size, chemical composition, surface area, shape, solubility and aggregation may also contribute for NPs toxicity. The results from lipid peroxidation showed an increase according to tested concentrations suggesting that TiO2 NPs is able to cause cell damage and is in agreement with biochemical and histological findings. After 14 days of depuration, GST and lipid peroxidation levels were not significant different from controls suggesting that cells are able to recover in a certain degree. The results from LM (Fig. 1) showed that exposure to TiO2 NPs affected liver structure, with more pronounced changes detected in livers from fish exposed to higher concentrations. Observed changes include tissue degeneration, inflammation and pyknosis among others. The TEM analysis revealed also severe changes in liver cells compatible with oxidative stress. Hepatocytes of treated fish showed glycogen depletion, swollen mitochondria and increased lysosomes, compared to controls. After depuration, some cells recovered nearly normal morphology, but retained the lysosomes, while others underwent necrotic changes (Fig. 2). Differences among the two species studied were of a quantitative nature, and more pronounced in Danio rerio.

The results suggest that potential risk to fish health exist related to the TiO2 NPs release to the aquatic environment and may cause deleterious effects in aquatic organisms. It is evident that the effects of TiO2 NPs on environment is a matter of great concern and the precise mechanisms of toxicity of this and other types of NPs must be clarified.

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.