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.

Correlative fractography is a new expression proposed here to describe a new method for the association between scanning electron microscopy (SEM) and light microscopy (LM) for the qualitative and quantitative analysis of fracture surfaces. This article presents a new method involving the fusion of one elevation map obtained by extended depth from focus reconstruction from LM with exactly the same area by SEM and associated techniques, as X-ray mapping. The true topographic information is perfectly associated to local fracture mechanisms with this new technique, presented here as an alternative to stereo-pair reconstruction for the investigation of fractured components. The great advantage of this technique resides in the possibility of combining any imaging methods associated with LM and SEM for the same observed field from fracture surface.

In a previous article, we studied the influence of spectral noise on a new method for three-dimensional X-ray photoelectron spectroscopy (3D XPS) imaging, which is based on analysis of the XPS peak shape [Hajati, S., Tougaard, S., Walton, J. & Fairley, N. (2008). Surf Sci602, 3064–3070]. Here, we study in more detail the influence of noise reduction by principal component analysis (PCA) on 3D XPS images of carbon contamination of a patterned oxidized silicon sample and on 3D XPS images of Ag covered by a nanoscale patterned octadiene layer. PCA is very efficient for noise reduction, and using only the three most significant PCA factors to reconstruct the spectra restores essentially all physical information in both the intensity and shape of the XPS spectra. The corresponding signal-to-noise improvement was estimated to be equivalent to a reduction by a factor of 200 in the required data acquisition time. A small additional amount of information is obtained by using up to five PCA factors, but due to the increased noise level, this information can only be extracted if the intensity of the start and end points for each spectrum are obtained as averages over several energy points.

Vickers microindentation and Berkovich nanoindentation tests were carried out on a polycrystalline nickel (Ni) bulk specimen. Electron channeling contrast imaging (ECCI) in conjunction with electron backscattered diffraction was used to image and characterize plastic deformation inside and around the indents using a field emission scanning electron microscope. The ECCI was performed with a 5 keV beam energy and 0° tilt specimen position. The strain field distribution, slip lines, and Taylor lattices were imaged on an indented surface. Orientation mapping was used to investigate the local crystallographic misorientation and identify specific ⟨110⟩ slip systems. An ion milling surface preparation technique was used to remove materials from the surface which permitted the study of deformed microstructure below the indent. A dislocation density of 1011 cm−2 was calculated based on the curvature of bend contours observed in the ECCI micrographs obtained from the Vickers indents. A yield strength of 500 MPa was calculated based on the size of the strain field measured from the ECCI micrographs of the nanoindents. The combination of ion milling, ECCI, and electron backscattered diffraction was shown to be beneficial to investigate the indentation-induced plastic deformation in a polycrystalline Ni bulk specimen.

Preparation of high-quality polished sample surfaces is an essential step in the collection of microanalytical data on the microstructures of minerals and alloys. Poorly prepared samples can yield insufficient or inconsistent results and, in the case of gold, potentially no data due to the “beilby” layer. Currently, preparation of ore samples is difficult as they commonly contain both hard and soft mineral phases. The aim of our research is to produce suitably polished sample surfaces, on all phases, for electron backscatter diffraction analysis. A combination of chemical–mechanical polishing (CMP) and broad ion-beam polishing (BIBP) was used to tackle the problem. Our results show that it is critical to perform CMP first, as it produces a suitable polish on the hard mineral phases but tends to introduce more damage to the soft mineral surfaces. BIBP is essential to produce a high-quality polish to the soft phases (gold). This is a highly efficient method of sample preparation and is important as it allows the complete quantification of ore textures and all constituent mineral phases, including soft alloys.

The electron energy loss spectroscopy (EELS) technique was applied to investigate the local variation in the phase of barium titanate (BaTiO3) ceramics. It was found that the fine structure of the titanium L2,3 edge and their satellite peaks were sensitively varied with the tetragonal–cubic phase transition. The peak splitting of Ti-L3 edge of tetragonal-phased BaTiO3 ceramics was widened because of the increased crystal field effect compared with that of cubic-phased BaTiO3. In case of nanoscale BaTiO3 powders, the L3 edge splitting of the core region was found to be smaller than that of the shell region. The energy gap between peaks t2g and eg varied from 2.36 to 1.94 eV with changing the probe position from 1 to 20 nm from the surface. These results suggest that the EELS technique can be used to identify the local phase of sintered BaTiO3 ceramics.

Sea anemones species identification of can be difficult, especially in the field. The taxonomic keys currently utilised are based mainly on histological differences and therefore require collection of whole animals. Furthermore, histological analysis of sea anemones requires considerable expertise and worldwide sea anemones histological descriptions are still limited. Here it is reported a detailed histological description of the internal structure of the two most conspicuous species in tidal pool communities in European shores, Actinia equina and Anemonia sulcata. This species description makes them excellent models for studying reproductive strategies that are instrumental in establishing and maintaining new populations. Therefore they are potential indicator species important both for micro and macro scale monitoring programs. The anatomy and histology of main organs of the studied sea anemones were described. The different colour morphs of the species were studied. Fine histological details of their muscular and digestive systems and appendages, down to the cellular level, were studied. They are two-germinal layer animals and possess radial symmetry in body form. Samples of the main organs of sea anemones including epidermis, tentacles, mesentery, gonads and digestive tract (mouth, stomodaeum and gastrovascular cavity) were processed for histological examination according to procedures described elsewhere [1,2]. The epithelia were composed of epithelio-muscular cells, in which many gland cells were distributed. Three types of cnidoblasts (nematooysts) were observed in the epithelioma of the tentacles, mesentery filament and stomodaeum. Cnidoblasts in different places have different functions. The gonads were of the follicular type and were composed of germ cells and nutritional cells. The investigation of the internal organization of invertebrate communities provides important information on the organism’s biology with special interest for the study of ecological processes to evaluate their reproductive modes in order to understand how they may reflect their dispersal strategies [1, 2]. Traits that influence reproduction in animal and plant species have been a central focus of evolutionary biology. Therefore, considering their basal position in evolution, the Anthozoa (Cnidaria) provide important models for further understanding processes affecting reproductive strategies in the eumetazoan (i.e., cnidarian-bilaterian) ancestor and in modern vertebrates.

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.

This study quantitatively investigated the immediate effects of a photooxidative collagen cross-linking treatment with photosensitizer riboflavin (RF) and 370 nm UVA light in in vitro human corneoscleral collagen fibrils using histology, thickness, scanning electron microscopy, and atomic force microscopy analyses. Twenty 8 × 2 mm corneoscleral strips were dissected sagittally from donor tissue using a scalpel. Four parameters were investigated, including the density, thickness, adhesion force, and stiffness of corneoscleral tissues before and after the collagen cross-linking treatment. The RFUVA-catalyzed collagen cross-linking treatment led to an increase in the density of both corneal (8%) and scleral (23%) stromal collagens. However, there was no difference in corneoscleral thickness. Furthermore, RFUVA-catalyzed collagen cross-linking treatment led to an increased biomechanical response of corneosclera: 25 and 8% increases in corneoscleral stiffness, and 24 and 22% increases in corneoscleral adhesion force. The collagen cross-linking treatment through RF-sensitized photoreaction may cause structural and biomechanical changes in the collagen fibril network of the cornea and the sclera. This is due to narrowing of the interfibrillar spacing and the stromal edema.

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

An optimum method is proposed to prepare thin foil transmission electron microscopy (TEM) lamellae of multiphase porous functional ceramics: prefilling the pore space of these materials with an epoxy resin prior to focused ion beam milling. Several advantages of epoxy impregnation are demonstrated by successful preparation of TEM specimens that maintain the structural integrity of the entire lamella. Feasibility of the TEM alignment procedure is demonstrated, and ideal TEM analyses are illustrated on solid oxide fuel cell and solid oxide electrolysis cell materials. Some potential drawbacks of the TEM specimen preparation method are listed for other samples.

On the basis of the suggestion that bone nanostructure bears “tissue age” information and may reflect surface deposition/modification processes, we performed nanoscale characterization of the external cortical bone surface at the femoral neck in women using atomic force microscopy (AFM). The specific aims were to assess age-related differences in bone nanostructure and explore the existence of nanostructural traces of potential bone apposition at this surface. Our findings revealed that the external cortical surface represents a continuous phase composed of densely packed mineral grains. Although the grains varied in size and shape, there was a domination of small grains indicative of freshly deposited bone (mean grain size: young, 35 nm; old, 37 nm; p > 0.05). Advanced quantitative analysis of surface morphological patterns revealed comparable roughness and complexity of the surface, suggesting a similar rate of mineral particle deposition at the surface in both groups. Calcium/phosphorus ratio, a measure of bone tissue age, was within the same range in both groups. In summary, our AFM analyses showed consistent nanostructural and compositional bone features, suggesting existence of new bone at the periosteal bone surface in both young and elderly women. Considering observed age-related increase in the neck diameter, AFM findings may support the theory of continuous bone apposition at the periosteal surface.

Carbon nanotubes (CNT) are proper tips for atomic force microscopes (AFMs) as a result of their small tip diameter, high aspect ratio, and high flexibility. For nanoscale imaging of soft biological specimens, a CNT tipped AFM is an ideal tool. In this article we review the application of CNTs as AFM tips and present related research about the forces applied from liquids on nanotubes. Then a dynamic mode CNT tipped AFM in liquid is modeled and simulated. The simulation results are compared with experimental results. For modeling and simulation, a continuous beam model and a forward-time simulation method are used. The simulation results show that when a CNT tip vibrates in liquid, the oscillation amplitude and resonance frequency are changed compared to the state of oscillation in air. The small structure of CNTs reduces the hydrodynamic forces, and the liquid environment reduces the adhesive forces between the CNT tip and the sample. These two factors make CNTs a good choice as an AFM tip.

The dislocation distribution of high-quality single-crystal gallium nitride (GaN) films grown by the hybrid vapor phase epitaxy was analyzed. This study examined the domain structure of GaN from the dislocation distribution on the macroscale by optical microscopy. The surface structure of GaN consisted of domains with microcolumns as the substructure. The inner domains contained a lower density of dislocations but a large number of these dislocations were observed along the domain boundaries. The existence of a domain boundary structure doubly increased the total dislocation density.

Microorganisms, that evolve to acquire resistance to environmental amoeba, are likely to become human pathogens due to the physiological similarities of free-living amoebae to human macrophages. In this sense free-living amoebae can be regarded as nurseries of pathogenic microorganisms. Due to the widespread distribution of amoebae in the environment and their resistance to current disinfection procedures, they may constitute important reservoirs of pathogenic microorganisms. Striking examples are Legionella pneumophila and several mycobacteria including Mycobacterium tuberculosis. This work represents the first attempt to detect and characterize amoebae and their associated microorganisms in Portugal.

Detection, isolation and in vitro culture of amoebae from several environmental sources, including lagoon and estuarine water and sediments, were performed according to previously developed methods. Identification of the isolated amoebae was done by optical microscopy based on morphological criteria and by DNA sequence analysis of PCR products amplified with one of the following two sets of primers: EUK and ITS. Transmission electron microscopy (TEM) studies and PCR/sequencing approaches were used to detect and identify amoeba-associated microorganisms (AAMs). Bacterial 16S region was amplified with the primers 616V/630R. TEM studies were performed according to standard procedures. In short, samples were fixed sequentially in glutaraldehyde, osmium tetroxide and uranyl acetate, dehydrated in ethanol and embedded in Epon-Araldite. Thin sections contrasted with uranyl acetate and lead citrate were observed with a JEOL 100-SX electron microscope.

Several species of amoeba (Acanthamoeba lenticulata, A. polyphaga, A. rhyzodes, Platyamoeba oblongata, Saccamoeba limax, Vannella simplex, Vannella sp. and other unidentified amoebae) were found and AAMs were detected both by TEM and PCR/sequencing. The bacteria species Variovorax paradoxus, previously found in association with Saccamoeba and Arcella, were isolated and identified by the PCR/sequencing approach, which also allowed the detection of an unidentified species with about 85% identity with Marivirga tractuosa, a species not yet associated with amoebae. These results point to the existence of AAMs in the environments subjected to this study and the need to evaluate their pathogenic potential. This is an important issue particularly in environments related to human recreational, nutritional and public health activities.

The influence on alloy 413.0 of the refinement and modification of its microstructure was analyzed by means of several microscopy techniques, as well as the effect of the application of high pressure during solidification. For each treatment and solidification pressure condition employed, the most suitable microscopy techniques for identifying and characterizing the phases present were investigated. Color metallography and electron microscopy techniques were applied to the qualitative microstructural analysis. Volume fraction and grain size of the primary α-Al were characterized by quantitative metallographic techniques. The results show that the effect caused by applying high pressure during solidification of the alloy is more pronounced than that caused by modification and refinement of the microstructure when it solidifies at atmospheric pressure. Furthermore, it has been shown that, for Al–Si alloy characterization, when aiming to characterize the primary α-Al phase, optical color metallography observed under crossed polarized light plus a sensitive tint filter is the most suitable technique. When the goal is to characterize the eutectic Si, the use of optical color metallography or electron microscopy is equally valid. The characterization of iron-rich intermetallic compounds should preferably be performed by means of backscattered electron imaging.

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

This work establishes a multiscale and multitechnique nondestructive approach as valid methodology for monitoring surface properties and evaluating the effectiveness of enzymatic removal of varnishes from paintings/polychrome artefacts.

Mock-up samples (documented reconstructions of oil, tempera, and gilded layers on canvas and wooden supports) were covered with different proteinaceous varnishes (egg white, animal and fish glue, casein) and then characterized before and after the removal of these coatings with enzyme-based solutions. The varnish was cleaned in several steps (two dry swabs and two wet swabs) with a clearance step for removing the residues from proteinaceous varnish or from enzyme solution.

Microscopy [stereomicroscopy (SM), optical microscopy (OM), atomic force microscopy (AFM), and scanning electron microscopy (SEM)] and colorimetric (CIE L*a*b* system) techniques were used for characterization of the reconstruction surfaces at different scales (macro-scale by SM and OM; micro-scale by SEM and nano-scale by AFM). These techniques were also used to monitor the cleaning treatment.

Although results presented in this work were obtained for the specific treatment of enzyme removal, the methodology could be extended to other types of materials and cleaning. Further experiments on real works of art are needed for a complete validation of the methodology.