Paget Disease of Bone is a disorder characterized by abnormal bone remodeling, associated with morphological and functional abnormalities of osteoclasts although its etiology remains unproven. Ion beam analysis (IBA) techniques may be useful to characterize the abnormal distribution and concentration of the major and trace elements that can be associated topagetic bone condition. In order to access the viability of this approach, healthy femoral head of cat has been investigated by Particle Induced X-Ray Emission (PIXE),Rutherford Backscattering Spectrometry (RBS) and Scanning Electron Microscopy (SEM).

The bone section was irradiated, under vacuum conditions, with a 2.0 MeV alpha particle beam produced by the 2.5 MV Van de Graaff Accelerator of CTN/IST (Lisbon, Portugal). An Oxford Microbeams-type nuclear microprobe was used (OM150 triplet system), which allowed the proton beam to be focused on the bone section with a spatial resolution of 3x4 µm2 and a mean beam current of ~90pA. The X-rays generated by the bone were recorded using an 80 mm2 Link X-ray detector (with a 145 eV energy resolution), that is positioned at 135º to the beam direction. The backscattered particles were collected by a 200 mm2 silicon surface barrier radiation detector with a 15 keV energy resolution that is positioned at 40º to the beam direction in a cornell geometry. SEM observations have been carried out with backscattered electrons (BSE) using a JEOL JSM 7001F microscope equipped with an INCA Oxford Instruments EDS spectrometer for point analyses and X-ray mapping. Standard petrographic procedures have been employed to prepare the samples for SEM observation: the specimens were partly encapsulated in epoxy resin, ground with 800 and 1000-grit SiC paper, polished with 6 μm and 1 μm diamond suspensions and coated with carbon.

A selected area of the sample has been scanned with the proton beam, allowing to generateelemental distribution maps for Ca and P (Figure 1). From X-line scans it was possible to extractline profiles of these elements. RBS and PIXE were used to determine the matrix and trace element composition in order to normalize the elemental concentrations. The results are shown in Table 1.Structural information on the Haversian Systems, includingbone lacunaeandosteocytes localization have been obtained by SEM (Figure 2).

The combination of these techniques proved to be a viable approach for the Pagetic bone characterization and will be used to analyze the structure of trabecular and cortical bones as well as the distribution and morphology of the pagetic bone cells, which can contribute to clarify the differences between pagetic and healthy bone.

V. Corregidor acknowledges the support from FCT-Ciência program. The work was also supported by FCT PEST-OE/CTM-UI0084/2011 grant.

Laser scanning fluorescence confocal microscope (LSFCM) imaging is an extensively used modality in biological research. However, these images present low signal to noise ratio and a time intensity decay effect due to the so called photoblinking/photobleaching (PBPB) phenomenon that corresponds to an intensity fading of a fluorescent probe along the time, as shown in Figure 1. This effect is caused by quantum phenomena associated with the electronic excitation and photochemical reactions among the fluorescent and the surrounding molecules induced by the incident radiation that temporarily or irreversibly destroy their ability to fluoresce. Since illumination is needed to excite and observe the tagging fluorescent proteins in the specimen and all the fluorophores will eventually photobleach upon extended excitation, the acquisition of this type of images becomes a hard task for long exposures.

There are in the literature several proposals to model this fading effects and among them the single and double exponential are the most used. However, simple and tractable theoretical models based on the physics of the observation process to support these empirical laws are not available. In this work, that theoretical model, supported on the underlying physics of the process, is derived to describe the PBPB effect (see Figure 2).

From a fluorescence point of view, tagging molecules can be in three main states (see Figure 3), i) ON-state, where they are able to fluoresce and be observed, ii) OFF-state, where they are temporarily not able to fluoresce and therefore are not visible and finally at the iii) BLEACHED-state where they become permanently OFF. Here, a continuous time differential equation dynamic model is proposed to describe the number of molecules at the ON-state, nON, along the time. The model is based on the underlying quantum mechanic physics theory of the observation process associated with this type of images and the common empirical weighted sum of two decaying exponentials (DExp), usually used in the literature, is derived from the model. The parameters βON and βOff are the transitions rate from and to the ON-state respectively and ξis the decay rate associated with the transitions for the permanent BLEACHED state.

Experiments with synthetic and real data are presented to validate the PBPB model and estimate the physical variables associated with the process. Intensity decay from real data and the corresponding theoretical curve are compared and displayed in Figure 4.

This work was supported by the FCT project [PEst-OE/EEI/LA0009/2011]. The authors thank Dr. José Rino and Profa Carmo Fonseca, from the Molecular Medicine Institute (IMM) of Lisbon, for providing biological technical support and the real data used in this paper.

The increase in CO2 emissions from anthropogenic sources may not only result in temperature increase on a global scale but also in increased ocean acidification (OA), by lowering the ocean's capacity to absorb additional atmospheric CO2. As a consequence, changes in ocean chemistry are prone to occur through the imbalancing of sea-atmosphere gas exchange, thus affecting O2 absorption as well.

There are many studies on the potential effects of OA and increased ocean temperatures on the physiology of marine organisms but little is known about changes at histological level and there are still several gaps at molecular level which must be studied for a better understanding of all biological mechanisms involved. With regard to hypoxia, alterations to the stress response can provide information on the organisms’ physiological effects and coping strategies triggered by anoxia. For instance, it is known that organisms respond by reducing protein synthesis. Once temperature affects physiological, behavioral and ecological processes, there is a need to understand what mechanisms are behind the organisms’ response to stress, enhancing our predictive and environmental management capacities considering a climate change scenario. This is of great importance, in particular to countries with a sea-based economy.

The aim of the present study is to assess the stress response of a marine fish, sea bream (Sparus aurata) exposed to increasing water temperature and different water acidity (alone or in combination). Here we present preliminary data on temperature effects on S. aurata at a cellular and molecular level. In addition, tissue samples from muscle, livers, gills and intestine are examined to evaluate any alterations caused by altering this physical parameter.

Fish were distributed randomly in tanks (n=96) and allowed to acclimate at 18ºC (the same temperature of the hatchery) before the beginning of the bioassays. After assessment of the upper thermal limits (UPL), water temperature was increased at a rate of 1ºC per hour using a thermostatized bath with a constant rate of water-temperature until reaching the endpoint, following the dynamic method of Critical Thermal Maximum (CTM). Every 2ºC step, fish were euthanized by cervical transection and the selected organs removed and stored at -80ºC until further analysis. Sub-samples were taken and processed for histological examination following standard techniques. Frozen samples were analysed for heat stress proteins (HSP70) as described by Madeira et al. The histological observations were carried out using a Leica microscope (DMLB model). Preliminary results of the histological examination showed changes in the cellular structure, with visible damage at higher temperatures in liver and gills (Fig. 1). Regarding HSP70, significant changes were observed throughout the temperature assay. The results indicate that elevated water temperature can be a major stressor that will affect fish due to potential climate changes, thus compelling the need to perform these studies to enhance our predictive and environmental management capacities.

The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through grant PTDC/MAR/119068/2010 and through project no. PEst-C/EQB/LA0006/2011 granted to Requimte.

The genus Flavivirus (Flaviviridae) includes over seven dozen known enveloped viruses with ssRNA genomes, the evolution, geographic dispersion and epidemiology of which seems to have been shaped both by environmental constraints (involving ecological factors and human activities), and by their replication in vertebrate and invertebrate hosts. Most bona-fide flaviviruses have been classified as mosquito or tick-borne viruses, although for some no known invertebrate vector has yet been found. Over time, a divergent cluster including viral agents that seem to be specific of insect (Insect-Specific Flaviviruses-ISF) has been associated with the genus. The classification of ISF as flaviviruses is supported by their genomic organization, protein hydropathy plots, conserved polyprotein cleavage sites and enzyme domains, which are similar to those of classical flaviviruses. However, they are antigenically distinct, and share approximately the same level of nucleotide sequence identity with other members of the genus, as when compared to the members of the two other genera in the Flaviviridae family.

This work reports the initial characterization, including near full-length sequence analysis, of an ISF which seems to form a divergent lineage within the ISF radiation (Figure 1), as indicated by phylogenetic analysis based on ac multiple sequence alignment of the NS5 flavivirus coding sequence. This virus, tentatively designated OCFV (Ochlerotatus caspius flavivirus) was isolated from clarified macerates of a pool of Aedes (Ochlerotatus) caspius (Pallas, 1771) adult mosquitoes (lab code number 174) using the Ae. albopictus-derived C6/36 cell line. The mosquitoes from which OCFV was isolated were collected using CDC-traps baited with CO2, and are amongst those found in high densities in the coastal, and estuarine, districts of Setúbal and Faro. Detection of viral genomes by RT-PCR using RNA directly extracted from supernatants of C6/36 infected cultures, collected 24h post-infection, indicated that the virus replicates rapidly in these cells. Nevertheless, but as expected for an ISF, it does not replicate in Vero cells. Unexpectedly, unlike most ISF OCFV seems to cause overt cytopathic effect in C6/36 cells, which rapidly detach from a solid support and round-up soon (24-48h) after infection. Electron-microscope analysis of thin-sections of C6/36 cells at 48h post-infection with OCFV revealed nuclear hyperplasy, and an evident enlargement of the intracisternal space of the nuclear envelope, which is filled by multiple sized vesicles. In some cells, a complex network of apparent membrane trabeculae was also clearly evident (Figure 2A). Viral particles seem to assemble at the endoplasmatic reticulum, from which viruses reach the cell surface by vesicular transport (Figure 2B). Unexpectedly for a flavivirus, but as previously observed, viral particles also seem to gemulate directly from the cytoplasmic membrane (Figure 2C).

Gastropods statoliths are biogenic structures of CaCO3, analogous to fish otoliths. Their application in sclerochronology is recent and may be advantageous comparing to the most traditional organisms used hitherto. Moreover, since elements bind within calcified structures, statoliths theoretically incorporate chemical information from the surrounding environment over time. So, besides being used for age estimation and growth studies, statoliths elemental fingerprinting is recognized as natural tag to track larval dispersal and may also be considered as a potential marker of environmental change and/or pollution. However there is a lack of information on the elemental composition of adult gastropod statoliths. Here we characterize Nassariusreticulatus (Gastropoda: Caenogastropoda) statoliths elemental composition by electron microprobe analysis (EMPA).

One statolith from each of 3 adults was prepared for EMPA (Figure 1) and showed the same structural pattern: a nucleus circled by a metamorphic ring and, in addition to this core, 4 other rings delineating 5 increments (Figure 1F). General composition was assessed by Energy and Wavelength Dispersive Spectroscopy revealing C, Ca, O, Sr, Na, S and Mg as the most representative elements. These were then quantified in 3 points per increment. Carbon was not determined since surfaces were C-coated and the amount of oxides of Ca, Sr, Na, S and Mg was estimated from the valence. In addition, Ba – trace element in statoliths of some gastropods larvae – and Al, Cu, Hg, Sn – known as environmental pollutants – were also sought in one of the statoliths. For such, mean values for Ca, Sr, Na, S, Mg and O were set as the sample matrix. Standards and mean detection limits by element and statolith are indicated in Table 1.

Concentrations of Ca, O, Na, Sr and S varied between 3.7x105-4.2x105, 1.5x105-1.7x105, 2.5x103-6.1x103, 1.3x103-8.9x103 and 4.7x102-1.3x103 ppm, respectively; Mg, which was not always detected, ranged from 2.0x102-8.6x102 ppm. No significant differences in these elements mean concentrations were found between increments, except Na in one statolith between increments 1 and 5 (higher at the edge; Dunn’s test s=2.014, p>0.05). Thus the general elemental composition is nearly constant throughout lifespan (i.e. along statoliths radiuses). Regarding trace elements: Sn was never detected; Al and Hg were at 22.2% of the punctual analyses conducted while Cu was at 66.7% and Ba at 88.9%. Concentrations of Al, Hg, Cu and Ba varied between 55-71, 176-180, 183-302 and 647-969 ppm, respectively.

EMPA proved to be a useful technique in sclerochronology, allowing the characterization of the overall composition of gastropods statoliths and the quantification of elements in specific locations with high resolution.

This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the grant SFRH/BPD/70368/2010.

Squid statoliths are paired calcareous concretions essentially composed of calcium carbonate crystallised as aragoniteand found inside cranial fluid-filled cavities, the statocysts - the main organ of detection of gravity and movement. Statolith use as an ageing tool was validated in the 1980s, when the daily periodicity of the growth increments was confirmed by chemically marking these structures in animals maintained in aquaria. Although statolith analysis has brought major advances in the knowledge of squid population-dynamics, little is known about the processes involved in statolith’s calcification and increment deposition (e.g. during embryogenesis). According to Villanueva (2000), embryonic statolith development involves the formation of structures such as the focus, nucleus, postnuclear zone and natal ring.

The aim of this study was to investigatethe microstructure of the embryonic statolith, considering all main structures,in recently-hatched squids using optical (fluorescence) microscopy, scanning electron microscopy (SEM) and confocal microscopy.

Recently-spawned egg masses of the European squid, Loligo vulgaris, were collected in the west coast of Portugal. After hatching at 13-15ºC, statoliths were removed from the paralarvae andpreserved at -20ºC. Prior to confocal analysis, statoliths were submitted to Alizarin red S (ARS) staining (5% of ARS in 0.1% KOH) to verify the presence of calcium content. In addition, images from optical microscopy and scanning electron microscopy (SEM) were acquired.

The findings obtained by auto-fluorescence observation (Figure 1 A, B) and SEM (Figure 1 C), show that the embryonic statolith is hollow in some of the area between the nucleus and the natal ring. ARS stained samples analysed in confocal microscopy (Figure 2 A-G), confirmed the previous observation.

This surprising structural feature in the origin of CaCO3 sensorial structures has never been described before and suggests a production, with minimum of material and energy expenditure. This is fundamentally interesting not only in biological terms but also considering biotemplating orbiomimetics approaches (i.e. synthetic approaches), where hollow calcium carbonate structures have attracted considerable attention owing to their unique structural, optical, and surface properties that lead them to a wide range of applications, including as templates for functional architecture composite materials.

The Portuguese Foundation for Science and Technology (FCT) supported this study through project grant PTDC/BIA-BEC/103266/2008 to R. Rosa.

1

The seasonal fluctuations in zooplankton densities in temperate climates have been long known and the multiplicity of performed studies identified a vast number of factors responsible for these phenomena, such as changes in the physico-chemical factors and other such types of environmental forces governing them. Acartia clausi is a euryaline temperate-boreal species very common in the Portuguese coastal ecosystems, in both estuarine and coastal waters. It is usually described as a temperate water species of neritic calanoid copepod, which is associated with warmer water regions, and as a result becomes more abundant in the summer months, reaching a biomass maximum during the months of July and August. Growth and egg production have been studied extensively in some Acartia species. In the present study histology and histochemistry were selected to determine the reproductive potential of A. clausi.

The ovigerous females were identified with a binocular microscope, isolated and fixed in the Bouin’s solution for histological (5 om thickness, mounting and Haematoxylin - Eosin staining) and histochemical analysis (Periodic Acid Schiff method (PAS) with Haematoxylin as a counter stain for the identification of the carbohydrate content and vitellogenic oocytes. The size of the oocytes was evaluated through measurements made with a micrometer.

The microscopic studies and Image analysis indicated that, in the month of September, the majority of the oocytes were immature and had reduced or almost negligible carbohydrate contents with very few vitellogenic oocytes (Figure 1 A and B), while the specimens from the month of March exhibited a large difference in the oocyte dimensions. These were mature and more vitellogenic and occupied almost half the volume of the body (Figure 1 C, D and E). This shows that, during the month of September, the environmental conditions are not favorable for the maturation of gonads in these species while in the month of March they proliferate and the species shows a high degree of reproductive potential.

This work forms a valid approach in understanding the population fluctuations and reproductive status in a key species of copepod showing a particular temporal variation associated with its reproductive strategy.

Over the last decade, with the advent of aberration correctors and energy-filters, transmission electron microscopes (TEMs) have seen phenomenal developments both in regards to spatial and spectral resolutions. Today, the TEM is a characterisation tool pervasive to nanoscaled sciences and technologies. Imaging and identifying single atoms, including low Z elements such as B or C, is within reach of a skilful operator with access to this new generation of microscopes.

Parallel to these advances, sample holders have also been evolving. Instead of just fixing and orienting the sample for observation, these ancillary components of TEMs have gradually been morphing into full-fledged instruments. Increasingly sophisticated devices enable a multitude of experiments performed inside the column of the TEM which includes the manipulation and exposure of discrete nanostructures to a variety of stimuli (heat, pressure, electrical current…). An example is the real-time observation of solid-vapour phase transitions in filled carbon nanotubes acting as interconnects and experiencing the dynamical effects of Joule heating. Together with resolution improvements, these added capabilities represent a paradigmatic shift in how we perceive and work with TEMs. No longer is the electron microscopist limited to simply analyse specimens in “post-mortem” state but (s)he can now take hold of a full set of physical and chemical experiments that encompass a much broader range of scientific interests.

Despite all progress, there is a crucial part of the TEM capabilities that is still awaiting a similar leap in resolution: the time-domain. In result of the past dominance of “post-mortem” analysis, a temporal resolution enhancement has been overlooked by most instrument manufacturers and researchers. However, with the growing interest on in situ methodologies, the ability to perform time-resolved experiments has suddenly become a fundamental necessity. Classically, registering tools such as photographic films, charge-coupled devices or video-rated cameras have been adequate to acquire data on events that take place on timescales ranging from seconds to hours. Such temporal resolution is not sufficient for an immensity of life- and materials science-related processes. To illustrate this, Figure 1 shows the before and after states of a ZnS-filled carbon nanotube that was subjected to a pulse of electrical current. Although it is possible to observe clearly the end result, one can only speculate on how the confined semiconductor was released, transported and delivered to the receiving substrate. Considering that the pulse lasted for 250 ms, a temporal resolution of at least 50 ms (or 20 frames per second) for the registering device would be necessary to build a reasonable impression of the mechanism behind this delivery-procedure.

PMFJC acknowledges the support from the Fundação para a Ciência e Tecnologia (Ciencia2007 Fellowship and project grant PTDC/EME-PME/112073/2009) and Alexander von Humboldt Foundation (Advanced Researcher Fellowship).

Ion Beam Analytical techniques (IBA) using MeV charged particles are powerful techniques for the study of different type of samples in several science fields such as Material Science, microelectronics or biology/biomedicine due to its fine sensitivity, versatility and “non-destructive” characteristics. The possibility of beam focusing and beam scanning adds spatial resolution down to the dm level and imaging capabilities then allowing the IBA techniques to become microscopy techniques.

In the Nuclear Microprobe installed at IST/ITN several IBA techniques can be routinely used for materials characterization, the most common ones being PIXE (Particle Induced X-ray Emission), RBS (Rutherford Backscattering Spectrometry) and STIM (Scanning Transmission Ion Microscopy). Whether through their independent or combined use the most important thing of these techniques is the complementary information that they can grant. As any other X-ray spectroscopic technique pPIXE can also provide elemental identification (for Z>12) but further present their spatial distribution in the sample as well as, for thin biological samples (<20 <m), calculate their areal mass density. RBS on the other hand is able to probe sample in depth then allowing obtaining, for instance, elemental depth profile and at the same time sample matrix areal mass density. The combined use of PIXE and RBS then allows determining elemental concentration for thin biological samples. For the mentioned thin biological samples the ion beam energy loss when crossing them (base of the STIM technique), contains information on their density or thickness allowing unique information on its structure and morphology.

Advantages and draw backs can always be taken into account when comparing with similar or competitive techniques. This is the case of PIXE and SEM-EDS which is quite unfavorable for PIXE in the case of image spatial resolution, but quite favorable if elemental sensitivity is considered. Due to the much lower X-ray spectrum background, detection limits for PIXE reach the tg/g level.

As an example of application, some of the results obtained for PE-g-HEMA films are here shown. To allow their utilization as biomaterials for biomedical applications (e.g. drug delivery) apart from the needed mechanical properties and surface characteristics, biocompatibility of these materials is of fundamental importance. Regarding biocompatibility one important parameter to be assessed is its cytotoxicity that strongly depends on the contamination level at the surface. As shown in Fig. 1, MeV ion beam microscopy not only can provide major and trace element spatial distribution (combining PIXE and RBS data) but also valuable information on its near-surface structure (STIM). Furthermore, quantitative elemental analysis can be performed through the analysis of the PIXE spectra with sensitivity down to the rg/g level as also revealed in Figure 1.

V. Corregidor acknowledges the funding support from the FCT-Ciência program.

The reuse of waste fluid catalytic cracking (FCC) catalyst, from oil-refinery industry, as a partial substitute of cement is a promising way of reducing the environmental impact of cement production and, therefore, contributes for industrial sustainability. On the other hand partial replacement of cement by oil-cracking catalysts up to 10-15% (w/w) results in improved mechanical properties as the waste catalyst tends to react with Ca(OH)2 - liberated upon cement hydration - and forms additional strength-providing reaction products. Nevertheless, the pozzolanic reaction mechanisms between cement and the waste FCC catalyst, which involve Ca migration from the cementitious matrix into the FCC particles is still not well understood.

In the present study waste FCC catalyst has been incorporated in cement paste mortars with a 15:85 weight ratio. The microstructural evolution of the blended materials has been investigated by X-ray diffraction and scanning electron microscopy coupled with X-ray energy dispersive spectroscopy after curing times of 2, 7 and 28 days. Namely, the evolutions of the catalyst crystal structure and of the calcium content in the dispersed catalyst particles have been evaluated.

The results show that the Ca/(Si,Al) ratio in the catalyst particles tends to increase during the cure. However, the inward migration does not occur in a continuous front but rather tends to follow high-diffusivity paths resulting in Ca depleted rims (Fig. 1). On the other hand the presence of heavy metals resulting from oil-cracking (such as La) remains essentially unaltered after a cure of 28 days. The present study demonstrates that the diffusion of Ca from the surrounding cementitious matrix into the dispersed catalyst particles plays an important role in the pozzolanic activity of the system.

The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through PTDC/ECM/113115/2009 and PEST-OE/CTM-UI0084/2011 grants.

Smart materials like piezoelectrics and ferroelectrics play a crucial role in applications such assensors and actuators,radio-frequency switching, drug delivery, chemicals detection, and power generation and storage. K0.5Na0.5NbO3 (KNN) is one of the leading lead free piezoelectric materials being considered as an alternativeto Pb(Zrx,Ti1-x)O3 (PZT), which is currently the most widely used material for electromechanical applications. Although pure KNN has inferior electromechanical properties compared to PZT,efforts are on going to tailor and improve its piezoelectric coefficients by doping and texturing.Although the piezoelectric constant (d33) of undoped KNN is unsuitable for practical electromechanical applications, properties comparable to PZT at room temperature (d33>400 pC/N) have been reported for modified KNN ceramics. Electromechanical properties are however, very much dependent on the crystalline phase content, crystallographic orientation, microstructure, interfaces and domain configuration.

Mn is an indispensable dopant for both PbO-based as well as PbO-free ceramics like BaTiO3, SrTiO3, KNbO3 and KTaO3. It has been reported to improve the density, mechanical quality factor, electromechanical properties and to reduce dielectric loss. Mn has been successfully used to reduce the leakage current and lower the orthorhombic to tetragonal phase transition temperature (TO-T) in KNN single crystals. It has also been shown to improve the density and properties of KNN–LiTaO3–LiSbO3. However, the effect of Mn on the KNN domain structure and phase assemblage has not yet been reported. In this work, KNN ceramics doped with Mn on the B-site (Mn content was 0.5, 1.0. 1.5 and 2 mole%) were synthesized by a conventional mixed oxide method. Transmission electron microscopy (TEM)(Hitachi 9000) studies were carried out to analyse the effect of B-site Mn doping on the ferroelectric domain structure and phase assemblage.

Undoped KNN ceramics had large grains (>30 >m) which contained large (>1 >m wide) wedge shaped ferroelectric domains. KNN doped with 0.5 mole % Mn exhibited a smaller grain size (~2 mm) in which a well defined domain structure was observed with widths approximately an order of magnitude smaller than those in undoped KNN. For KNN doped with 2 mole % Mn, the presence of a second phase, Figure 1c, was often observed. Electron diffraction patterns from the second phase were consistent with a tetragonal tungsten bronze (TTB) structured compound although more work is required to definitively determine the phase assemblage. The domain structure became increasing complex as Mn concentration increased, suggesting that the presence of Mn on the B-site disrupts polar order.

In conclusion, TEM analysis demonstrated that Mn doping changes the domain structure of KNN ceramics: for low Mn content, well defined ferroelectric domains and for high Mn content, tangled domains and second phase were the main features.These microstructure details elucidate reasons that may account for the inferior piezoelectric properties of KNN at higher Mn concentration.

The design and synthesis of high-efficiency materials to convert solar to electrical energy is an increasingly important research field. Within the photovoltaic technologies, crystalline Si have an 80% share while the remaining 20% are mostly thin film solar cells based on Cu(In,Ga)(S,Se)2 (CIGSSe) and CdTe. However, the cost, the abundance and the environmental impact of the elemental components cannot be neglected. For these reasons, Cu2ZnSnS4 (CZTS), Cu2ZnSnSe4 (CZTSe) and their solid solutions CZTSSe has attracted much attention recently since they can provide the development of cost competitive solar cells. The CZTS-based solar cells consist of earth abundant and relatively inexpensive elements and represent an environmentally friendly alternative compared to the above mentioned systems. The energy conversion efficiency of the CZTS-based solar cells has increased from 0.66% in 1996 to 11.1% recently.

The present work shows preliminary results that are related to the characterization of CZTSSe monograin powders by scanning electron microscopy. High purity metal compounds, S and Se powders were used as precursors for the synthesis of the Cu2ZnSn(SSe)4 monograin powders. The precursor powders were mixed to the desired composition and, additionally, KI was added as a flux material. Afterwards, the powders were blended in a mixer and encapsulated in quartz ampoules. The blended powders were degassed under dynamic vacuum at room temperature, sealed and annealed isothermally between 700 ºC and 780 ºC for a time ranging between 44 h and 136 h. After synthesis the flux material was removed with deionized water and the powders were sieved into several fractions. The morphology, microstructure and chemical composition of the synthesized powders was obtained with a Philips XL30 field-emission scanning electron microscope (FE-SEM) equipped with a backscattered electron (BSE) detector and an integrated EDAX energy dispersive X-ray spectroscopy (EDS) microanalysis system.

The typical morphology obtained for the CZTSSe powders can be seen in Figure 1. Basically, the particles show a polyhedral morphology with some of them showing a needle shape, i.e. a large shape factor (L/D>>1). Moreover, it was also observed a slight increase of the median particle size with the increase of the synthesis temperature. Due to the complexity of the synthesis of CZTSSe monograins, the formation of binary or ternary phases is a common feature. A very good control over the synthesis parameters is then required not only to obtain the desired phase but also to have a tight control over the stoichiometry of the material. Taking this into account, SEM/BSE observations and EDS analysis are two powerful techniques for evaluate the degree of the compositional homogeneity of the CZTSSe monograins. Figure 2 puts in evidence the degree of the homogeneity of the CZTSSe monograins in relation to their size. Overall, and independently of the synthesis conditions, the powder particles consisted predominantly of CZTSSe monograins and, in a much smaller extent, of particles having a ZnS or ZnS(Se) core coated by a CZTSSe layer. The presence of undesired phases occurred more frequently in the larger powder particles (> 100 µm) and the degree of homogeneity was lower for the powders synthesized at lower temperature and for a shorter time. However, when the EDS results obtained for CZTSSe monograins belonging to different size fractions are compared no major variations in the content of the five elements can be inferred.

Zinc Oxide is a wide band-gap compound semiconductor that has been used in optoelectronic and photovoltaic applications due to its good electrical and optical properties. Aluminium has been an efficient n-type dopant for ZnO to produce low resistivity films and high transparency to visible light. In addition, the improvement of these properties also depends on the morphology, crystalline structure and deposition parameters. In this work, ZnO:Al films were produced by d.c. pulsed magnetron sputtering deposition from a ZnO ceramic target (2.0 wt% Al2O3) on glass substrates, at a temperature of 250 ºC.

The crystallographic orientation of aluminum doped zinc oxide (ZnO:Al) thin films has been studied by Electron Backscatter Diffraction (EBSD) technique. EBSD coupled with Scanning Electron Microscopy (SEM) is a powerful tool for the microstructural and crystallographic characterization of a wide range of materials.

The investigation by EBSD technique of such films presents some challenges since this analysis requires a flat and smooth surface. This is a necessary condition to avoid any shadow effects during the experiments performed with high tilting conditions (70º). This is also essential to ensure a good control of the three dimensional projection of the crystalline axes on the geometrical references related to the sample.

Crystalline texture is described by the inverse pole figure (IPF) maps (Figure 1). Through EBSD analysis it was observed that the external surface of the film presents a strong texture on the basal plane orientation (grains highlighted in red colour). Furthermore it was possible to verify that the grain size strongly depends on the deposition time (Figure 1 (a) and (b)). The electrical and optical film properties improve with increasing of the grain size, which can be mainly, attributed to the decrease in scattering grain boundaries which leads to an increasing in carrier mobility (Figure 2).

The authors kindly acknowledge the financial support from the Portuguese Foundation for Science and Technology (FCT) scientific program for the National Network of Electron Microscopy (RNME) EDE/1511/RME/2005.

One way to improve the photophysical and photochemical properties of NIR probes is to incorporate them in nanoparticles (NPs). The characteristics of NP-based imaging agents, such as the ability to carry large payloads of optical probes and multiple targeting groups, together with the possibility to tune their pharmacokinetics, have motivated the development of different systems for use in combined diagnostic and therapeutic (theranostic) applications. The use of silica nanoparticles (SiNP) in nanomedicine, as carriers for drug delivery and imaging agents has increase remarkably in the last decade. On the other hand, it is well known that the use of near infrared (NIR) light (750-1400 nm) in optical bioimaging presents several advantages over lower wavelength light (visible), namely higher tissue penetration, lower interference from autofluorescence in biological specimens, and reduced light scattering. These factors have greatly increased the interest in NIR fluorescent dyes for use in biological imaging. An additional advantage of NIR dyes is that they can be used with inexpensive laser diode excitation sources in laser scanning microscopy.

In this communication we evaluate the performance of new fluorescent SiNP in laser scanning microscopy. The particles are labelled with two different perylenediimide (PDI) derivatives, one with emission in the visible region of the spectrum (PDIvis) and the other emitting in the NIR (PDInir). The PDI derivatives were covalently incorporated in the SiNPs during their synthesis by a modified Stöber method. The labelled SiNP were characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), fluorescence emission spectroscopy and laser scanning fluorescence confocal microscopy (LSFCM).

The excellent photophysical properties and stability of the fluorescently labelled SiNP encouraged us to evaluate their performance in vivo, as markers for LSFCM. The nanoparticles were internalized in HEK293 cells co-stained with plasma membrane (AF594-WGA) and/or nucleus (Hoechst 33258) markers. Confocal microscopy images show that both SiNP-PDInir and SiNP-PDIvis were efficiently internalized in the cells (Figure 1 and 2, respectively). The major difference between the two sets of SiNPs is that while SiNP-PDIvis is excited at 488 nm (argon laser) showing emission in the green region of the spectra, SiNP-PDInir can be excited at 633 nm (using a low power/low cost HeNe laser) with the emission observed in the NIR. The in vivo photostability of the labelled nanoparticles was compared with that of a commercial membrane marker, DiIC18(5), showing much higher photostability during laser scanning, due to the shielding effect of the silica nanoparticles.

This work was partially supported by Fundação para a Ciência e a Tecnologia (FCT-Portugal) and COMPETE (FEDER) within projects PTDC/CTM/101627/2008 and PEst-OE/CTM/LA0024/2011. T.R., R.S. and F.F. also thank FCT for Ph.D. (SFRH/BD/64702/2009) and Pos-Doc (SFRH/BPD/71249/2010; SFRH/BPD/64320/2009) grants.

Liposomes or lipid vesicles are self-closed structures formed by one or several concentric lipid bilayers with an aqueous phase inside, which may incorporate almost any molecule, namely proteins, hormones, enzymes, antibiotics, anticancer agents, antifungical agents, gene transfer agents, DNA, and whole viruses. Scientific evidences prove that unprotected liposomes containing drugs are easily released from the endoplasmic reticulum of the cell. To increase the vesicles lifetime and to activate a controlled drug release with an external stimulus, the vesicles immobilization on a surface and the factors which create conditions to the liposome rupture have to be analyzed. A number of studies have identified some of the critical stages of vesicle adsorption (adhesion), fusion, deformation, rupture, and spreading of the lipid bilayer. Nevertheless, the formation mechanisms of well-controlled continuous supported bilayers or adsorption of whole liposomes are still not fully understood. As yet it was demonstrated that a controlled adsorption of vesicles containing a small fraction of charged lipids occurs without rupture and their subsequent embedding in polyelectrolyte multilayer (PEM) films, meaning vesicles may be immobilized in an intact or slightly deformed state, which can act as drug reservoirs. Moreover, depending on the nature of the physicochemical conditions of the vesicle solution and the substrate surface, a flat lipid bilayer can be formed, known as supported lipid bilayers, which can incorporate membrane proteins and keep the native dynamics of the lipid bilayer mimicking a biological membrane. In this study, a layer of 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DPPG) liposomes adsorbed onto PEMs cushions based on poly(ethylenimine) (PEI), poly(sodium 4-styrenesulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) polyelectrolytes was analyzed by atomic force microscopy (AFM) technique in non-contact mode and quartz crystal microbalance (QCM).

Sequential heterostructures of Si/PEI(PSS/PAH)4 and Si/PAH, also designated cushions, were prepared onto silicon substrates using the layer-by-layer (LbL) technique with polyelectrolyte solutions of PEI, PSS and PAH of monomeric concentrations of 0.01M. Topographic images of 1×1μm2 area of Si/PAH/DPPG (Figure 1 a), and Si/PEI(PSS/PAH)4/DPPG (Figure 1 b) LbL films were acquired by AFM. The root mean square roughness (RMS) calculated from topographies data are listed in table I. As shown, when a DPPG layer is adsorbed onto Si/PAH the RMS keeps an approximately equal value meaning that the liposome disrupted and spread onto the surface forming a planar lipid bilayer. But when a DPPG layer is adsorbed onto Si/PEI(PSS/PAH)4 the RMS value doubled, indicating that the structural integrity of the liposomes is maintained, even though there has been any deformation during adsorption. The adsorbed amount of the two PEMs and DPPG-liposomes layers was measured using a QCM and is displayed in table I. The DPPG adsorbed amount obtained on the PAH cushion was approximately equal to a planar lipid bilayer, while the adsorption onto PEI(PSS/PAH)4 was higher than the predicted for a planar lipid bilayer. This behavior suggests that the DPPG liposomes on the second PEM remained intact during adsorption. Both confirm the AFM results. Therefore we conclude that the initial roughness of the surface is a primordial factor to determine the adsorption or not of intact vesicles.

The authors acknowledge the “Fundação para a Ciência e Tecnologia” (FCT-MEC) by the post-graduate scholarship SFRH/BD/62229/2009 and the “Plurianual” funding.

Microstructural characterisation of an archaeological collection of Protohistoric bronze artefacts attributed to around the VIII century B.C. and has been carried out using optical microscopy (OM) and scanning electron microscopy combined with energy dispersive spectroscopy (SEM-EDS). The collection was found in Medronhal cave (Condeixa-a-Nova, Portugal) and is composed by 1 fibula, 5 bracelets and 31 rings.

Small areas on the surfaces of the artefacts were cleaned from corrosion and were metallographically prepared by a manual polishing with several diamond suspensions in a cotton swab until 1 micron diamond size. OM observations were performed in bright field (BF) illumination and under polarized light (Pol), in unetched and etched conditions. SEM-EDS was performed without a conductive coating for a minimum external elemental interference.

OM observations allowed the study of the method and sequence of manufacture involved in the production of the various types of artefacts. Results show that the fibula and the bracelets have equiaxed (recrystallised) grain structures resulting from cyclic thermo-mechanical treatments performed to a pre-form cast bar until requested shape and surface finishing was attained. The rings, which are of diverse sizes, have various types of microstructures as a result of diverse manufacturing techniques. Some show a dendritic structure that resulted from the casting, and others show recrystallised structures that resulted from cycles of deformation and annealing procedures posterior to their casting (Figure 1).

SEM-EDS analysis allowed the study of the presence of inclusions in the metallic matrix and the study of corrosion in some artefacts (Figure 2). Different types of inclusions were identified, namely copper sulphides, tin oxide, and lead rich inclusions. Both copper sulphides and lead inclusions are commonly found in archaeological bronzes, and can be a result of copper ores impurities. Tin oxide, on the other hand, is not so common, and its presence can be understood as a result of preferential oxidation of tin regarding copper during a melting or alloying operation. The study of the interface alloy/corrosion showed the presence of chlorides in internal corrosion layers, which can probably be related to an aggressiveness of the burial environment.

This research work has been financed by the Portuguese Science Foundation (FCT) through the EarlyMetal project (PTDC/HIS-ARQ/110442/2008), the grant SFRH/BPD/73245/2010 (to EF) and the Strategic Project-LA25-2011-2012 (PEst-C/CTM/LA0025/2011) (to CENIMAT/I3N).

Stony materials applied in the built environment interact with diverse pollutants and can originate INORGANIC neoformations that can contribute to depreciation of these materials.

Microscopy studies help to characterize the chemical and textural features of these neoformations hence contributing to the understanding of the decay processes. In a study of diverse modern constructions in towns of Northern and Central Portugal were identified diverse neoformation substances in stony materials that were sampled and subjected to scanning electron microscopy (SEM) studies. While, in a strict perspective, implying destructive procedures that require the removal of a sample, microscopy techniques require minute amounts and can study occurrences of neoformations that can be detected at the macroscopic level. Products clearly resulting of metals corrosion were not considered in this study.

The dominant inorganic coatings and stains in the studied constructions are carbonate-rich whitish ones that can present diverse morphological features. Some of these carbonate-rich coatings have showed textural features defining a kind of urban travertine and the existence of several layers suggesting diverse episodes of solutions circulation and crystals precipitation, indicating the recurrence of the neoformation processes. Phosphates aggregates were also detected in several built surfaces, including indoor areas (Figure 1a,b). Another relatively common occurrence are efflorescences of more soluble salts, dominated by alkaline sulphates and carbonates (especially sodium sulphate) but were other salts such as alkaline nitrates (Figure 1c,d) and chlorides and magnesium sulphates have also been occasionally found. In some buildings it was possible to observe black crusts resulting from the fixation of atmospheric particles by gypsum aggregates. A very rare neoformation are silica-rich stains that in the situation presented in Figure 1e,f occur associate with calcium carbonate stains and metal corrosion products. The observed neoformations show a great geochemical and textural diversity in the surfaces of stony materials of modern built works, being evidences of diverse possible pollution sources that can interact under variable environmental conditions. Besides an important role that seems to be attributable to mortars materials (namely in relation to calcium carbonates and alkaline sulphates but that might also be the case for the phosphates in inside portions), there are also possible contributions from atmospheric pollutions (in the case of the black crusts) and the presence of alkaline nitrates could be indicative of organic sources as this connection is well established in older works. It has been admitted that alkaline nitrates in these modern works could either represent the persistence of contamination conditions from the past in the soils or the recurrence of the same polluting conditions. Also of particular interest are the textural informations indicating recurrence of neoformation episodes (relevant for possible cleaning interventions) and the chemical associations observed even in some very small samples.

The author acknowledges the support of the Fundação para a Ciência e Tecnologia (PEST-OE/CTE/UI0697/2011, funding by the national budget of the Portuguese Republic).

Salt weathering is one of the main processes affecting rock materials applied in the built environment. Scanning electron microscopy (SEM) studies can contribute to the understanding of the susceptibility of petrographic features of rocks to salt weathering.

SEM studies were performed in limestones (grainstones with the commercial designations of Semi-rijo and Moca Creme and a travertine) submitted to salt weathering tests (EN 12370 using sodium sulphate solutions and cubic specimens) consisting of 15 cycles of immersion/drying followed by water washing after the 15th cycle. Fragments from small cubes of the grainstones subjected to salt weathering cycles without the final water cleaning were also studied. SEM observation before and after the tests allows the discussion of the petrographical characteristics of these rocks that contribute to erosive decay under salt weathering.

In the case of the grainstones specimens the texture can be described in general as allochemical particles cemented by sparry calcite (Figure 1 a) and it is visible from the preparation of the polished surfaces the interface between the sparry cement and the allochemical components. After the salt weathering tests, SEM studies show (Figure 1 b) extensive fissuring in the sparry cement and separation between grains and sparry cement. Observations of disaggregation products showed an important amount of apparently intact grains. These results are similar to those observed by with the sparry cement being affect by salt crystallisation and lesser impact on the grains. Studies in the unwashed specimens showed the presence of sodium sulphate in the interface between allochemical grains and the sparry cement (Figure 1 c,d). Travertine specimens show heterogeneous patterns of erosion attributed to the heterogeneous texture of this rock, with more terrigenous (especially clay-rich) portions that favour a higher erosive susceptibility and the presence of clays has been frequently connected with salt weathering susceptibility in limestones. In Figure 1e can be observed the heterogeneity of a polished surface, with the more irregular portions showing a chemical spectra (Figure 1 f) indicative of the terrigenous component and the presence of sodium and sulphate (resulting from the solutions used in these weathering tests).

The authors acknowledge the support of the Fundação para a Ciência e Tecnologia (POCTI/CTA/44940/2002; PEst-OE/CTE/UI0697/2011 and Pest-OE/CTE/UI0098/2011, funding by the European Union and national budget of the Portuguese Republic). Acknowledgments to Eng. Teresa Luís, Eng. Sónia Pereira and Enterprise Mármores Galrão for the rock blocks.

There are several problems regarding the dating of the works in the historical buildings (see a review in Sanjurjo-Sánchez, 2012). Mortars are a potential material for dating of the built environment works, being ubiquitous and not reusable. Optical stimulated luminescence (OSL) dating can be applied to mortars with and without lime and considering the age interval of mortars from thousands of years up to a few decades. OSL dating is based on measuring the accumulated luminescence signal of aliquots (of one or more mineral grains) of constituents from the mortars, resulting from the absorbed dose of surrounding radioactivity (at a constant dose rate), considering contributions both from environment and the radioactive phases of the mortars.

In this abstract is considered the relevance of microscopy studies as support for the preparation of samples for dating and the precise age calculations.

Microscopy studies can allow identifying samples that cannot be dated due to lack of enough grains of suitable phases as is illustrated in Figure 1a. It is also possible to assess the presence of datable phases. Usually quartz and potassium (K) feldspar are used for dating (phases that can be easily distinguished by scanning electron microscopy and generally by polarizing optical microscopy). For dating, the OSL signal of such datable phases of a mortar must have been bleached before the mortar manufacture and settling, typically by daylight exposure of such minerals. Quartz grains have an easy to bleach signal. K-feldspars grains have a significant internal dose making them less susceptible to environmental variations but they present signal anomalous fading and the luminescence signal is harder to bleach than in quartz grains. However, according to Li B. et al., 2007, the effect of anomalous fading in K-feldspar may be negligible for samples younger than 100 years and there also some proposals for overcoming signal fading and the combination of measurements on K-feldspars and quartz. On the preparation of the sample, microscopy studies can assess the risk of contamination by other phases and, in analogy with what is done in mining studies, define a liberation size that relates to the obtainment of monomineral aliquots. It can also contribute to the identification of the presence of different grainsize fractions of the phases to use in dating (see example in Figure 1b), information that can be used for comparative studies of dose in the different grainsize fractions, such as isochron measurement procedures. One of the main issues for dating is the assessment of the radioactive dose rate that affected the mortar constituents related to the distribution of the contents of U, Th, Rb and K that can occur disperse through the sample or concentrated on some grains. Most OSL studies on sediments relate K content to K-feldspar occurrences. However, in some mortars there could be an important presence of K-micas such as muscovite and the biotite series (see Figure 1b,c) that have a lower K content and a different morphology. The presence of these mica grains has also been considered as potentially disturbing of the signal measurements and procedures for its removal have been proposed by Kortekaas and Murray, 2005. The grainsize distributions of radioactive grains and dated grains can also affect the way the radioactive dose affect the measured dose given that the same amount of K could be distributed in grains with different volume and hence different relation area/mass. Additionally, K-bearing phases can have contents of other radioactive elements affecting the beta dose as is illustrated by Figure 1c showing the presence of halos resulting from radioactive decay of inclusions in biotite crystals. While in general it is admitted that quartz grains had a zero internal dose, the presence of inclusions such as rutile (that can be assessed by optical microscopy) can endanger this assumption. Microscopy studies can also be used to assess precipitation and dissolution process that occur after the preparation of the mortar and that can affect the beta dose for the grains (see Figure 1d). Finally, microscopy studies can be used after dose estimates and can help solve problems that have baffled researchers such as low content of feldspar separations. In the case of mortar samples, for which can be obtained representative sections, the previous considerations show that petrographic models can help in the definition of spatial models for dose rate modelling and the establishment of appropriated age models.

Support from Portuguese-Spanish collaboration Project “Ação Integrada E-141/10” (Fundação das Universidades Portuguesas)/“Acción Integrada PT2009-0077” (Ministerio de Ciencia e Innovación) and the Fundação para a Ciência e Tecnologia (pluriannual funding program for research units, PEst-OE/CTE/UI0697/2011, Portuguese funds).

Solid oxide fuel cells (SOFCs) are devices that allow direct conversion of chemical to electrical energy through an electrochemical reaction in a cleaner and more efficient way than conventional processes (eg. gas turbines). They are characterized by the use of a solid oxide material as the electrolyte. Yttria-stabilised zirconia (YSZ) has traditionally been used in SOFCs electrolytes at temperatures in the range of 850-1000 °C. Recent research is being focused on the development of new materials with increased ionic conductivity at intermediate temperatures (500-800ºC) as alternative materials to YSZ. Rare earth silicates with an apatite-type structure, such as doped lanthanum oxides of general formula La10(MO4)6O2, where M = Ge, Co, Si, Al, or P, are among these materials. The major limitation associated with the manufacture of these materials is their poor sinterability, which requires high sintering temperatures (1600ºC).

The present work concerns the production of La9.33Si2Ge4O26 dense materials from La2O3 (99.9%), SiO2 (99.4%) and GeO2 (99.9%) powders by mechanical alloying (MA) followed by conventional and microwave hybrid sintering at 1350ºC. Dry mechanical milling was carried out in protective atmosphere (argon at 200 kPa) by using a rotating speed of 350 rpm for 15h. Prior to the synthesis of the La9.33Si2Ge4O26 mixture, the starting materials were milled separately at 350 rpm in argon at 50 kPa in order to achieve low particle size distributions with a greater ability for sintering. The MA mixture was compacted by uniaxial pressing at 390 MPa followed by sintering for 1 h at 1350 °C. Density of the La9.33Si2Ge4O26 pellets was determined by the boiling test method. SEM/EDS, XRD and atomic force microscopy (AFM) were used for samples characterization.

The bulk densities and the open porosities of the pellets (EF and MW for conventional and microwave hybrid sintering, respectively) are presented in Table 1. Dense pellets were obtained in both cases. However, sample MW showed higher density and, consequently, lower porosity than sample EF. Both mechanically alloyed samples are formed by an apatite phase. This phase remains stable during sintering. However, traces of a second phase (La4GeO8) were detected after sintering. This phase was already referred in previous work (Serra et al., 2012). Figure 1 shows the morphology of the two samples obtained by SEM and AFM as well as the corresponding particles size distributions. The sintered samples were as-polished and thermal etched in air at 1300 ºC, for 10 min., for detailed microstructural observation. SEM images at low amplification confirm that sample EF is more porous than sample MW (Figure 1 (a) and (b), respectively). Although the same sintering temperature was used in both sintering processes, sample MW has a coarser grain size (Figure 1 (c) to (f)). This feature was already been reported in previous work (Fang et al., 1994). Mean values of 0.9 and 1.9 m were determined for samples EF and MW, respectively.

As conclusion, one may say that microwave hybrid sintering is a novel suitable process for the densification of La9.33Si2Ge4O26 powders for application in SOFCs electrolytes.

This research is partially sponsored by FEDER funds through the program COMPETE – Programa Operacional Factores de Competitividade – and by national funds through FCT – Fundação para a Ciência e Tecnologia – under the contract PTDC/EME-PME/102837/2008. The research fellowships granted to Cátia Alves, Márcio Santos and Mafalda Macatrão are also gratefully acknowledged.