SPMicros was founded in 1966 as Sociedade Portuguesa de Microscopia and since then has promoted Congresses dedicated to microscopy each year. For many of us this was the place where we delivered our first communications and received feedback by a panel of senior scientists devoted to the promotion of scientific quality; the Society was thus crucial to the rise of more than one generation of microscopists. In 2005 the Society's designation was changed to the “Portuguese Society for Microscopy” aka SPMicros, and incorporated a strong and growing group dedicated to materials science as well as to the life sciences.

Fluorescence images present low signal-to-noise ratio (SNR), are corrupted by a type of multiplicative noise with Poisson distribution, and are affected by a time intensity decay due to photoblinking and photobleaching (PBPB) effects. The noise and the PBPB effects together make long-term biological observation very difficult. Here, a theoretical model based on the underlying quantum mechanic physics theory of the observation process associated with this type of image is presented and the common empirical weighted sum of two decaying exponentials is derived from the model. Improvement in the SNR obtained in denoising when the proposed method is used is particularly important in the last images of the sequence where temporal correlation is used to recover information that is sometimes faded and therefore useless from a visual inspection point of view. The proposed PBPB model is included in a Bayesian denoising algorithm previously proposed by the authors. Experiments with synthetic and real data are presented to validate the PBPB model and to illustrate the effectiveness of the model in denoising and reconstruction results.

Previous studies have described promising antitumor activity of an organometallic Ru(II) complex, η5-Cyclopentadienyl(2,2′-bipyridyl)(triphenylphosphane) Ruthenium(II) triflate ([(η5-C5H5)Ru(2,2′-bipyridyl)(PPh3)][CF3SO3]) herein designated as TM34. Its broad spectrum of activity against a panel of human tumor cell lines and high antiproliferative efficiency prompted us to focus on its mode of action. We present herein results obtained with two human tumor cell lines A2780 and MDAMB231 on the compound distribution within the cell, the mechanism of its activity, and its cellular targets. The prospective metallodrug TM34 revealed: (a) fast antiproliferative effects even at short incubation times for both cell lines; (b) preferential localization at the cell membrane and cytosol; (c) cellular activity by a temperature-dependent process, probably macropinocytosis; (d) inhibition of a lysosomal enzyme, acid phosphatase, in a dose-dependent mode; and (e) disruption and vesiculation of the Golgi apparatus, which suggest the involvement of the endosomal/lysosomal system in its mode of action. These results are essential to elucidate the basis for the cytotoxic activity and mechanism of action of this RuII(η5-cyclopentadienyl) complex.

The toxicity of titanium dioxide nanoparticles (TIO2 NPs) and oxidative stress effects were studied in two freshwater fish species (Carassius auratus and Danio rerio) exposed for 21 days to different concentrations (0.01, 0.1, 1, 10, 100/mgL) of TiO2 NPs and to a control (tap water). Additional fish were transferred to clean water for 14 days to assess the ability to recover from exposure to TiO2 NPs. Activities of the enzyme glutathione-S-transferase (GST) and lipid peroxidation (LPO) (malondialdheyde) were measured as indicators of oxidative stress. Histological and ultra-structural changes in livers from bothspecies of fish were evaluated by light and electron microscopy. Results show a general GST activity increase according to TiO2 NPs concentrations, which is in agreement with data from LPO. After 21 days, GST activities decreased possibly caused by suppression of GST synthesis as a result of severe stress. Histological and ultra-structural analysis of livers from exposed fish show degeneration of the hepatic tissue and alterations in hepatocytes such as glycogen depletion and an increase in lipofucsin lysosome-like granules. After a depuration period a partial recovery for biochemical markers and cells was observed. The results suggest that TiO2 promotes alterations in hepatic tissues compatible with oxidative stress.

Azelaic acid (AzA) is used in the treatment of acne. However, side effects and low compliance have been associated with several topical treatments with AzA. Nanotechnology presents a strategy that can overcome these problems. Polymeric nanoparticles can control drug release and targeting and reduce local drug toxicity. The aim of this study was to produce and evaluate an innovative topical treatment for acne with AzA-loaded poly-dl-lactide/glycolide copolymer nanoparticles. A soft white powder of nanoparticles was prepared. The mean size of loaded nanoparticles was <400 nm and zeta potential was negative. Spherical nanoparticles were observed by scanning electron microscopy. Encapsulation efficiency was around 80% and a strong interaction between the polymer and the drug was confirmed by differential scanning calorimetric analysis. In vitro drug release studies suggested a controlled and pulsatile release profile. System efficacy tests suggested similar results between the loaded nanoparticles and the nonencapsulated drug against the most common bacteria associated with acne. Cytotoxicity of AzA-loaded nanoparticles was concentration dependent, although not pronounced. The occluded patch test seemed to indicate that the formulation excipients were safe and thus AzA-loaded nanoparticles appear to be an efficient and safe treatment for acne.

The taxonomy of fungal species, similar to that of many other microorganisms, suffers frequent revisions due to the discovery of new species and to the development and gathering of characterization data and morphological information. Morpho-taxonomy helps in the identification of many species. This work presents the macro, micro-morphological, and spectral mass analyses for phenotypic characterization of 13 species of Aspergillus section Nigri, showing that the characterization of spores (conidia) by scanning electron microscopy can be used as a tool to discriminate key morphological characteristics and separate closely related fungi. These results were corroborated by colony plates, stereomicroscopy, light microscopy, and spectral mass data.

Tuberculosis (TB) is a major health problem. The emergence of multidrug resistant (MDR) Mycobacterium tuberculosis (Mtb) isolates confounds treatment strategies. In Portugal, cases of MDR-TB are reported annually with an increased incidence noted in Lisbon. The majority of these MDR-TB cases are due to closely related mycobacteria known collectively as the Lisboa family and Q1 cluster. Genetic determinants linked to drug resistance have been exhaustively studied resulting in the identification of family and cluster specific mutations. Nevertheless, little is known about other factors involved in development of mycobacteria drug resistance. Here, we complement genetic analysis with the study of morphological and structural features of the Lisboa family and Q1 cluster isolates by using scanning and transmission electron microscopy. This analysis allowed the identification of structural differences, such as cell envelope thickness, between Mtb clinical isolates that are correlated with antibiotic resistance. The infection of human monocyte derived macrophages allowed us to document the relative selective advantage of the Lisboa family isolates over other circulating Mtb isolates.

A fully automatic approach to locate polyomavirus particles in transmission electron microscopy images is presented that can localize intact particles, many damaged capsids, and an acceptable percentage of superposed ones. Performance of the approach is quantified in 25 electron micrographs containing nearly 390 particles and compared with the interpretation of the micrographs by two independent electron microscopy experts. All parameterization is based on the particle expected dimensions. This approach uses indicators calculated from the local co-occurrence matrix of gray levels to assess the textured pattern typical of polyomavirus and prune the initial set of candidates. In more complicated backgrounds, about 2–10% of the elements survive. A restricted set of the accepted points is used to evaluate the typical average and variance and to reduce the set of survivors accordingly. These intermediate points are evaluated using (i) a statistical index concerning the radiometric distribution of a circular neighborhood around the centroid of each candidate and (ii) a structural index resuming the expected morphological characteristics of eight radial intensity profiles encompassing the area of the possible particle. This hierarchical approach attains 90% efficiency in the detection of entire virus particles, tolerating a certain lack of differentiation in the borders and a certain amount of shape alterations.

Icosahedral nucleo-cytoplasmic large DNA viruses (NCLDV)-like viruses, which form inclusions in the erythrocyte cytoplasm of reptiles, were previously presented as candidates for a new genus of the Iridoviridae family. The present work describes the distribution of infected lizard hosts and ultrastructural characteristics of the viral inclusions of NCLDV-like viruses from Portugal and adjacent locations in Spain. Giemsa-stained blood smears of 235 Lacerta schreiberi from Portugal and Spain, 571 Lacerta monticola from the mountain Serra da Estrela (Portugal), 794 Podarcis hispanica from several localities in Portugal and Spain, and 25 Lacerta dugesii from Madeira Island, were studied. Infection in L. schreiberi was only found in mountain populations, up to 30% in Serra da Estrela and 9–11% elsewhere. It was absent in lizards from lowlands. Prevalence of infection among L. monticola in Serra da Estrela was 10%; infected lizards were found during March to July and October but not in August and September. Infection in P. hispanica was below 3.3%. Only one infected specimen of L. dugesii was identified by light microscopy. Ultrastructural examination of infected samples revealed that the inclusions are virus assembly sites of icosahedral cytoplasmic iridovirus-like virions. Virions from different host species have different ultrastructural features and probably represent different related viruses.

Theories have been put forward on the etiology of sialoliths; however, a comprehensive understanding of their growth mechanisms is lacking. In an attempt to fill this gap, the current study has evaluated the internal architecture and growth patterns of a set of 30 independent specimens of sialoliths characterized at different scales by computed microtomography and electron microscopy. Tomography reconstructions showed cores in most of the sialoliths. The cores were surrounded by concentric or irregular patterns with variable degrees of mineralization. Regardless of the patterns, at finer scales the sialoliths consisted of banded and globular structures. The distribution of precipitates in the banded structures is compatible with a Liesegang–Ostwald phenomenon. On the other hand, the globular structures appear to arise from surface tension effects and to develop self-similar features as a result of a viscous fingering process. Electron diffraction patterns demonstrated that Ca- and P-based electrolytes crystallize in a structure close to that of hydroxyapatite. The organic matter contained sulfur with apparent origin from sulfated components of secretory material. These results cast new light on the mechanisms involved in the formation of sialoliths.

The UFeB4 phase present in different alloys of the B–Fe–U system was studied by powder X-ray diffraction (PXRD) and scanning electron microscopy complemented with energy-dispersive spectroscopy and electron backscattered diffraction (EBSD). The PXRD data showed that the ternary compound crystallized adopting essentially the YCrB4-type structure. However, microstructural observations revealed that under high undercooling conditions the UFeB4 phase exhibits a random distribution of defects parallel to, which are consistently associated with intense higher-order Laue zone rings in EBSD patterns. Indexation of the EBSD patterns showed that the defective structure is compatible with an intergrowth of YCrB4- and ThMoB4-type layers according to the (010)YCrB4//(110)ThMoB4 and [001]YCrB4//[001]ThMoB4 orientation relation previously reported for an analogous compound. Magnetic studies indicated that the annealed UFeB4 compound has a paramagnetic behavior in the 2–300 K temperature range.

Nd:11Fe:Ti alloys prepared by arc melting followed by splat quenching and annealing have been investigated by electron microscopy. The as-cast microstructure evidenced an α-Fe(Ti) → NdFe11Ti → Nd2(Fe,Ti)17 solidification sequence compatible with a cascade of peritectic reactions. The Nd2(Fe,Ti)17 phase was not detected in the microstructure of the splat-quenched materials, but after annealing the ternary compound grains consisted of a mixture of ThMn12-type and Th2Zn17-type structures exhibiting a consistent (020)1:12//(003)2:17 and [100]1:12//[110]2:17, orientation relation, with the invariant plane sitting at (022)1:12//(333)2:17. A series of 3D microdiffraction experiments carried out on grains presenting a random distribution of planar defects has been used to map the reciprocal space of the intergrown phases.

In this paper we describe the preparation of fluorescent mesoporous silica nanoparticles (MSNs) for traceable drug delivery systems. The nanoparticles were prepared following a sol–gel procedure, incorporating a modified perylenediimide dye in the silica structure. Transmission electron microscopy and scanning electron microscopy show that the nanoparticles are monodispersed, with a spheroid shape and a raspberry-type surface morphology. The hybrid MSNs are robust, maintaining the mesoporous structure after template removal, with a pore diameter above 2 nm. A polymer shell was synthesized from the external surface of the hybrid nanoparticles by atom transfer radical polymerization, showing temperature-switchable collapsed/expanded conformation control. The fluorescent properties of the perylenediimide dye incorporated in the MSN pore walls are intact, and internalization in HEK293 cells shows that the nanoparticles are efficiently dispersed in the cytosol. These results show that the mesoporous fluorescent hybrid nanoparticles are an excellent platform for development of a traceable drug delivery system.

Several authors have reported the workability, mechanical properties, and durability of concrete produced with construction waste replacing the natural aggregate. However, a systematic microstructural characterization of recycled aggregate concrete has not been reported. This work studies the use of fine recycled aggregate to replace fine natural aggregate in the production of concrete and reports the resulting microstructures. The used raw materials were natural aggregate, recycled aggregate obtained from a standard concrete, and Portland cement. The substitution extent was 0, 10, 50, and 100 vol%; hydration was stopped at 9, 24, and 96 h and 28 days. Microscopy was focused on the cement/aggregate interfacial transition zone, enlightening the effect of incorporating recycled aggregate on the formation and morphology of the different concrete hydration products. The results show that concretes with recycled aggregates exhibit typical microstructural features of the transition zone in normal strength concrete. Although overall porosity increases with increasing replacement, the interfacial bond is apparently stronger when recycled aggregates are used. An addition of 10 vol% results in a decrease in porosity at the interface with a corresponding increase of the material hardness. This provides an opportunity for development of increased strength Portland cement concretes using controlled amounts of concrete waste.

Salt weathering is a major erosive process affecting porous materials in buildings. There have been attempts to relate erosive mass loss to physical characteristics of materials, but in the case of natural stone it is necessary to consider the effect of petrographic features that are a source of heterogeneity. In this paper, we use scanning electron microscopy before and after salt weathering tests in cubic specimens of three limestone types (two grainstones and a travertine) in an attempt to built conceptual models that relate petrographic features and salt weathering susceptibility (represented by mass loss). In the grainstones, the most relevant feature in controlling salt weathering processes is the interface between micrite aggregates and sparry cement that constitute weakness surfaces and barriers to fluid migration. Given the small size of the heterogeneities in relation to the test sample dimension and their spatial distribution, the macroscopic erosive patterns are globally homogeneously distributed, affecting edges and corners. In the travertine specimens, there are macroheterogeneities related to the presence of detritic-rich portions that cause heterogeneous erosive patterns in the specimens. Petrological modeling helps to understand results of salt weathering tests, supporting field studies for natural stone selection.

The built environment is subjected to several pollutants under variable environmental conditions defined by diverse geochemical systems. These geochemical systems promote the occurrence of neoformations that can have a detrimental effect on surfaces of the building materials. Hence, the study of neoformations helps in the understanding of weathering processes that affect built structures. In the present paper we present a scanning electron microscopy study of macroscopic manifestations of neoformations detected during an extensive visual survey of several modern architectural works in urban areas of northern and central Portugal. The studies performed suggest that cementitious materials play an important role as a source of pollutants for the most common neoformations such as carbonate rich stains and coatings, as well as salt efflorescences of alkaline sulphates and carbonates. There are also indications of contributions from organic sources for alkaline nitrates and atmospheric pollution for gypsum-rich black crusts. Other less common neoformations include phosphate aggregates and silica stains that represent interesting indicators of the geochemical systems in built environments. In the case of carbonate-rich coatings, indications of recurrence related to the circulation of carbonate forming solutions relevant to the maintenance of built surfaces were detected.

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

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

A mean-shift clustering (MSC) algorithm is introduced as a valuable alternative to perform materials phase classification from multispectral images. As opposed to other multivariate statistical techniques, such as factor analysis or principal component analysis (PCA), clustering techniques directly assign a class label to each pixel, so that their outputs are phase segmented images, i.e., there is no need for an additional segmentation algorithm. On the other hand, as compared to other clustering procedures and classification methods, such as segmentation by thresholding of multiple spectral components, MSC has the advantages of not requiring previous knowledge of the number of data clusters and not assuming any shape for these clusters, i.e., neither the number nor the composition of the phases must be previously known. This makes MSC a particularly useful tool for exploratory research, assisting phase identification of unknown samples. Visualization and interpretation of the results are also simplified, since the information content of the output image does not depend on the particular choice of the content of the color channels. We applied MSC to the analysis of two sets of X-ray maps acquired in scanning electron microscopes equipped with energy-dispersive detection systems. Our results indicate that MSC is capable of detecting additional phases, not clearly identified through PCA or multiple thresholding, with a very low empirical reject rate.

Methods to determine the rotation axis using the rotation electron diffraction technique are described. A combination of rotation axis tilt, beam tilt, and simulated experimental diffraction patterns with nonintegers zone axis has been used. Accurate knowledge of the crystallographic direction of the incident beam for deducing the excitation error of reflections simultaneously near Bragg positions is essential in quantitative electron diffraction. Experimental patterns from CoP3 are used as examples.