Marine fungi occur either in Open Ocean or in the intertidal zone of sandy beaches, salt marshes and mangroves, where their hosts and substrates are found. The development of morphological adaptations like appendages and sheaths of the spores are vital to the settlement and attachment to substrate surfaces, floatation and dispersion on seawater. The morphological features of these appendages and sheaths of spores also have an important role in the identification of marine fungi. Differential interference contrast (DIC) microscopy is an essential tool for the observation of these mucilaginous structures in marine fungi spores and was therefore applied to marine mycota from surveys along the Portuguese coast.

Since 1991 Portuguese marine fungi have been studied and characterized based on morphological identifications. The studied environments included salt marshes, sandy beaches and marinas. On these environments different substrates were collected such as plants and baits of Spartina maritima, different categories of drift substrates and Fagus sylvatica and Pinus pinaster baits. These substrates, which had been exposed to different conditions of permanent and temporary submersion, were subjected to an initial examination under the stereoscope microscope in order to detect fruit bodies and spores of marine fungi. These structures were then observed in order to achieve the taxonomic identifications of these fungi, based on dichotomous keys for marine fungi.

To observe and characterize the wide variety of appendages and mucilaginous sheaths of the ascospores, basidiospores and conidia often invisible in the bright field of the light microscope, the use of DIC microscopy was implemented, because three-dimensional images are produced, highlighting them. The identified fungi were microphotographed with a Leica Wild MPS 52 with Fujichrome RTP- 135, 64T Tungsten.

The studies carried out with substrates subjected to conditions of permanent submersion highlighted the dominance of Ascomycota with unitunicate asci. The unitunicate asci are thin - walled, persistent or early deliquescing, favoring ascospores release on marine environmental conditions (fig 1a, 1b and 1c). On the other hand, Ascomycota with bitunicate asci, were mainly detected on temporary submersion conditions. These fungi presented asci with active spore discharge and mucilaginous sheaths that contributed to substrate attachment (fig. 2a and 2b). Ascospores and conidia presented morphological diversity on their appendages, which has particular importance on their success in marine environment (figs. 3, 4, 5, and 6).

Marine Mycologists are in agreement that the best technique to observe the appendages and sheaths of spores, often invisible in the bright-field microscope is the use of differential interference or phase contrast microscopy. DIC microscopy was then applied to the observation of Portuguese marine fungi enabling to thoroughly characterize the structures essential to their morphological identifications.

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

Acquiring and processing phase-contrast microscopy images in wide-field long-term live-cell imaging and high-throughput screening applications is still a challenge as the methodology and algorithms used must be fast, simple to use and tune, and as minimally intrusive as possible. In this paper, we developed a simple and fast algorithm to compute the cell-covered surface (degree of confluence) in phase-contrast microscopy images. This segmentation algorithm is based on a range filter of a specified size, a minimum range threshold, and a minimum object size threshold. These parameters were adjusted in order to maximize the F-measure function on a calibration set of 200 hand-segmented images, and its performance was compared with other algorithms proposed in the literature. A set of one million images from 37 myoblast cell cultures under different conditions were processed to obtain their cell-covered surface against time. The data were used to fit exponential and logistic models, and the analysis showed a linear relationship between the kinetic parameters and passage number and highlighted the effect of culture medium quality on cell growth kinetics. This algorithm could be used for real-time monitoring of cell cultures and for high-throughput screening experiments upon adequate tuning.

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Three-dimensional focused ion beam/scanning electron microscopy (FIB/SEM tomography) is currently an important technique to characterize in 3D a complex semiconductor device or a specific failure. However, the industrial context demands low turnaround time making the technique less useful. To make it more attractive, the following study focuses on a specific methodology going from sample preparation to the final volume reconstruction to reduce the global time analysis while keeping reliable results. The FIB/SEM parameters available will be first analyzed to acquire a relevant dataset in a reasonable time (few hours). Then, a new alignment strategy based on specific alignment marks [using tetraethoxylisane (TEOS) and Pt deposition] is proposed to improve the volume reconstruction speed. These points combined represent a considerable improvement regarding the reliability of the results and the time consumption (gain of factor 3). This method is then applied to various case studies illustrating the benefits of the FIB/SEM tomography technique such as the precise identification of the origin of 3D defects, or the capability to perform a virtual top-down deprocessing on soft material not possible by any mechanical solution.

Remineralization of hard dental tissues is thought to be a tool that could close the gap between prevention and surgical procedures in clinical dentistry. The purpose of this study was to examine the remineralizing potential of different toothpaste formulations: toothpastes containing bioactive glass, hydroxyapatite, or strontium acetate with fluoride, when applied to demineralized enamel. Results obtained by scanning electron microscopy (SEM) and SEM/energy dispersive X-ray analyses proved that the hydroxyapatite and bioactive glass-containing toothpastes were highly efficient in promoting enamel remineralization by formation of deposits and a protective layer on the surface of the demineralized enamel, whereas the toothpaste containing 8% strontium acetate and 1040 ppm fluoride as NaF had little, if any, remineralization potential. In conclusion, the treatment of demineralized teeth with toothpastes containing hydroxyapatite or bioactive glass resulted in repair of the damaged tissue.

This study evaluated the effects of nanoindentation on the surface of white mineral trioxide aggregate (WMTA), Bioaggregate and Nano WMTA cements. Cements were mixed according to the manufacturer directions, condensed inside glass tubes, and randomly divided into three groups (n = 8). Specimens were soaked in synthetic tissue fluid (pH = 7.4) and incubated for 3 days. Cement pellets were subjected to nanoindentation tests and observed by scanning electron microscopy. Then, the images were processed and the number of cracks and total surface area of defects on the surface were calculated and analyzed using ImageJ. Data were submitted to one-way analysis of variance and a post hoc Tukey's test. The lowest number of cracks and total surface of defects were detected in Nano WMTA samples; however, it was not significantly different from WMTA samples (p = 0.588), while the highest values were noticed in Bioaggregate specimens that were significantly different from Nano WMTA and WMTA (p = 0.0001). The surface of WMTA and Nano WMTA showed more resistance after exposure to nano-compressive forces which indicated a better surface tolerance against these forces and crack formation. This suggests these substances are more tolerant cement materials which can predictably withstand loaded situations in a clinical scenario.

We report the direct visualization of point defect clustering in {113} planes of silicon crystal using a transmission electron microscope, which was supported by structural modeling and high-resolution electron microscope image simulations. In the initial stage an accumulation of nonbonded interstitial–vacancy (I–V) pairs stacked at a distance of 7.68 Å along neighboring atomic chains located on the {113} plane takes place. Further broadening of the {113} defect across its plane is due to the formation of planar fourfold coordinated defects (FFCDs) perpendicular to chains accumulating I–V pairs. Closely packed FFCDs create a sequence of eightfold rings in the {113} plane, providing sites for additional interstitials. As a result, the perfect interstitial chains are built on the {113} plane to create an equilibrium structure. Self-ordering of point defects driven by their nonisotropic strain fields is assumed to be the main force for point defect clustering in the {113} plane under the existence of an energy barrier for their recombination.

Dragonfly eye beads are considered to be the earliest types of glass objects in China, and in the past have been considered as evidence of culture interaction or trade between West and East Asia. In this article, synchrotron radiation microcomputed tomography and μ-probe energy dispersive X-ray fluorescence were used to determine the chemical composition, microstructure, and manufacturing technology of four dragonfly eye beads, excavated from a Chu tomb at the Shenmingpu site, Henan Province, China, dated stylistically to the Middle and Late Warring State Period (475 bc–221 bc). First, a nondestructive method was used to differentiate the material types including faience (glazed quartz), frit, glazed pottery (clay ceramic), and glass. Three beads were identified as faience and one bead as glazed pottery. The glaze recipe includes quartz, saltpeter, plant ash, and various copper, and is classified as belonging to the K2O-CaO-SiO2 glass system, which indicates that these beads were not imported from the West. Based on computed tomography slices, the manufacturing technology of the faience eye beads appears to include the use of an inner core, molding technology, and the direct application glazing method. These manufacturing features are consistent with the techniques used in China during this same time period for bronze mold-casting, proto-porcelain, and glass.

Roughness of a positively charged poly(allylamine hydrochloride) (PAH) polyelectrolyte surface was shown to strongly influence the adsorption of 1.2-dipalmitoyl-sn-3-glycero-[phosphorrac-(1-glycerol)] (DPPG) liposomes on it. The adsorption kinetic curves of DPPG liposomes onto a low-roughness PAH layer reveal an adsorbed amount of 5 mg/m2, pointing to liposome rupture, whereas a high-roughness surface leads to adsorbed amounts of 51 mg/m2, signifying adsorption of intact liposomes. The adsorption kinetic parameters calculated from adsorption kinetic curves allow us to conclude that the adsorption process is due to electrostatic interactions and also depends on processes such as diffusion and reorganization of lipids on the surface. Analysis of the roughness kinetics enabled us to calculate a growth exponent of 0.19 ± 0.07 and a roughness exponent of around 0.84, revealing that DPPG liposomes adsorbed onto rough surfaces follow the Villain self-affine model. By relating self-affine surfaces with hydrophobicity, the liposome integrity was explained by the reduction in the number of water molecules on the PAH surface, contributing to counterion anchorage near PAH ionic groups, reducing the liposome/PAH layer electrostatic forces and, consequently, avoiding liposome rupture.

The aim of this study was to evaluate the efficacy of smear layer removal from teeth following root canals using lasers (Er:YAG and Nd:YAG), NaOCl, 17% EDTA, and MTAD by scanning electron microscopy (SEM). Root canals were irrigated with 3 mL of 5.25% NaOCl after each file. Teeth were randomly divided into five groups. In the final irrigation, canals were irrigated with 5.25% NaOCl (Group 1, control), 17% EDTA (Group 2), or BioPure MTAD (Group 3). Laser groups were irradiated with Er:YAG laser (1.8 W, 120 mJ, 15 Hz) (Group 4) or Nd:YAG laser (1 W, 100 mJ, 15 Hz) (Group 5). The smear layer at the apical, middle, and coronal thirds of each root canal was imaged using SEM. Smear layer removal by EDTA was the most effective in all regions. The difference between EDTA and MTAD was statistically significant in the coronal and middle (p < 0.05), but not the apical (p > 0.05), regions. The difference between the control and laser groups was not statistically significant (p > 0.05). In all regions, the difference between 17% EDTA–MTAD and the control–laser groups was statistically significant (p < 0.05). Although improvement was observed in removal of the smear layer using alternative materials and techniques, application of a combination of EDTA and NaOCl remains an effective technique.

Ion channeling contrast (iCC) and electron channeling contrast (eCC) are caused by variation in signal resulting from changes in the angle of the incident beam and the crystal lattice with respect to the target. iCC is directly influenced by the incident ion range in crystalline materials. The ion range is larger for low-index crystal orientated grains, resulting in the emission of fewer secondary electrons at the surface yielding a lower signal. Ions are stopped closer to the surface for off-axis grains, resulting in the emission of many secondary electrons yielding a higher signal. Conversely, backscattered electrons (BSEs) are the primary contribution to eCC. BSEs are diffracted or channeled to form an electron channeling pattern (ECP). The BSE emission of the ECP peaks when the electron beam is normal to the surface of an on-axis grain, and therefore a bright signal is observed. Thus, iCC and eCC images yield inverse contrast behavior for on-axis oriented grains. Since there is a critical angle associated with particle channeling, accurately determining grain boundary locations require the acquisition of multiple images obtained at different tilt conditions.

The purpose of this study was to investigate the in vitro biocompatibility of two dental composites (namely A and B) with similar chemical composition used for direct restoration using three-dimensional confocal laser scanning microscopy (CLSM) time-lapse imaging. Time-lapse imaging was performed on cultured human HGF-1 fibroblast-like cells after staining using Live/Dead®. Image analysis showed a higher mortality rate in the presence of composite A than composite B. The viability rate decreased in a time-dependent manner during the 5 h of exposure. Morphological alterations were associated with toxic effects; cells were enlarged and more rounded in the presence of composite A as shown by F-actin and cell nuclei staining. Resazurin assay was used to confirm the active potential of composites in cell metabolism; results showed severe cytotoxic effects in the presence of both no light-curing composites after 24 h of direct contact. However, extracts of polymerized composites induced a moderate decrease in cell metabolism after the same incubation period. Composite B was significantly better tolerated than composite A at all investigated end points and all time points. The finding confirmed that the used CLSM method was sufficiently sensitive to differentiate the biocompatibility behavior of two composites based on similar methacrylate monomers.

The lateral and axial resolution of three-dimensional (3D) focal series aberration-corrected scanning transmission electron microscopy was studied for samples of different thicknesses. The samples consisted of gold nanoparticles placed on the top and at the bottom of silicon nitride membranes of thickness between 50 and 500 nm. Atomic resolution was obtained for nanoparticles on top of 50-, 100-, and 200-nm-thick membranes with respect to the electron beam traveling downward. Atomic resolution was also achieved for nanoparticles placed below 50-, 100-, and 200-nm-thick membranes but with a lower contrast at the larger thicknesses. Beam broadening led to a reduced resolution for a 500-nm-thick membrane. The influence of the beam broadening on the axial resolution was also studied using Monte Carlo simulations with a 3D sample geometry.

In situ transmission electron microscopy was performed on the electromigration in platinum (Pt) nanowires (14 nm thick, 200 nm wide, and 300 nm long) with and without feedback control. Using the feedback control mode, symmetric electrodes are obtained and the gap usually forms at the center of the Pt nanowire. Without feedback control, asymmetric electrodes are formed, and the gap can occur at any position along the wire. The three-dimensional gap geometries of the electrodes in the Pt nanowire were determined using high-angle annular dark-field scanning transmission electron microscopy; the thickness of the nanowire is reduced from 14 nm to only a few atoms at the edge with a gap of about 5–10 nm.

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

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The identification of genome-wide expression profiles that discriminate different molecular subtypes of diseases became a recent field of research, with a visible impact in cancer research. In breast cancer, molecular immunophenotyping of the tumor is critical to support therapeutic decisions.

Over the last years, some studies revealed that a high percentage of Feline Mammary Tumours (FMTs) display fHER2 overexpression followed by a very short survival period, like in woman HER2-positive breast cancer. Usually, FMTs are malignant and very aggressive (85% to 93%), showing bad prognosis. In feline oncology, the histopathological classification is the only possible option, meaning that little data about molecular expression profiles is available. Given these facts, our study aims to confirm if primary tumor immunophenotype is conserved at regional and distant metastasis.

Two primary mammary carcinomas, their regional and distant metastasis were collected during necropsy examination of two queens. The immunohistochemical analysis measured HER2 status, progesterone receptor (PR), estrogen receptor (ER), cytokeratin (CK) expression levels and the Ki67 index in all samples using the antibodies: anti-HER2 (A0485, DAKO); anti-PR (1E2, Ventana); anti-ER (SP1, Ventana); anti-CK (AE1/AE3, DAKO) and anti-Ki67 (MM1, Leica). The fixation and the interpretation criteria followed the American Society of Clinical Oncology guidelines.

The first tumor was an anaplastic mammary carcinoma with high malignancy grade (III) both in the primary tumor and in metastatic lesions (Fig.1). Immunophenotyping revealed HER2 and PR positivity in both primary mass and regional metastasis (Fig.1 - A4, B4; A5, B5), but ER negative staining in these locations (Fig.1- A6, B6). Distant metastasis showed a lack of PR expression (Fig.1 - C5), but maintained equal status of the two other markers (Fig.1 - C4, C6). Similar Ki-67 index values were determined in primary tumor (0.26) and in metastasis (0.19). The second FMT was a tubulopapillary carcinoma (Fig.2) with high malignancy grade (III) and, unlike, the first tumor, immunolabeling for PR was positive both in the primary tumor and in the different metastasis, revealing molecular pattern conservation. The Ki-67 index was always high either in primary tumor (0.33) as in regional (0.48) and distant metastases (0.41) (Fig.2 - A3, B3, C3). All tissue samples were positive for CK, confirming their epithelial nature (Fig.1 - A2, B2, C2 and Fig.2 - A2, B2, C2).

Our study points out that FMTs molecular expression patterns may not be preserved in primary tumor and metastatic lesions. If we just looked to the immunophenotype of the primary mass and the regional metastasis, both carcinomas would be classified as luminal B subtype (ER-, PR+ and HER2+). However, when we evaluated the distant metastasis, while the immunophenotype of the second FMT was conserved, the PR expression in the first FMT was lost, which implied a tumor reclassification to HER2 subtype. In women, both subtypes have anthracycline-based regimens prescription but luminal B subtype also benefits with hormonal therapy, while the HER-2 subtype has indication for specific immunotherapy. Similarly to women breast cancer, the molecular classification of feline mammary tumors should be performed to improve treatment-response and animal live quality.

The authors acknowledge the funding by Fundação para a Ciência e Tecnologia through PhD fellowship SFRH/BD/70720/2010.