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

In Romanesque wall paintings in Aragon (Spain), the pigment used for creating blue was a very characteristic mineral, aerinite, which came from local ores in the southern Pyrenees. Optical and scanning electron microscopy (SEM), with energy-dispersive X-ray (EDX) analysis, X-ray diffraction, and reflectance spectroscopy were used to make a detailed microcharacterization of this rare blue pigment in order to improve the knowledge of its composition and possible variability, from samples of medieval paintings and some mineral ores. New analytical data on the chemical composition of the blue pigment are reported here, together with the characterization of its microstructure, and the heterogeneity of the natural pigment made by the features of the ore itself. X-ray diffraction pattern and color parameters of the mineral ores are also included. The data obtained by SEM-EDX will assist identification of this pigment by electron microscopy. The natural variability in composition observed in the samples may be used to explain formation of the extracted mineral and to compare several ore sources. Connection of the ore composition with the pigments used in Romanesque wall paintings will help both provenance and attribution studies.

Step height is defined as the vertical spacing between two plane-parallel planes comprising an elevation or an indentation and the substrate. In atomic force microscopy (AFM), there are many algorithms for determining feature dimensions such as step height and width. One common problem of many algorithms is the difficulty for users to accurately determine the corner positions needed to properly implement the said algorithms. A new algorithm based on ISO 5436-1 is proposed that determines the necessary corner positions along with two examples illustrating the implementation of this algorithm. We propose calling this new method the determinant method. Since the corner positions are automatically decided, feature dimensions such as step height of an AFM image are easily determined. Comparative experiments carried out to compare the step height measurement using this algorithm and the SPIP software from Image Metrology show encouraging results.

In situ scanning transmission electron microscopy (STEM) through liquids is a promising approach for exploring biological and materials processes. However, options for in situ chemical identification are limited: X-ray analysis is precluded because the liquid cell holder shadows the detector and electron energy-loss spectroscopy (EELS) is degraded by multiple scattering events in thick layers. Here, we explore the limits of EELS in the study of chemical reactions in their native environments in real time and on the nanometer scale. The determination of the local electron density, optical gap, and thickness of the liquid layer by valence EELS is demonstrated. By comparing theoretical and experimental plasmon energies, we find that liquids appear to follow the free-electron model that has been previously established for solids. Signals at energies below the optical gap and plasmon energy of the liquid provide a high signal-to-background ratio regime as demonstrated for LiFePO4 in an aqueous solution. The potential for the use of valence EELS to understand in situ STEM reactions is demonstrated for beam-induced deposition of metallic copper: as copper clusters grow, EELS develops low-loss peaks corresponding to metallic copper. From these techniques, in situ imaging and valence EELS offer insights into the local electronic structure of nanoparticles and chemical reactions.

Since contaminants such as metals, POPs (Persistent Organic Pollutants) and PAHs (Polycyclic aromatic compounds represent a risk to human health and to the environment, it is therefore extremely necessary to study their biological effects. Man-made chemicals endocrine disruptors such as estrogens pose the potential to modulate endocrine function and thus adversely affect humans and other animal’s reproductive development. In this work, sublethal toxicity tests were carried out with the sea anemones Actinia equina and Anemonia sulcata exposed to 17-β-Estradiol. A. equina and A. sulcata are species that present a wide geographic distribution and might possibly be effective pollution indicators. Histological and histochemical techniques were used to detect morphological changes in sea anemones in order to find histological parameters that could be useful as early biomarkers of environmental contamination. The histological and histochemical procedures followed by standard methods to Hematoxylin and eosine (H & E), Periodic acid Shiff reagent (PAS) and Masson`s Tricrome (TMass) stained adaptated to Actiniidae conditions. Such as, the fixation (formalin and alcohol) time is high (96 hours), because this organisms are almost 98% of water body constitution. The slides obtained were observed by light microscopy means. The assemblage of methodologies described permitted the identification of several anomalies/pathologies in different parts of the sea anemones body, with special attention to reproductive structures. Results obtained for A. sulcata showed vitellogenic oocytes with anomalous dimensions, altered cytoplasm or without cellular membrane limits (Figure 1). It can also be observed lipid accumulations and cells membranes not always preserved. In certain areas oocytes presents small reactivity with atypical PAS low basophilic patterns. In the mesoglea the amoebocytes showed more eosinophilic cytoplasm or extracellular bodies suggesting necrosis or protein content.  The effects at 10 µg/L concentrations show a considerable number of oocytes with germinal vesicles membranes and indistinct cytoplasm boundaries (Figure 1). Results obtained for A. equina showed some morphological changes in the spermatocytes of male gonads and in the germinal vesicles the female gonads. The effects observed at higher concentrations shows oocytes and ovarian tissues disintegration. The morphologic alterations observed suggested a delay in spermatogenesis and although there have been no alterations in female vitellogenic granules, there are changes in their maturation. The whole effects lead to verify the role that the estradiol in the Anthozoan reproductive system.

Acknowledgments: This work was supported by the Portuguese Foundation for the Science and Technology – Portugal and FEDER funds, through the Projects: PTDC/MAR/464729/2006 and FCT/CNPq (Brazil), Project 6818, Programme 19/ 004.

This article reports the structure and morphology of the in vivo interface between implants composed of either α tricalcium phosphate (αTCP) or αTCP doped with 3.0 wt% dicalcium silicate (αTCPss) ceramic, and natural bone of rabbit tibias. Both interfaces developed a new bone layer in direct contact with the implants after 4 and 8 weeks of implantation. The specimens were examined using analytical scanning and transmission electron microscopy, up to the lattice plane resolution level. Degradation processes of the implants developed at the interfaces encouraged osseous tissue ingrowth into the periphery of the material, changing the microstructure of the implants. The ionic exchange initiated at the implant interface with the environment was essential in the integration process of the implant, through a dissolution–precipitation–transformation mechanism. The interfaces developed normal biological and chemical activities and remained reactive over the 8-week period. Organized collagen fibrils were found at the αTCPss/bone interface after 4 weeks, whereas a collagen-free layer was present around the Si-free αTCP implants. These findings suggest that the incorporation of silicate ions into αTCP ceramic promotes processes of the bone remodeling at the bone/αTCPss interface, hence the solubility rate of the αTCPss material decreased.

The effects of polymer substrates on the interfacial structure and the thermal stability of Ga-doped ZnO (GZO) thin films were investigated. The GZO thin films were deposited on polyethylene terephthalate (PET) and polyethylene naphthalate (PEN) substrates by rf-magnetron sputtering at room temperature, and thermal stability tests of the GZO thin films on the polymer substrates were performed at 150°C up to 8 h in air. Electrical and structural characterizations of the GZO thin films on the PET and the PEN substrates were carried out, and the origins of the stable interfacial structure and the improved thermal stability of the GZO thin film on the PEN substrate were discussed.

In this work, the interactions between tungsten (W) and silicon carbide (SiC) in SigmaTM SiC fibers at high temperatures were characterized using scanning and transmission electron microscopy. These fibers could have the potential for use in fusion-related applications owing to their high thermal conductivity compared with pure SiC-based fibers. The as-received fibers were composed of a 100-μm-thick shell of radially textured β-SiC grains and a 15-μm-thick tungsten core, composed of a few hundreds of nm-sized elongated tungsten grains. The interfaces between the tungsten and the SiC and the SiC and the outer coatings were sharp and smooth. After heat treatment at 1,600°C for 3 h in Ar, the tungsten core reacted with SiC to form a rough interface surface. Inside the core, W5Si3, W3Si, and W2C phases were detected using energy-dispersive X-ray spectroscopy and electron-diffraction techniques. The mechanical properties of the fibers deteriorate after the heat treatment.

Finding a faster alternative to tilt-series electron tomography is critical for rapidly evolving fields such as the semiconductor industry, where failure analysis could greatly benefit from higher throughput. We present a theoretical and experimental evaluation of scanning confocal electron energy-loss microscopy (SCEELM) using valence-loss signals, which is a promising technique for the reliable reconstruction of materials with sub-10-nm resolution. Such a confocal geometry transfers information from the focused portion of the electron beam and enables rapid three-dimensional (3D) reconstruction by depth sectioning. SCEELM can minimize or eliminate the missing-information cone and the elongation problem that are associated with other depth-sectioning image techniques in a transmission electron microscope. Valence-loss SCEELM data acquisition is an order of magnitude faster and requires little postprocessing compared with tilt-series electron tomography. With postspecimen chromatic aberration (Cc) correction, SCEELM signals can be acquired in parallel in the direction of energy dispersion with the aid of a physical pinhole. This increases the efficiency by 10×–100×, and can provide 3D resolved chemical information for multiple core-loss signals simultaneously.

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

As part of an ongoing effort to increase image reproducibility and fidelity in addition to improving cross-instrument consistency, we have proposed using four separate instrument quality tests to augment the ones we have previously reported. These four tests assessed the following areas: (1) objective lens quality, (2) resolution, (3) accuracy of the wavelength information from spectral detectors, and (4) the accuracy and quality of spectral separation algorithms. Data were received from 55 laboratories located in 18 countries. The largest source of errors across all tests was user error which could be subdivided between failure to follow provided protocols and improper use of the microscope. This truly emphasizes the importance of proper rigorous training and diligence in performing confocal microscopy experiments and equipment evaluations. It should be noted that there was no discernible difference in quality between confocal microscope manufactures. These tests, as well as others previously reported, will help assess the quality of confocal microscopy equipment and will provide a means to track equipment performance over time. From 62 to 97% of the data sets sent in passed the various tests demonstrating the usefulness and appropriateness of these tests as part of a larger performance testing regiment.

Azelaic acid, a saturated dicarboxylic acid, is used in the treatment of acne. However, some side effects and low patient compliance have been associated with several topical formulations of azelaic acid. Thus, nanotechnology presents an innovative strategy that can overcome these problems. Polymeric nanoparticles are generally applied to control drug release and to reduce local drug toxicity. In this study, we used the polymer PLGA (acid poly (DL-lactic-co-glycolic)) which is a biocompatible and biodegradable polymer that provides a slow and controlled drug release. The aim of this study was to evaluate a new topical treatment for acne with azelaic acid-loaded PLGA nanoparticles in terms of size, surface charge, encapsulation efficiency, morphology, drug-polymer interactions, efficacy, toxicity and safety of excipients.

Nanoparticles were produced by a modified spontaneous emulsification/solvent diffusion method and included into a gel of carbopol 940.  Size and zeta potential were determined by photon spectroscopy and electrophoretic mobility, respectively. Efficiency encapsulation was accessed by HPLC analysis using an adaptation of a previously described method. Loaded and blank nanoparticles morphology was observed by SEM. Drug-polymer interactions and thermal characteristics were evaluated by DSC. Staphylococcus epidermidis and Propionibacterium acnes susceptibility to the azelaic acid-loaded nanoparticles were tested by the broth microdilution method (efficacy test) and the minimum inhibitory concentration (MIC) values were determined using vancomycin as a positive control. The cytotoxicity of the nanoparticles was investigated on a Saccharomyces cerevisiae model. Excipients safety was accessed by occluded patch tests in humans (blank nanoparticles). The tests were performed in 12 healthy, female volunteers with skin type II whose ages were in between 22-38 years. All the tests were carried out according to the Declaration of Helsinki and received the approval of local Ethical Committee.

Nanoparticles formed a soft white powder (Figure 1A). The loaded nanoparticles mean size was 378.63±60.86 nm (0.09±0.03, P.I.) (Figure 1B) and zeta potential was -7.82 ± 9.01 mV. The encapsulation efficiency was 76±3.81%. Entrapped nanoparticles were visible in the empty nanoparticles sample (Figure 1C). Monodispersed and spherical loaded nanoparticles were observed by SEM (Figure. 1D). System efficacy test suggested similar results of azelaic acid-loaded nanoparticles and azelaic acid in the free form against S. epidermidis and P. acnes (Table 1). DSC analysis suggested an interaction between the polymer (PLGA) and the drug. Cytotoxicity of azelaic acid-loaded nanoparticles on S. cerevisiae was concentration-dependent although not pronounced. Occluded patch test seemed indicate that the formulation excipients were safe.

Small and anionic PLGA nanoparticles demonstrated to be efficient in the encapsulation of azelaic acid.

Nanoparticles proved to be efficient against the most common bacteria associated with acne. Further studies will include additional bacteria susceptibility tests.

Xenopus laevis oocytes are an interesting model for the study of many developmental mechanisms because of their dimensions and the ease with which they can be manipulated. In addition, they are widely employed systems for the expression and functional study of heterologous proteins, which can be expressed with high efficiency on their plasma membrane. Here we applied atomic force microscopy (AFM) to the study of the plasma membrane of X. laevis oocytes. In particular, we developed and optimized a new sample preparation protocol, based on the purification of plasma membranes by ultracentrifugation on a sucrose gradient, to perform a high-resolution AFM imaging of X. laevis oocyte plasma membrane in physiological-like conditions. Reproducible AFM topographs allowed visualization and dimensional characterization of membrane patches, whose height corresponds to a single lipid bilayer, as well as the presence of nanometer structures embedded in the plasma membrane and identified as native membrane proteins. The described method appears to be an applicable tool for performing high-resolution AFM imaging of X. laevis oocyte plasma membrane in a physiological-like environment, thus opening promising perspectives for studying in situ cloned membrane proteins of relevant biomedical/pharmacological interest expressed in this biological system.

Electron tomography is becoming one of the most used methods for structural analysis at nanometric scale in biological and materials sciences. Combined with chemical mapping, it provides qualitative and semiquantitative information on the distribution of chemical elements on a given sample. Due to the current difficulties in obtaining three-dimensional (3D) maps by energy-filtered transmission electron microscopy (EFTEM), the use of 3D chemical mapping has not been widely adopted by the electron microscopy community. The lack of specialized software further complicates the issue, especially in the case of data with a low signal-to-noise ratio (SNR). Moreover, data interpretation is rendered difficult by the absence of efficient segmentation tools. Thus, specialized software for the computation of 3D maps by EFTEM needs to include optimized methods for image series alignment, algorithms to improve SNR, different background subtraction models, and methods to facilitate map segmentation. Here we present a software package (EFTEM-TomoJ, which can be downloaded from http://u759.curie.fr/fr/download/softwares/EFTEM-TomoJ), specifically dedicated to computation of EFTEM 3D chemical maps including noise filtering by image reconstitution based on multivariate statistical analysis. We also present an algorithm named BgART (for background removing algebraic reconstruction technique) allowing the discrimination between background and signal and improving the reconstructed volume in an iterative way.

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

Rheumatoid arthritis is a chronic, systemic inflammatory illness that predominantly affects the joints. Recent works tested inductor agents of arthritis models and their effects when associated to low intensity laser therapy. In this work the effects of the biological agent Infliximab and low intensity laser therapy were investigated in an animal experimental model of Zymosan induced arthritis. This model is widely used in pharmacological studies because it enhances the hypernociception of inflammation, detected throughout the observation of joint articular incapacitation, increase of vascular permeability, release of inflammatory mediators in the joint space and histological changes.

A laser instrument (GaAlAs) DMC brand, model Photon Laser III was used, and the radiation was applied at wavelength 808 nm, power density of 200 mW/cm2, energy density of 2000 mJ/cm2, dose of energy or fluence 2 J/cm2, spot area of 0.02 mm, length of 5 dots per second. Rats with 250g were separated in 5 groups; the control (GI) received intra-articular 50µl of 0.9% saline solution in the right knee. The experimental group GII received intra articular injections of Zymosan (AZy) in the right knee at day zero (1mg, in 50µl of saline solution 0.9%); group GIII received daily doses of the same solution in the medial face of the right knee and 10 applications of laser (904 nm); Group GIV received 3 doses of 1,6ml of endovenous Infliximab in the vein of tail; Group GV received 3 doses of infliximab and daily doses of laser (904 nm). In the 14th day of the experiment the group GII was not treated. The organisms were sacrificed 24 hours after each protocol was completed and submitted to histopathological analysis using standard histological procedures.

Results showed that, with 14 days of AZy, the groups presented a moderate to intense inflammation, presence of giant cell granulomas and synovial hyperplasia. When treatment was associated with laser therapy the inflammation was substantially reduced. The results suggest the efficiency of laser therapy associated or not with the biological agent application, to the treatment of induced chronical synovitis.