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Polymer-based nanocomposites (PNCs) have gained popularity in the last two decades due to their exciting bulk and surface properties. However, the current bottleneck for the exploitation of the theoretical excellent properties of PNCs is the complete dispersion of the nanoparticles in the matrix and the consequent development of a huge interfacial area. This complete dispersion will allow maximizing the available matrix–particle interphase optimizing then the organic–inorganic interaction, responsible of the enhanced properties of the final material. So, most of the research efforts in this area have been focused on developing rational processing strategies for PNCs and in promoting better matrix–particle interactions. In this scenario, we present our combined experimental/computational approach to the dispersion of modified Fe2O3 nanoparticles in a lamellar PS-PMMA copolymeric matrix. Specifically, we synthesized and characterized several of these PNCs at different nanoparticle concentrations; contextually, multiscale molecular simulations were employed to predict their phase behavior.

This paper presents the design of a Scanning Near-field Optical Microscopy (SNOM) probe for the use with specimen in liquid environment. The approach is based on a first electro-mechanical characterization of existing SNOM probes. A modeling stage has permitted to identify and optimize critical probe parameters in order to improve SNOM sensitivity: development and implementation of SNOM probes for air and in liquid environments have been verified with imaging of both standard and soft samples.

Ce chapitre est organisé autour de la question d’une double responsabilité : d’une part, la responsabilité du personnel de santé d’être informé et compétent sur le sujet de violences masculines envers les femmes (VeF); de l’autre, la responsabilité de ceux/celles qui interviennent dans le social d’évaluer leur interventions, puisque une intervention, même bien construite, pourrait avoir des effets négatifs ou n’avoir pas d’effets du tout. La violence a des sérieuses conséquences sur la santé. Les médecins devraient être en mesure d’aider les victimes, mais ils manquent souvent des connaissances, ou ne se sentent pas concernés : sensibiliser les étudiant-e-s est donc une étape nécessaire. En 2009, un cours sur la VeF a été proposé aux étudiant-e-s en médicine à Trieste (Italie). Pour l’évaluation du cours nous avons utilisé une approche quasi-expérimentale, avec des questionnaires pré et posttest, et avec un groupe expérimental (N=75) et un groupe de contrôle (N=63). Les résultats montrent que, au départ, il n’y avait pas de différences entre les deux groupes concernant connaissances et attitudes relatives à la VeF. Après le cours, les étudiant-e-s du groupe expérimental avaient plus de connaissances, se sentaient plus concernées et présentaient moins de préjugés, alors que aucun changement n’a été observé pour le groupe de contrôle. Les commentaires des étudiant-e-s confirment que la participation au cours a modifié leur perception de la VeF. Tous/toutes ont qualifié ces changements de très positifs; toutefois une minorité dit avoir été mal à l’aise face à ce qu’ils considéraient une approche féministe. Cette étude indique que qu’il est possible de mettre en œuvre une évaluation rigoureuse et que ce cours sur la VeF a eu un impact positif.

In this study, we have performed a morphological analysis of crocidolite fibres interaction with mesothelial cells (MET5A) by combining conventional electron microscopy with atomic force (AFM) and scanning near-field optical microscopy (SNOM). After 6-h exposure at a crocidolite dose of 5 μg cm(-2), 90% of MET5A cells interact with fibres that under these conditions have a low cytotoxic effect. SEM images point out that fibres can be either engulfed by the cells that lose their typical morphology or they can accumulate over or partially inside the cells, which preserve their typical spread morphology. By using AFM we are able to directly visualize the entry-site of nanometric-sized fibres at the plasma membrane of the spread mesothelial cells. More importantly, the crocidolite fibres that are observed to penetrate the plasma membrane in SNOM topography can be simultaneously followed beneath the cell surface in the SNOM optical images. The analysis of SNOM data demonstrates the entrance of crocidolite fibres in proximity of nuclear compartment, as observed also in the TEM images. Our findings indicate that the combination of conventional electron microscopy with novel nanoscopic techniques can be considered a promising approach to achieve a comprehensive morphological description of the interaction between asbestos fibres and mesothelial cells that represents the early event in fibre pathogenesis.

Pulmonary toxicity induced by asbestos is thought to be mediated through redox-cycling of fiber-bound and bioavailable iron (Fe).This impacts an oxidative stress which potentially mediates a cascade of events culminating in inflammation, fibrosis and cancer (Ghio et al, 2008 and 2009). The present study aimed to elucidate in a murine model of asbestos exposure the distribution of ferric and ferrous iron and its relation with the development of fibrosis. C57/Bl/6 mice were exposed to saline or crocidolite asbestos in saline (55 and 110 µg intra-tracheally) and examined at 1 and 6 months for lung pathology. The left lung was embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E) and Masson’s trichrome. A modified Perl’s or Turnbull’s blue histochemical stain was used to demonstrate Fe3+ and Fe2+ respectively (Wang et al, 2002). One month following exposure, mice instilled with crocidolite displayed a significant inflammatory injury on staining with H&E (relative to animal exposed to saline). Crocidolite fibers could be observed most frequently in the region of distal bronchioles. The retention of these fibers was associated with an accumulation of inflammatory cells, predominantly macrophages. This response persisted 6 months following asbestos instillation. Increased collagen was observed at one and 6 months after asbestos exposure. One important observation made in 1-month H&E-stained slides of asbestos-exposed mice was the presence of focal atypical hyperplastic lesions. These lesions did not progress in severity from 1 to 6 months. Histological lung sections from saline exposed mice did not stain for both ferric and ferrous Fe. However, one month following crocidolite instillation, a number of Fe3+-positive (but no Fe2+-positive) cells (predominantly macrophages) were present, in the lower respiratory tract. At 6 months postinjection, an increase number of Fe3+-positive cells was observed and also some Fe2+ positive cells were present. The observation of some Fe2+ positive cells in the lung of asbestos exposed mice at 6 months postinjection suggests a shift in the oxidation state of the accumulated iron, with more cells becoming Fe2+-positive at a late stage. These growing amounts of Fe2+ could support a continuated generation of oxidative stress resulting in clinical manifestations, which culminate in inflammarory, fibrotic and hyperplastic lesions., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

L'applicazione della microscopia SNOM al campo delle bio-nano tecnologie è limitata dalla mancanza di soluzioni tecnologiche che permettano di effettuare misure (topografiche e ottiche) ad alta risoluzione su cellule in condizioni fisiologiche (cellule sia blandamente fissate che in vivo). Con lo sviluppo e la realizzazione di nuovi trasduttori secondari, caratterizzati da elevati fattori di qualità Q ed idonei per le misure in liquido, con specifiche superiori all'attuale stato dell'arte, e una nuova tecnica di controllo della distanza punta campione, sono state ottenute delle immagini SNOM in fase liquida, sia su campioni di riferimento che su campioni biologici blandamente fissati. In questo lavoro vengono presentati i trasduttori sviluppati.

The interaction of asbestos fibers with human mesothelial cells is described by the use of SEM, AFM and SNOM techniques. Particularly SNOM discloses details about such a interaction not observed up to now.

Introduction. The cell membrane has a dynamic role that enables the reorganization of receptors and signal molecules in response to signaling processes. To visualize these phenomena we can nowadays benefit from techniques that allow subdiffraction optical resolution.(1) Among these, scanning near-field optical microscopy (SNOM) exploits the evanescent field exiting at the probe fibre apex. The lateral resolution depends essentially on the subwavelength aperture size of the optical fibre (typically better than 100 nm). This makes SNOM particularly suited for nanoscale study on intact biological membrane. Recently, we demonstrated that SNOM combined with immunolabelling and diaminobenzidine (DAB) staining is a valuable non-invasive approach for investigating nanostructures components within intact oligodendrocites.(2) Here we extend this approach to the study of reorganization of FcεRI immunoglobulin E (IgE) receptor in intact mast cells upon antigen-induced degranulation by IgE cross-linking. Materials and Methods. Rat basophilic leukemia RBL-2H3 cells were grown on coverslips, incubated overnight with anti-2,4-Dinitrophenol (DNP) IgE and degranulated by adding DNP. After 30’ cells were fixed and immunolabelled with anti-FcεRI monoclonal antibody. DAB staining was performed with VECTASTAIN® UNIVERSAL Elite ABC kit. TriA-SNOM microscope (A.P.E. Research, Trieste) was used for near-field measurements. Results and discussions. Resting and activated cells with and without DAB staining immunolabelling for the internal portion of FcεRI are analyzed by SNOM. Topography of activated cells shows membrane ridges over surface and in some cases a considerable cell flattening, in general accordance with the morphology observed as result of the degranulation process. The optical transmission images of DAB stained activated mast cell display numerous very dark circular areas, not observed in the unlabelled activated cells. Such features are likely due to a strong light adsorption for the presence of localized DAB reaction. These areas (lateral size about 300 nm) appear to be in agreement with IgE receptor FcεRI patches observed on cytoplasmatic side of membrane sheets of activated mast cells.(3) In conclusion these results demonstrate that SNOM combined with immunolabelling and DAB staining holds great potential for investigating the organization of proteins into micro- or nanodomains in cell membrane. 1. Bioch. Biophys. Acta 2010, 1798: 77. 2. Neuroimage 2010, 49: 517. 3. Biophys. J. 2006, 90: 2404., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Italian Journal of Anatomy and Embryology, Vol 115, No 1/2 (Supplement) 2010