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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

Introduction. The exposure to asbestos fibres is associated with the development of severe diseases such as lung cancer and pleural mesothelioma. The interaction mechanism of these fibres with the mesothelial cells is still debated.(1) This work aims at obtaining information about the interaction of crocidolite fibres with mesothelial cells, for a better understanding of the processes that trigger cell transformation. For this reason we combine optical microscopy and SEM, with nanoscopic techniques as near-field optical (SNOM) and atomic force microscopy (AFM). These two latter techniques, thanks to their high sensitivity and non-invasiveness, are suitable for investigating phenomena occurring at the cell membrane with nanometric resolution.(2) In addition, SNOM provides simultaneous topography and optical image with a resolution beyond the light diffraction limit. This allows a direct coupling of the morphological features with the optical properties of the sample. Materials and Methods. Mesothelial cell line (MET5A from ATCC) are grown in RPMI with FCS 10%, 2 mM glutamine. Cells are exposed to 5µg/cm2 crocidolite for 3, 6 or 12 h. For optical microscopy cells are stained with Diff-Quick. The samples after fixation with PFA 4% are prepared for SEM, SNOM and AFM observations that are carried out by using a Leica Stereoscan 430i, a A-100 AFM and TriA-SNOM microscope (A.P.E.Research, Trieste, Italy). Results and Discussion. By analysing the optical data we estimate that fibres are associated with 75% of mesothelial cells. SEM images confirm these results and allow distinguishing that some fibres are on cell surface, while others appears to be clearly inside the cells, in some cases even deforming the cell morphology. A deeper investigation is achieved by SNOM and AFM. By comparing the SNOM topography with the simultaneous transmission and reflection images, we can define the position of the fibres respect to the cell membrane, owing to difference in optical properties between the crocidolite and the cell material. In addition, high-resolution AFM images highlight the entrance site of the nanometre-size fibres at cell membrane. In conclusion the combination of our findings provides an accurate description about the interaction of mesothelial cells with crocidolite fibres having different size. Importantly, SNOM optical images can disclose details about such interaction not observed up to now. 1. Arch. Biochem. Biophys. 2010, 502: 1. 2. J. Cell Sci. 2001, 114: 4153., Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Recent studies have shown that protein kinase C (PKC) plays a pivotal role in many cellular functions, i.e. cell proliferation and differentiation, exocytosis, ion-exchange regulation, hormone and neurotransmitter release, programmed cell death. Up to now the tissue and organ distribution of PKC isoenzymes have been studied in various mammalian adults and it has been suggested that each of them could play a specific role in the regulation of various cellular functions. However, few works have been performed on the expression of the enzyme in actively proliferating and differentiating tissues, i.e. during embryonal and fetal developmental stages. In the present study we have performed an immunohistochemical analysis by using polyclonal antibodies in order to verify the distribution of nine PKC isoenzymes in the vertebral column of human fetuses in a key period (8th developmental week), when the most relevant chondrogenetic and osteogenetic processes occur. The detection of the various PKC isoenzymes and their different distribution in the vertebral bodies and in the intervertebral spaces lead to speculate that the appearance, localization and activation of PKC isoforms in chondrocytes might depend on the stage of chondrogenesis and suggest that cartilage and developing bone represent an appropriate cell system to understand the mechanism of signal transduction, which involves the enzyme.

In this report we describe a simple and rapid staining technique for cartilage and bone embedded in Araldite. Semithin sections of embryonic vertebrae obtained from 15 to 17 day mouse fetuses were stained using an aqueous solution 0.25% with respect to methylene blue, 0.25% with respect to azure A, and 0.5% with respect to Na2 CO3, then counterstained with 1% aqueous pararosaniline chloride (MAP). Results were compared with toluidine blue stained sections. MAP permitted good discrimination of developmental stages of both cells and extracellular matrix within vertebral ossification centers during endochondral ossification. The technique is simple, rapid and applicable to plastic embedded sections, and can be used prior to ultrastructural examination.

This data article contains data related to the research article entitled, “Synchrotron X-ray microscopy reveals early calcium and iron interaction with crocidolite fibers in the lung of exposed mice” [1]. Asbestos fibers disrupt iron homeostasis in the human and mouse lung, leading to the deposition of iron (Fe) onto longer asbestos fibers which forms asbestos bodies (AB) [2]. Similar to Fe, calcium (Ca) is also deposited in the coats of the AB. This article presents data on iron and calcium in the mouse lung after asbestos exposure detected by histochemical evaluation. Keywords: Asbestos, Animal model, Iron, Calcium, Histochemistry, Histopathology

The high cost and environmental impact of fossil-based organic carriers represent a critical bottleneck to their use in large-scale industrial processes. The present study demonstrates the applicability of rice husk as inexpensive renewable carrier for the immobilization of enzymes applicable sectors where the covalent anchorage of the protein is a pre-requisite for preventing protein contamination while assuring the recyclability. Rice husk was oxidized and then functionalized with a di-amino spacer. The morphological characterization shed light on the properties that affect the functionalization processes. Lipase B from Candida antarctica (CaLB) and two commercial asparaginases were immobilized covalently achieving higher immobilization yield than previously reported. All enzymes were immobilized also on commercial epoxy methacrylic resins and the CaLB immobilized on rice husk demonstrated a higher efficiency in the solvent-free polycondensation of dimethylitaconate. CaLB on rice husk appears particularly suitable for applications in highly viscous processes because of the unusual combination of its low density and remarkable mechanical robustness. In the case of the two asparaginases, the biocatalyst immobilized on rice husk performed in aqueous solution at least as efficiently as the enzyme immobilized on methacrylic resins, although the rice husk loaded a lower amount of protein.

Cholesterol metabolism is crucial for cells and, in particular, its biosynthesis in the central nervous system occurs in situ, and its deregulation involves morphological changes that cause functional variations and trigger programmed cell death. The pathogenesis of rare diseases, such as Mevalonate Kinase Deficiency or Smith–Lemli–Opitz Syndrome, arises due to enzymatic defects in the cholesterol metabolic pathways, resulting in a shortage of downstream products. The most severe clinical manifestations of these diseases appear as neurological defects. Expanding the knowledge of this biological mechanism will be useful for identifying potential targets and preventing neuronal damage. Several studies have demonstrated that deregulation of the cholesterol pathway induces mitochondrial dysfunction as the result of respiratory chain damage. We set out to determine whether mitochondrial damage may be prevented by using protective mitochondria-targeted compounds, such as MitoQ, in a neuronal cell line treated with a statin to induce a biochemical block of the cholesterol pathway. Evidence from the literature suggests that mitochondria play a crucial role in the apoptotic mechanism secondary to blocking the cholesterol pathway. Our study shows that MitoQ, administered as a preventive agent, could counteract the cell damage induced by statins in the early stages, but its protective role fades over time.

Carbon nanotubes (CNTs) are promising products in industry and medicine, but there are several human health concerns since their fibrous structure resembles asbestos. The presence of transition metals, mainly iron, in the fibres seems also implicated in the pathogenetic mechanisms. To unravel the role of iron at mesothelial level, we compared the chemical changes induced in MeT-5A cells by the exposure to asbestos (crocidolite) or CNTs at different content of iron impurities (raw-SWCNTs, purified- and highly purified-SWCNTs). We applied synchrotron-based X-Ray Fluorescence (XRF) microscopy and soft X-ray imaging (absorption and phase contrast images) to monitor chemical and morphological changes of the exposed cells. In parallel, we performed a ferritin assay. X-ray microscopy imaging and XRF well localize the crocidolite fibres interacting with cells, as well as the damage-related morphological changes. Differently, CNTs presence could be only partially evinced by low energy XRF through carbon distribution and sometimes iron co-localisation. Compared to controls, the cells treated with raw-SWCNTs and crocidolite fibres showed a severe alteration of iron distribution and content, with concomitant stimulation of ferritin production. Interestingly, highly purified nanotubes did not altered iron metabolism. The data provide new insights for possible CNTs effects at mesothelial/pleural level in humans.

Research question Does synchrotron X-ray fluorescence (XRF) provide novel chemical information for the evaluation of human ovarian tissue cryopreservation protocols? Design Tissues from five patients undergoing laparoscopic surgery for benign gynaecological conditions were fixed for microscopic analysis either immediately or after cryopreservation. After fixation, fresh and slowly frozen samples were selected by light microscopy and transmission electron microscopy, and subsequently analysed with synchrotron XRF microscopy at different incident energies. Results The distributions of elements detected at 7.3 keV (S, P, K, Cl, Fe, and Os) and 1.5 keV (Na and Mg) were related to the changes revealed by light microscopy and transmission electron microscopy analyses. The light elements showed highly informative findings. The S distribution was found to be an indicator of extracellular component changes in the stromal tissues of the freeze–stored samples, further revealed by the transmission electron microscopy analyses. Low-quality follicles, frequent in the freeze–thawed tissues, showed a high Na level in the ooplasm. On the contrary, good-quality follicles were detected by a homogeneous Cl distribution. The occurrence of vacuolated follicles increased after cryopreservation, and the XRF analyses showed that the vacuolar structures contained mainly Cl and Na. Conclusions The study demonstrates that elemental imaging techniques, particularly revealing the distribution of light elements, could be useful in establishing new cryopreservation protocols.

Purpose: To report on the combined use of trypan blue (TB) and brilliant blue G (BBG) for staining the epiretinal membrane (ERM) and internal limiting membrane (ILM) during vitrectomy and to describe the histopathological findings. Methods: 10 surgical specimens were removed from 10 eyes with macular pucker during vitrectomy using a commercially available combination of TB and BBG for ERM and ILM staining and peeling. Specimens were evaluated using light and transmission electron microscopy. Results: In all cases the combination of TB and BBG was useful for identifying and delineating ERM and ILM. No complications related to the use of the dye were observed during or after surgery. Glial cells were present in all specimens. Hyalocytes were observed in 6 cases and myofibroblasts in 3 of them. In 7 cases native vitreous collagen fibrils were found on the ILM, while in 5 specimens newly formed collagen was present. No clinical evidence of toxicity was observed during the 3-month follow-up. Conclusion: The combined use of TB and BBG appeared to be very useful intraoperatively to improve the visualization of ERM and ILM, thus facilitating their complete removal. Anatomical and histopathological findings demonstrated the safety and the efficacy of this vital dye.

La preservazione della fertilità è un ambito della ricerca in continua evoluzione, che permette a soggetti che rischiano di divenire sterili di congelare i loro gameti, per poi nel futuro consentire loro di procreare e divenire genitori.Le tecniche che si usano per preservare la fertilità nelle donne sono il congelamento degli ovociti, il congelamento degli embrioni e, da qualche anno, il congelamento del tessuto ovarico.Mentre le prime due metodiche risultano ormai standardizzate e sono utilizzate quotidianamente nei reparti di Procreazione Medicalmente Assistita, il congelamento del tessuto ovarico rimane ancora una tecnica considerata sperimentale.In questo lavoro di tesi si è inteso confrontare due protocolli, utilizzando i tessuti ovarici donati da nove pazienti seguite dall’I.R.C.S.S. Burlo Garofolo di Trieste, che sono stati congelati in parallelo con il congelamento lento e la vitrificazione. Lo stato di conservazione e gli eventuali danni indotti dalla criopreservazione sono stati valutati mediante microscopia ottica ed elettronica. Dal confronto tra i due protocolli è emerso che entrambi mantengono un’ottima preservazione degli ovociti ed influenzano in parte le cellule della granulosa; è risultato inoltre che alterano in maniera importante le cellule dello stroma.Su parte dei tessuti, alle microscopie tradizionali è stata affiancata una tecnica di microscopia avanzata, la microscopia a fluorescenza a raggi X con luce di sincrontrone (XRF); questa microscopia è stata usata per meglio comprendere da un punto di visto biochimico i danni del tessuto indotti da congelamento.Detta microscopia, che consente la mappatura degli elementi chimici contenuti nel campione, ha evidenziato che, nei campioni di tessuto criopreservato con il congelamento lento, la distribuzione dello zolfo risulta disomogenea e diminuita rispetto a quella dei campioni di tessuto fresco. Il segnale dello zolfo nei campioni istologici di tessuto deriva principalmente dai glicosamminoglicani solforati che compongono la sostanza amorfa del tessuto connettivo. Una distribuzione non uniforme conferma i risultati morfologici e ultrastrutturali che evidenziano una rarefazione del connettivo e la presenza di vacuoli nel tessuto congelato.Un’ulteriore dimostrazione dell’aumentata permeabilità dei tessuti congelati viene dall’osservazione che questi tessuti, quando sono preparati per il TEM, trattengono quantità di osmio molto maggiori (quasi 50 volte) rispetto al tessuto fresco: un effetto rilevato sempre grazie alla microscopia XRF.In questi tesi ci si è occupati anche di preservazione della fertilità del sesso maschile, dove la metodica più utilizzata è la crioconservazione del liquido seminale.Durante il lavoro di tesi è stato effettuato uno studio preliminare per valutare se il danno al DNA degli spermatozoi può essere valutato con la spettroscopica vibrazionale. Con la spettroscopia a infrarossi (FTIR) sono stati analizzati i liquidi seminali capacitati di 10 pazienti, afferenti al reparto di Procreazione Medicalmente Assistita dell’I.R.C.S.S. Burlo Garofolo di Trieste, che sono stati trattati per indurre un danno ossidativo. La FTIR è risultata una tecnica molto promettente per identificare in modo rapido il danno al DNA negli spermatozoi, anche se sono state rilevate delle problematiche legate alle condizioni sperimentali da usare.Infine è stato valutato, con l’arancio di acridina, il danno al DNA indotto dal congelamento e dal danno ossidativo sugli spermatozoi. Dal confronto, fatto sugli stessi campioni, è emerso che il danno rilevabile al 20% di Fenton è simile al danno dopo congelamento/scongelamento. Ci si attende quindi che il danno da congelamento possa essere valutato efficacemente mediante protocolli di spettroscopia vibrazionale FTIR e possa diventare una tecnica per la pratica clinica.

Deregulation of the cholesterol pathway is an anomaly observed in human diseases, many of which have in common neurological involvement and unknown pathogenesis. In this study we have used Mevalonate Kinase Deficiency (MKD) as a disease-model in order to investigate the link between the deregulation of the mevalonate pathway and the consequent neurodegeneration. The blocking of the mevalonate pathway in a neuronal cell line (Daoy), using statins or mevalonate, induced an increase in the expression of the inflammasome gene (NLRP3) and programmed cell death related to mitochondrial dysfunction. The morphology of the mitochondria changed, clearly showing the damage induced by oxidative stress and the decreased membrane potential associated with the alterations of the mitochondrial function. The co-administration of geranylgeraniol (GGOH) reduced the inflammatory marker and the damage of the mitochondria, maintaining its shape and components. Our data allow us to speculate about the mechanism by which isoprenoids are able to rescue the inflammatory marker in neuronal cells, independently from the block of the mevalonate pathway, and about the fact that cell death is mitochondria-related.

Human exposure to asbestos can cause a wide variety of lung diseases that are still a current major health concern, even if asbestos has been banned in many countries. It has been shown in many studies that asbestos fibers, ingested by alveolar macrophages, disrupt lung iron homeostasis by sequestering iron. Calcium can also be deposited on the fibers. The pathways along which iron and above all calcium interact with fibers are still unknown. Our aim was that of investigating if the iron accumulation induced by the inhaled asbestos fibers also involves calcium ions accumulation. Lung sections of asbestos-exposed mice were analyzed using an extremely sensitive procedure available at the synchrotron facilities, that provides morphological and chemical information based on X-ray fluorescence microspectroscopy (μ-XRF). In this study we show that (1) where conventional histochemical procedures revealed only weak deposits of iron and calcium, μ-XRF analysis is able to detect significant deposits of both iron and calcium on the inhaled asbestos fibers; (2) the extent of the deposition of these ions is proportionally directly related and (3) iron and calcium deposition on inhaled asbestos fibers is concomitant with the appearance of inflammatory and hyperplastic reactions.