The application of scanning near field optical imaging to the study of human sperm morphology

BackgroundThe morphology of spermatozoa is a fundamental aspect to consider in fertilization, sperm pathology, assisted reproduction and contraception. Head, neck, midpiece, principal and terminal part of flagellum are the main sperm components to investigate for identifying morphological features and related anomalies. Recently, scanning near-field optical microscopy (SNOM), which belongs to the wide family of nanoscopic techniques, has opened up new routes for the investigation of biological systems. SNOM is the only technique able to provide simultaneously highly resolved topography and optical images with a resolution beyond the diffraction limit, typical of conventional optical microscopy. This offers the advantage to obtain complementary information about cell surface and cytoplasmatic structures.ResultsIn this work human spermatozoa both healthy and with morphological anomalies are analyzed by SNOM, to demonstrate the potentiality of such approach in the visualization of sperm morphological details. The combination of SNOM topography with optical (reflection and transmission) images enables to examine typical topographic features of spermatozoa together with underlying cytoplasmic structures. Indeed the head shape and inner components as acrosome and nucleus, and the organization of mitochondria in the midpiece region are observed. Analogously for principal tract of the tail, the ridges and the columns are detected in the SNOM topography, while their internal arrangement can be observed in the corresponding SNOM optical transmission images, without requiring specific staining procedures or invasive protocols.ConclusionsSuch findings demonstrate that SNOM represents a versatile and powerful tool to describe topographical and inner structural details of spermatozoa simultaneously. This analysis could be helpful for better characterizing several morphological anomalies, often related to sperm infertility, which cannot be examined by conventional techniques all together

Mapping of FcεRI immunoglobulin E receptor in activated mast cells by scanning near-field optical microscopy

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

The interaction of asbestos fibres with human mesothelial cells: a combined investigation exploiting microscopic and nanoscopic techniques

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

Goodbye Hartmann trial: a prospective, international, multicenter, observational study on the current use of a surgical procedure developed a century ago

Background: Literature suggests colonic resection and primary anastomosis (RPA) instead of Hartmann's procedure (HP) for the treatment of left-sided colonic emergencies. We aim to evaluate the surgical options globally used to treat patients with acute left-sided colonic emergencies and the factors that leading to the choice of treatment, comparing HP and RPA. Methods: This is a prospective, international, multicenter, observational study registered on ClinicalTrials.gov. A total 1215 patients with left-sided colonic emergencies who required surgery were included from 204 centers during the period of March 1, 2020, to May 31, 2020. with a 1-year follow-up. Results: 564 patients (43.1%) were females. The mean age was 65.9 ± 15.6 years. HP was performed in 697 (57.3%) patients and RPA in 384 (31.6%) cases. Complicated acute diverticulitis was the most common cause of left-sided colonic emergencies (40.2%), followed by colorectal malignancy (36.6%). Severe complications (Clavien-Dindo ≥ 3b) were higher in the HP group (P < 0.001). 30-day mortality was higher in HP patients (13.7%), especially in case of bowel perforation and diffused peritonitis. 1-year follow-up showed no differences on ostomy reversal rate between HP and RPA. (P = 0.127). A backward likelihood logistic regression model showed that RPA was preferred in younger patients, having low ASA score (≤ 3), in case of large bowel obstruction, absence of colonic ischemia, longer time from admission to surgery, operating early at the day working hours, by a surgeon who performed more than 50 colorectal resections. Conclusions: After 100 years since the first Hartmann's procedure, HP remains the most common treatment for left-sided colorectal emergencies. Treatment's choice depends on patient characteristics, the time of surgery and the experience of the surgeon. RPA should be considered as the gold standard for surgery, with HP being an exception

Effect of methylene blue photodynamic therapy on human neutrophil functional responses.

Photodynamic therapy (PDT) has become an emerging novel therapeutic approach for treating localized microbial infections, particularly those sustained by multidrug-resistant strains. Given the irreplaceable role played by professional phagocytes in limiting infections, such as polymorphonuclear neutrophils, any newly designed antimicrobial therapeutic approach must not interfere with their function. The present investigation presents a detailed analysis of the effect of PDT on the viability and several functional responses of human polymorphonuclear neutrophils loaded with methylene blue (MB), one of the more commonly used photosensitizers in antimicrobial PDT. Taking advantage of the use of a specifically-designed optical LED array for illuminating MBloaded human polymorphonuclear neutrophils, a number of cell functions have been assayed under miniaturized, strictly controlled and reproducible experimental conditions. The major findings of this study are the following: (1) MB-PDT increases human neutrophils adhesion and does not modify myeloperoxidase release; (2) MB-PDT markedly enhances reactive oxygen species generation that is independent of superoxide-forming phagocytic oxidase and very likely ascribable to LED-dependent excitation of accumulated methylene blue; (3) MB-PDT almost abolishes human neutrophils candidacidal activity by hindering the engulfing machinery. This in vitro study may represent a valuable reference point for future research on PDT applications for treating localized microbial infections. 1. Introduction Two main therapeutic approaches are nowadays available to counteract infectious diseases, i.e. (i) eliminate the microbes causing the infection and, (ii) potentiate the immune response of the affected patient. In some instances, due to the pressing need for quickly containing the ongoing infection, both therapeutic interventions are adopted a

Représentations sociales de la laïcité et leurs relations avec la dominance sociale etl'expression des préjugés

13e Conférence Internationale sur les Représentations Sociales : Epistémologies de la vie quotidienne, Marseille, FRANCE, 14-/09/2016 - 17/09/2016Laïcité is the political principle ensuring secularity of the French State through its neutral stance towards religion. This allows for freedom of religion in France. However, historians (Baubérot 2010, 2014) have noticed a shift in the understanding and use of Laïcité among both politicians and the public. They called this new version of Laïcité 'Counterfeited Laïcité' (CL). Now, individuals are expected to become laïc, and religion is progressively excluded from the public sphere. Social psychological investigations have demonstrated that CL is correlated with expression of prejudice towards people from north african descent (Kamiejski & al., 2012; Nugier & al., 2016), which indicates that CL may serve as a legitimizing myth (Sidanius & Pratto, 1994). Nevertheless, these studies use scales that were developed on an expert understanding of Laïcité, and links with Social Dominance were not found. Therefore, we sought to 1) investigate Representations of Laïcité (RoL) among the french population 2) use this content to create a scale for measuring RoL 3) explore the links between RoL, Social Dominance and expression of prejudice. Results show that a) RoL is made of 7 core themes (including the State, school, freedom, religion...) b) RoL is a 3 dimensional construct (KMO=.82; R²=.63, ± =.73) including CL, Rejection of Religion and Republican Laïcité c) RoL's subscales correlate differently with expression of prejudice, and mediation analyses using boostrap methods revealed significant partial mediations of RoL subscales effects on expression of prejudice by Social Dominance Orientation (pLa Laïcité est le principe politique qui assure la position de neutralité de l'Etat français envers la religion et qui permet la liberté de culte en France. Cependant, les historiens (Baubérot 2010, 2014) ont noté un changement dans l'appréciation et l'utilisation de la Laïcité parmi les politiciens et le public. Ils ont appelé cette nouvelle conception de la Laïcité "Laïcité Falsifiée" (LF). Dès lors, il est attendu des individus qu'ils deviennent laïcs, et la religion se trouve progressivement exclue de la sphère publique. Les études psycho-sociales ont démontré que la LF est corrélée à l'expression de préjugés envers les personnes d'origine nord-africaine (Kamiejski & al., 2012; Nugier & al., 2016), ce qui indique que LF pourrait servir de mythe légitimateur (Sidanius & Pratto, 1994). Néanmoins, ces études utilisent des échelles qui ont été développées à partir d'un savoir expert de la Laïcité, et les liens avec l'Orientation à la Dominance Sociale (ODS) n'ont pas été trouÎs. Par conséquent, nous avons cherché 1) à étudier les Représentations de la Laïcité (RdL) parmi la population française 2) utiliser ce contenu pour créer une échelle permettant d'appréhender les RdL 3) explorer les liens entre RdL, ODS et expression de préjugés. Les résultats montrent que, a) la RdL est composé de 7 thèmes centraux (incluant l'État, l'école, la liberté, la religion ...), b) la RdL est un construit tridimensionnelle (KMO = 0,82; R² = 0,63, ± = 73) incluant la LF, le rejet des religions et la Laïcité républicaine, c) Les sous-échelles de la RdL corrèlent différemment avec l'expression des préjugés, et les analyses de médiation en boostrap ont réÎlé des médiations partielles des effets des sous-échelles de la RdL sur l'expression des préjugés par l'ODS (