Stefano Prato

SNOM on cell thin sections: observation of Jurkat and MDAMB453 cells

In this study a comparison of scanning near‐field optical microscopy with a traditional, well‐known microscopic technique like transmission electron microscopy is discussed. To establish a reliable and comparable method for high‐resolution scanning near‐field optical microscopy imaging of biological samples, the attention is focussed on cell sections. In particular, we present a study of ultrathin sections of Jurkat T‐cells and MDAMB453 cells. We show the relationship among the scanning near‐field optical microscopy (topographic and optical) images and the kind of embedding medium (resin), the sections thickness and the staining of the sample. For a complementary investigation atomic force microscopy measurements are carried out, as well. The study reveals that scanning near‐field optical microscopy technique on opportunely prepared thin sections can be applied successfully for investigation of the interior of the cells. Scanning near‐field optical microscopy and transmission electron microscopy allow to obtain different, however comparable, and complementary information of the cell sample.

Conductive atomic force microscopy of InAs∕GaAs quantum rings

The properties of self-assembled InAs∕GaAs quantum rings are investigated by conductive atomic force microscopy. Our two-dimensional current maps and current-voltage curves show a lower conductivity of the central ring hole as compared to rim and surrounding planar region. This result is quite surprising if we take into account the compositional profile of quantum rings: being the region with the highest In concentration, one would expect the central hole to be the region with the highest conductivity. However, including the presence of a surface oxide into numerical simulations yields consistent results, which show the same qualitative behavior as the measured conductivities.

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.

The crocidolite fibres interaction with human mesothelial cells as investigated by combining electron microscopy, atomic force and scanning near-field optical microscopy

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.

Novel approaches for scanning near-field optical microscopy imaging of oligodendrocytes in culture.

Newborn rat oligodendrocyte cultures were investigated by scanning near-field optical microscope (SNOM), a versatile new tool able to map cell membranes in 3D and simultaneously obtain images of the cytoplasm. Topography, error, transmission and reflection signals were acquired to describe cell morphology with nanometer-scale resolution. Oligodendrocytes were studied as a model because their extensive membrane processes (typical of their physiological role in myelination) made them particularly suitable to test the sensitivity of the new method. Furthermore, we combined a classical histochemical method with SNOM, to identify specific intracellular proteins at high definition. In particular, with this technique, cytoskeleton elements of oligodendrocytes, such as microtubules, were observed with tubulin antibodies. Images obtained with SNOM were also compared with those from conventional scanning electron microscopy (SEM) and optical microscopy. Our results showed that SNOM allowed to observe cell nanostructures otherwise undetectable all together with other microscopies. In conclusion, SNOM, combined with rapid and non-invasive methods of specimen preparation, appears to be a powerful tool that can offer new possibilities in the field of neuroscience imaging at nano-scale level.

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 FceRI 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-FceRI 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 FceRI 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 FceRI 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.

SNOM fluorescence techniques applied to cytoskeleton study in cell cultures

Introduction Scanning near field optical microscope (SNOM) is a tool which overcomes the diffraction limit of conventional optical microscopy and allows optical imaging with a resolution of less than 100 nm without having to go through any special sample preparation. Moreover one of the best advantages of SNOM is to produce topographic, transmission and reflection images simultaneously [1]. Several studies have been carried out by SNOM in the last years on biological specimens, but the fluorescence SNOM technique has been rarely applied on cell culture investigations. Here we set up a TriA-SNOM with a specific fluorescence equipment to develop the performance of this probe microscope and identify cytoskeleton elements in cell cultures, mainly primary rat podocytes cultures. Materials and methods The microscope used were a TriA-SNOM (APE Research, Trieste, Italy) equipped with a specific fluorescence set-up that allowed to obtain different SNOM images of the samples simultaneously: topography, SNOM fluorescence in reflection and SNOM-fluorescence in transmission. Cell cultures: Primary culture of rat podocytes were treated by immunofluorescence techniques with TRITC-phalloidin to visualize cytoskeletal elements such as actin filaments. Actin filaments were analyzed by SNOM-fluorescence and compared with conventional fluorescence. Results SNOMfluorescence, unlike all other techniques, may reconstruct a fluorescence mapping of the sample, and obtain a direct measure in height of the fluorescence structures. The major technical problem in this kind of microscopy is to reduce of the optical background noise. We actually obtained a SNOM-fluorescence set-up to adequately visualize the specimens, producing images with low background noise and high resolution. SNOM moreover allowed to discriminate the superficial actin filaments from the deep filaments by reflection system. Conclusions SNOM with fluorescence technique may be suitable method for detection of specific molecules in cell cultures, providing an overlapping of the fluorescence image on the 3D mapping of the sample. These studies open further possibilities for application of SNOM to biological materials, especially in cells observed in liquid environment.