“…In this case the SNOM reflection image (Figure 6 B) does not highlight particular differences in optical contrast. These structures may be positioned in the tail likely more deeply than 50–100 nm below the cell membrane, and consequently undetectable by reflection, as already described for SNOM reflection imaging [ 39 ]. Figure 6 SNOM image of the principal part of the human sperm tail.…”
Section: Resultsmentioning
confidence: 85%
“…These detectors generate SNOM reflection and transmission images simultaneously with high optical resolution in xy axes (<100 nm) higher than conventional optical microscopies (i.e. confocal, phase contrast, differential interference contrast) [ 39 , 40 ]. As result, SNOM allows us to detect simultaneously different signals: Topography: it is an accurate real three-dimensional image of the sample surface.…”
Section: Resultsmentioning
confidence: 99%
“…As result, subdiffraction resolution optical images (reflection, transmission, back-reflection and fluorescence) and high-resolution (nanometer level) topographic image are simultaneously generated. Such technique has been successfully applied for the investigation of cell membranes at molecular level [ 35 - 38 ], subcellular structures [ 39 , 40 ], thin-sections [ 41 ] and chromosomes [ 42 ]. In the present work, we exploit the advantage of SNOM technique to describe simultaneously different superficial and internal morphological details of human spermatozoa from normozoospermia and teratozoospermia, without needing invasive or expensive sample preparation.…”
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-014-0061-5) contains supplementary material, which is available to authorized users.
“…In this case the SNOM reflection image (Figure 6 B) does not highlight particular differences in optical contrast. These structures may be positioned in the tail likely more deeply than 50–100 nm below the cell membrane, and consequently undetectable by reflection, as already described for SNOM reflection imaging [ 39 ]. Figure 6 SNOM image of the principal part of the human sperm tail.…”
Section: Resultsmentioning
confidence: 85%
“…These detectors generate SNOM reflection and transmission images simultaneously with high optical resolution in xy axes (<100 nm) higher than conventional optical microscopies (i.e. confocal, phase contrast, differential interference contrast) [ 39 , 40 ]. As result, SNOM allows us to detect simultaneously different signals: Topography: it is an accurate real three-dimensional image of the sample surface.…”
Section: Resultsmentioning
confidence: 99%
“…As result, subdiffraction resolution optical images (reflection, transmission, back-reflection and fluorescence) and high-resolution (nanometer level) topographic image are simultaneously generated. Such technique has been successfully applied for the investigation of cell membranes at molecular level [ 35 - 38 ], subcellular structures [ 39 , 40 ], thin-sections [ 41 ] and chromosomes [ 42 ]. In the present work, we exploit the advantage of SNOM technique to describe simultaneously different superficial and internal morphological details of human spermatozoa from normozoospermia and teratozoospermia, without needing invasive or expensive sample preparation.…”
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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12951-014-0061-5) contains supplementary material, which is available to authorized users.
“…Trevisan et al have explored the cell membrane of oligodentrocytes (ODC) and their underlying microtubules by SNOM [180]. The authors have set up a new method to visualize peculiar elements of the ODC cytoskeleton, using˛-tubulin antibodies and DAB (3-3,3-diaminobenzidine tetrahydrochloride)-based immunocytochemistry technique.…”
New-generation biomaterials are information-rich and incorporate biologically active components derived from Nature. The chapter reviews current approaches for the realization of biomimetic coatings for tissue engineering applications, via functionalization with bioactive molecules such as native long chains of extracellular matrix (ECM) proteins as well as short peptide sequences derived from intact ECM proteins. Biomimetic materials provide cues for cell-matrix interactions, favouring cell adhesion, proliferation, spreading, and differentiation. The recently developed scanning probe techniques for optical and spectroscopic characterization of surfaces are also described, providing advanced topographical imaging and sensitive force measurements. The spectroscopic imaging of surfaces at the nanoscale provides insight into the function and structure of biomimetic molecules and represents a tool for tailoring the surface design.
“…cells . However, the TEM needs an extensive sample preparation, as well as the SEM analysis demands invasive protocols, like strong sample dehydration and metal coating . Moreover, the low speed and high costs are additional limitations of electron microscopy techniques to test biological specimens, including live cells.…”
The scanning near-field optical microscopy (SNOM) shows a potential to study details of biological samples, since it provides the optical images of objects with nanometric spatial resolution (50-200 nm) and the topographic information at the same time. The goal of this work is to demonstrate the capabilities of SNOM in transmission configuration to study human endothelial cells and their morphological changes, sometimes very subtle, upon inflammation. Various sample preparations were tested for SNOM measurements and promising results are collected to show: 1) the influence of α tumor necrosis factor (TNF-α) on EA.hy 926 cells (measurements of the fixed cells); 2) high resolution images of various endothelial cell lines, i.e. EA.hy 926 and HLMVEC (investigations of the fixed cells in buffer environment); 3) imaging of live endothelial cells in physiological buffers. The study demonstrate complementarity of the SNOM measurements performed in air and in liquid environments, on fixed as well as on living cells. Furthermore, it is proved that the SNOM is a very useful method for analysis of cellular morphology and topography. Changes in the cell shape and nucleus size, which are the symptoms of inflammatory reaction, were noticed in TNF-α activated EA.hy 926 cells. The cellular structures of submicron size were observed in high resolution optical images of cells from EA.hy 926 and HLMVEC lines.
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