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

Abstract

SummaryIn 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.

Iron-related toxicity of single-walled carbon nanotubes and crocidolite fibres in human mesothelial cells investigated by Synchrotron XRF microscopy

“…After a careful analysis we have noticed that the iron-rich regions (shown in Fe map) are in proximity of these vesicles, which seem to be a cellular response to the nanomaterial toxicity. These morphological observations are in line with previous reports 55 .…”

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

“…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.…”

“…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.…”

Xenopus laevisOocytes as a Model System for Studying the Interaction Between Asbestos Fibres and Cell Membranes

“…Asbestos fibres can also be found free in the cytosol of macrophages (Palomä ki et al, 2011) or within the phagosomes, protruding from them into the cytosol (Blake et al, 2007;Johnson and Davies, 1983), but they were also found in the nuclear compartment, suggesting a passive piercing (Johnson and Davies, 1983;Malorni et al, 1990). Other findings obtained by studying carcinoma and mesothelial cells in culture supported this possibility (Andolfi et al, 2013;Malorni et al, 1990), even if in the latter case, the fibre entry into the cell appears to be dependent on the phagocytic process (Liu et al, 2000).…”

“…The procedure to analyze the samples by scanning electron microscope (SEM) was previously described elsewhere (Andolfi et al, 2013). Control and treated oocytes were fixed with 2.5% glutaraldehyde (Serva, Heidelberg, Germany) in Ringer's solution at room temperature for 20 min, rinsed in Ringer, and postfixed in 1% osmium tetroxide in PBS for 30 min.…”