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.