Background

Nematodes, both free-living and parasitic, have been extensively studied due to their remarkable biology and effects on agriculture and human well-being. Apparently, the most studied and famous species among nematodes is a free-living soil nematode Caenorhabditis elegans (Sterken et al., 2015). This round worm has been successfully employed as a versatile model organism in a number of investigations (Fire et al., 1998; Kenyon, 2010; Swierczek et al., 2011; O’Reilly et al., 2014; Stroustrup et al., 2016), securing eventually a Nobel prize for Sidney Brenner, John Sulston and Robert Horvitz in 2002. C. elegans nematode is a tiny (~1 mm long) transparent animal which can be easily visualised using an optical microscope. Its cuticle, though thin and transparent, serves as a principal protective barrier between the worm and its habitat. Cuticle is an important marker of disease, when pathogenic bacteria colonize it, leading to the death of the infected animal. In addition, the structure of the cuticle may indicate the ageing in the nematodes. From the human health care and industrial point of view, monitoring of the cuticle of nematodes might be helpful in elucidation of novel antinematode chemicals affecting the cuticle. As a result, nanoscale imaging of the nematode epicuticle in its natural liquid environments may open new avenues in biomedical research.

Previously, the cuticle of nematodes (mostly employing C. elegans as a model organism) has been studied using electron microscopy, both transmission and scanning. Unfortunately, electron microscopy does not allow imaging the worms in liquid, which is their native environment. Sample preparation for electron microscopy requires fixation, dehydration, contrast staining or surface sputtering, and also resin embedding for ultrathin slicing. As a result, nematode cuticle may exhibit secondary artifacts preventing from evaluation of native surface structure and mechanical characterisation. Atomic force microscopy has been established as a potent tool in imaging biological samples in situ, including imaging live cells in liquid media (Beaussart et al., 2015). Recently, the application of tapping mode AFM to visualise C. elegans nematodes in air has been reported (Allen et al., 2015). Imaging in air suffered from the same drawback as scanning electron microscopy, for example the images demonstrated the typical shrunk and collapsed cuticle surfaces apparently caused by dehydration and air imaging. We envisaged a different technique, based on PeakForce Tapping AFM mode (Alsteens et al., 2012) for successful visualisation of C. elegans nematode cuticle in water (Fakhrullina et al., 2017). Here we report a detailed protocol for this technique.