Background

Networks of non-toxic amyloid fibrils assembled from common globular proteins (Jung et al., 2008; Lara et al., 2011) adsorbed to solid supports have applications in a wide variety of fields (Dharmadana et al., 2017; Wei et al., 2017). Particularly interesting is their applications in eukaryotic cell culture and biomaterials in general (Reynolds et al., 2013, 2014 and 2015; Gilbert et al., 2017b). This is largely due to the fact that networks of amyloid fibrils have morphologies and mechanical properties that closely resemble the local microenvironment of many cell types (the ECM). Such amyloid fibril networks have the added attraction that they are simple to fabricate, inexpensive and possess well-defined chemistries that can be easily reproduced.

As expected, the response of cells grown on these amyloid fibril networks is highly dependent on the nanoscale properties of the fibril network itself. For instance, small changes in fibril diameter, nanoscale roughness, surface coverage and fibril morphology have been shown to affect cell attachment and spreading (Reynolds et al., 2014 and 2015). Thus, it is important to accurately characterize the nanotopography and surface roughness of the immobilized networks before using them for cell culture applications. AFM is a powerful technique to perform this analysis as it requires little sample preparation, possesses nanoscale lateral resolution and sub-nanometer vertical resolution (Reynolds et al., 2014 and 2015; Gilbert et al., 2017a and 2017b; Reynolds et al., 2017). Additionally, parameters such as nanoscale roughness can be extracted by post imaging analysis. In this protocol, we will describe the process of imaging a dense network of amyloid fibrils (fabricated from the protein Hen Egg White Lysozyme) on solid (mica) substrates by AFM. We will also describe the most common steps of post-processing analysis, namely flattening (removal of sample tilt and bowing artefacts) and roughness analysis.