Resolving physical interactions between bacteria and nanotopographies with focused ion beam scanning electron microscopy

The nanostructures found on cicada and dragonfly wings have been shown to mediate antibacterial effects, which have since been reproduced on many synthetic materials, providing candidates for anti-infective surfaces. To robustly assess the antibacterial mechanisms of synthetic nanotopographies, it is critical to analyse the bacteria-nanotopography adhesion interface. Here, we utilize focused ion beam milling combined with scanning electron microscopy to generate three-dimensional reconstructions of whole bacteria (Staphylococcus aureus and Escherichia coli), enabling bacteria-nanotopography interactions to be resolved with nanometer resolution, from any perspective. For the first time, 3D morphometric analysis has been exploited to quantify the intrinsic contact area between each nanostructure and the bacterial envelope. This revealed that nanotopography density is crucial for bacterial envelope penetration and cell impedance. Surfaces with nanostructure densities between 36-58 per µm2 and tip diameters between 27-50 nm mediated envelope deformation and penetration, while surfaces with higher nanostructure densities (137 per µm2) induced envelope penetration and mechanical rupture, leading to marked reductions in cell volume due to cytosolic leakage. On nanotopographies with densities of 8 per µm2 ¬and tip diameters greater than 100 nm, bacteria predominantly adhered between nanostructures, resulting in cell impedance. The FIB-SEM method shown here allows multiple aspects of bacteria-nanotopography interactions to be quantified, such as the proportion of nanostructures penetrating the bacterial envelope, the effective contact surface area for each interaction and changes in cell dimension. In turn, these metrics provide an objective framework from which to derive the possible mechanisms driving the antibacterial activity of synthetic nanotopographies.