Unveiling the structure of protein-based hydrogels by overcoming cryo-SEM sample preparation challenges
Dimitra Katrantzi, Stuart Micklethwaite, Nicole Hondow, Andy Brown, Lorna Dougan
Abstract
gelation, high pressure freezing (HPF), plasma focused ion beam (pFIB) milling, sublimation, and low dose secondary electron imaging. Cryo-SEM of folded BSA protein hydrogels prepared in this way reveals a heterogeneous network with nanoscale porosity (∼60 nm pores) surrounded by high secondary electron emission regions (∼30 nm diameter) interconnected by narrower, lower emission regions (∼20 nm length). This heterogeneous network structure is consistent with small angle scattering studies of folded protein hydrogels, with fractal-like clusters connected by intercluster regions. We further test the potential of cryo-SEM to detect the impact of protein unfolding on hydrogel network formation and reveal nanoscale differences in cluster sizes consistent with those derived from scattering data. Importantly, cryo-SEM directly images pores for sizing in both systems, with initial results on BSA suggesting protein unfolding induces an increase of ∼10 nm in pore sizes. Our findings on cryo-SEM sample preparation challenges and solutions provide new opportunities to link hydrogel structure to function.