Unveiling the structure of protein-based hydrogels by overcoming cryo-SEM sample preparation challenges

Abstract

Protein-based hydrogels have gained significant attention for their potential use in applications such as drug delivery and tissue engineering. Their internal structure is complex, spans across multiple length scales and affects their functionality, yet is not well understood because of folded proteins sensitivity to physical and chemical perturbations and the high water content of hydrogels. Cryo-scanning electron microscopy (cryo-SEM) has the potential to reveal such hierarchical structure when hydrated hydrogels are prepared with appropriate cryofixation. We show for photochemically cross-linked, folded globular bovine serum albumin (BSA) protein hydrogels that preparation artefacts are reduced by in-situ 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.