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

Area of Science:

• Materials Science
• Biotechnology
• Microscopy

Background:

• Protein-based hydrogels are crucial for drug delivery and tissue engineering.
• Understanding their hierarchical structure is vital but challenging due to protein sensitivity and high water content.
• Cryo-scanning electron microscopy (cryo-SEM) offers potential for visualizing hydrated hydrogel structures.

Purpose of the Study:

• To optimize cryo-scanning electron microscopy (cryo-SEM) sample preparation for protein hydrogels.
• To investigate the hierarchical structure of bovine serum albumin (BSA) hydrogels using cryo-SEM.
• To assess the impact of protein unfolding on hydrogel network formation and pore size.

Main Methods:

• Photochemically cross-linked, folded globular bovine serum albumin (BSA) protein hydrogels were prepared.
• Optimized cryo-SEM preparation involved in situ gelation, high pressure freezing (HPF), plasma focused ion beam (pFIB) milling, sublimation, and low dose imaging.
• Cryo-SEM was used to image both folded and unfolded BSA hydrogels.

Main Results:

• Optimized cryo-SEM preparation minimized artefacts in hydrated protein hydrogels.
• Cryo-SEM revealed a heterogeneous network structure in BSA hydrogels with nanoscale porosity (∼60 nm pores).
• Protein unfolding was observed to increase pore sizes by approximately 10 nm, consistent with scattering data.

Conclusions:

• Advanced cryo-SEM techniques enable detailed visualization of protein hydrogel hierarchical structures.
• The study provides a method to link protein hydrogel structure, including the effects of unfolding, to function.
• This approach opens new avenues for designing and characterizing protein-based biomaterials.