Exploring the microstructure of hydrated collagen hydrogels under scanning electron microscopy

Merryweather, Daniel J.; Weston, Nicola; Roe, Jordan; Parmenter, Christopher; Lewis, Mark P.; Roach, Paul

Abstract

Collagen hydrogels are a rapidly expanding platform in bioengineering and soft materials engineering for novel applications focused on medical therapeutics, medical devices and biosensors. Observations linking microstructure to material properties and function enables rational design strategies to control this space. Visualisation of the microscale organisation of these soft hydrated materials presents unique technical challenges due to the relationship between hydration and the molecular organisation of a collagen gel. Scanning electron microscopy is a robust tool widely employed to visualise and explore materials on the microscale. However, investigation of collagen gel microstructure is difficult without imparting structural changes during preparation and/or observation. Electrons are poorly propagated within liquid-phase materials, limiting the ability of electron microscopy to interrogate hydrated gels. Sample preparation techniques to remove water induce artefactual changes in material microstructure particularly in complex materials such as collagen, highlighting a critical need to develop robust material handling protocols for the imaging of collagen hydrogels. Here a collagen hydrogel is fabricated, and the gel state explored under high-vacuum (10−6Pa) and low-vacuum (80–120Pa) conditions, and in an environmental SEM chamber. Visualisation of collagen fibres is found to be dependent on the degree of sample hydration, with higher imaging chamber pressures and humidity resulting in decreased feature fidelity. Reduction of imaging chamber pressure is used to induce evaporation of gel water content, revealing collagen fibres of significantly larger diameter than observed in samples dehydrated prior to imaging. Rapid freezing and cryogenic handling of the gel material is found to retain a porous 3D structure following sublimation of the gel water content. Comparative analysis of collagen hydrogel materials demonstrates the care needed when preparing hydrogel samples for electron microscopy.

Citation

Merryweather, D. J., Weston, N., Roe, J., Parmenter, C., Lewis, M. P., & Roach, P. (2023). Exploring the microstructure of hydrated collagen hydrogels under scanning electron microscopy. Journal of Microscopy, 290(1), 40-52. https://doi.org/10.1111/jmi.13174

• Journal Article Type: Article
• Acceptance Date: Jan 26, 2023
• Online Publication Date: Jan 30, 2023
• Publication Date: 2023-04
• Deposit Date: Apr 12, 2023
• Publicly Available Date: Apr 18, 2023
• Journal: Journal of Microscopy
• Print ISSN: 0022-2720
• Electronic ISSN: 1365-2818
• Publisher: Wiley
• Peer Reviewed: Peer Reviewed
• Volume: 290
• Issue: 1
• Pages: 40-52
• DOI: https://doi.org/10.1111/jmi.13174
• Keywords: Histology; Pathology and Forensic Medicine
• Public URL: https://nottingham-repository.worktribe.com/output/16802146
• Publisher URL: https://onlinelibrary.wiley.com/doi/10.1111/jmi.13174