Exploring the micro-structure of hydrated collagen hydrogels under scanning electron microscopy.

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 micro-structure to material properties and function enables rational design strategies to control this space. Visualisation of the micro-scale organization of these soft hydrated materials presents unique technical challenges due to the relationship between hydration and the molecular organization of a collagen gel. Scanning electron microscopy is a robust tool widely employed to visualize and explore materials on the micro-scale. However, investigation of collagen gel micro-structure 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 micro-structure 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 -6 Pa) and low-vacuum (80 - 120 Pa) 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. This article is protected by copyright. All rights reserved.