Preparation artefacts mask the detectability of crosslinking-induced structural changes in electron microscopy of...

Collagen hydrogels are widely used as biomimetic three-dimensional matrices whose mechanical behaviour depends strongly on fibrillar micro- and nanoarchitecture and crosslinking state. Electron microscopy offers high spatial resolution for structural analysis, but sample preparation can substantially distort highly hydrated collagen networks, thereby confounding interpretation. Here, we compared common SEM and TEM preparation workflows for their ability to preserve structurally subtle, mechanically validated differences in collagen type I hydrogels. Collagen hydrogels containing riboflavin were photochemically crosslinked by blue-light exposure, and the resulting increase in bulk stiffness was first confirmed by rotational rheometry. Samples were then prepared using various workflows for SEM and TEM analysis based on classical glutaraldehyde fixation or high-pressure freezing. As control experiments, we performed nitrogen plunge freezing to visualise the effect of ice crystal formation, and high-pressure freezing with ethanol cryoprotection to rule out the influence of ice crystal artefacts. The results show that sample preparation-induced artefacts strongly influence structural interpretation in electron microscopy of collagen hydrogels. The methodology based on glutaraldehyde fixation altered network morphology, while the nitrogen plunge freezing based process introduced severe freezing artefacts attributed to ice crystal formation, obscuring the subtle pore-size changes associated with riboflavin/blue-light crosslinking. The high-pressure freezing based process with and without cryoprotectant retained the ability to resolve crosslinking-associated structural differences in these soft collagen hydrogels. This indicates that high-pressure frozen samples of the collagen hydrogels used in this study are already free of visible ice crystal damage and that the use of additional cryoprotectants with unknown structural effects is unnecessary. More broadly, the study supports combining mechanical validation with electron microscopy when assessing soft hydrogel architectures to minimise the potential to interpret preparation-based artefacts as structural features.