{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T14:44:43Z","timestamp":1792593883901,"version":"4.5.0"},"reference-count":77,"publisher":"Wiley","license":[{"start":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T00:00:00Z","timestamp":1792540800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T00:00:00Z","timestamp":1792540800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Journal of Microscopy"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Collagen hydrogels are widely used as biomimetic three\u2010dimensional matrices whose mechanical behaviour depends strongly on fibrillar micro\u2010 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\u2010light 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\u2010pressure freezing. As control experiments, we performed nitrogen plunge freezing to visualise the effect of ice crystal formation, and high\u2010pressure freezing with ethanol cryoprotection to rule out the influence of ice crystal artefacts. The results show that sample preparation\u2010induced 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\u2010size changes associated with riboflavin\/blue\u2010light crosslinking. The high\u2010pressure freezing based process with and without cryoprotectant retained the ability to resolve crosslinking\u2010associated structural differences in these soft collagen hydrogels. This indicates that high\u2010pressure 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\u2010based artefacts as structural features.<\/jats:p>","DOI":"10.1111\/jmi.70190","type":"journal-article","created":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T14:28:31Z","timestamp":1792592911000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Preparation artefacts mask the detectability of crosslinking\u2010induced structural changes in electron microscopy of collagen hydrogels"],"prefix":"10.1111","author":[{"given":"Lydia","family":"Rebehn","sequence":"first","affiliation":[{"name":"Institute of Experimental Physics Ulm University  Ulm Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Clarissa","family":"Read","sequence":"additional","affiliation":[{"name":"Central Facility for Electron Microscopy Ulm University  Ulm Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul","family":"Walther","sequence":"additional","affiliation":[{"name":"Central Facility for Electron Microscopy Ulm University  Ulm Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kay\u2010E.","family":"Gottschalk","sequence":"additional","affiliation":[{"name":"Institute of Experimental Physics Ulm University  Ulm Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,10,21]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1016\/0142\u20109612(96)87280\u20102"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biomaterials.2010.07.046"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1002\/pat.70645"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1002\/bit.22361"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1177\/1087057117696795"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1021\/acsami.8b22343"},{"key":"e_1_2_8_8_1","doi-asserted-by":"crossref","unstructured":"Chen Z. 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