Modulating Hydrogel Architecture via Cross-Linker Length for High-Resolution Tissue Imaging and Photochemical Sectioning.

The recent development of Volumetric Imaging via Photochemical Sectioning (VIPS) has enabled nanoscale imaging of whole-mount tissue samples of virtually any size by embedding intact tissue in a photocleavable, superabsorbent hydrogel. However, the efficacy of sample embedding, imaging, and photochemical sectioning is fundamentally governed by the mechanical stiffness, structural stability, and photodegradation performance of the photocleavable hydrogel (PC-gel) polymer network. To elucidate the effect of the photosensitive cross-linker design on these critical properties, we synthesized a set of photocleavable cross-linkers (PCs) with varying polyethylene glycol (PEG) backbone lengths and prepared the corresponding PC-gels under a fixed monomer formulation and polymerization condition. We quantified and compared the viscoelastic properties of the formed PC-gels in their swollen states, and found that the cross-linker length markedly reshaped the PC-gel mechanics. In addition, we evaluated the light-triggered degradation of the PC-gels using both wide-field and spatially controlled illumination. We found that PC-1000, PC-1500, and PC-2000 gels remained comparably photodegradable, all enabling on-demand, spatially confined decross-linking under such illumination. These results provide practical guidelines for modulating the cross-linker architecture of PC-gel polymer networks to achieve optimal physicochemical properties for whole-mount tissue imaging using VIPS.