In-situ observation of gel-to-gel transition in dipeptide hydrogel using SHG microscopy.

Peptide-based hydrogels formed by FmocFF (9-fluorenylmethoxycarbonyl-diphenylalanine) are established materials in tissue engineering, drug delivery, and bioelectronics. Although initial assembly processes and final states are relatively well known, the possibility of hidden, intermediate gel forms remains underexplored. Such "gel-to-gel" transitions may yield previously unnoticed polymorphs with distinct mechanical and structural features, expanding options for tuning gel functionality. Here, we combine conventional techniques with in-situ second harmonic generation (SHG) microscopy-a label-free, non-invasive technique sensitive to supramolecular chirality-to visualize these transitions in FmocFF/BPE hydrogels directly in their native state. We find that hydrogels initially presumed stable can reorganize into more thermodynamically favored "gelmorphs" when subjected to elevated temperature or increased concentration. This reorganization involves bundling nanoscale fibrils into helical, trigonal-symmetry microfibers with enhanced mechanical stability. Importantly, our novel SHG microscopy method provides unique structural sensitivity-revealing not only the helical pitch but also the local structural symmetry of the fibers in situ, a capability that conventional techniques lack. These results challenge the assumption that the first accessible gel structure is the end state. Instead, peptide hydrogels can traverse multiple metastable forms before settling into more robust configurations. Understanding and controlling these gel-to-gel transitions present a new strategy for directing hierarchical organization and properties in soft materials. Coupled with in-situ imaging, this approach can guide the design of biomimetic scaffolds, anisotropic materials, and responsive gels, advancing the rational engineering of next-generation functional systems.