Abstract Gelatin polymers made from partially degraded collagen are important biomaterials, but their in‐situ analysis suffers from uncontrollable covalent labelling and poor spatial‐temporal imaging resolution. Herein, three tetrazolate‐tagged tetraphenylethylene fluorophores ( TPE−TAs ) are introduced for practical fluorogenic labelling of gelatin in aqueous phase and hydrogels. These probes with aggregation‐induced emission characteristics offer negligible background and elicit turn‐on fluorescence by simply mixing with the gelatin in aqueous phase, giving a detection limit of 0.15 mg/L over a linear dynamic range up to 100 mg/L. This method does not work for collagens and causes minimal interference with gelatin properties. Mechanistic studies reveal a key role for multivalent electrostatic interactions between the abundant basic residues in gelatin (e. g., lysine, hydroxylysine, arginine) and anionic tetrazolate moieties of the lipophilic fluorophore synergistically in spatially rigid macromolecular encapsulation to achieve fluorogenic labelling. The AIE strategy by forming non‐covalent fluorophore‐gelatin complexes was developed for novel hydrogels that exhibited reversible fluorescence in response to dynamic microstructural changes in the hydrogel scaffold upon salting‐in/out treatments, and enabled high spatial‐temporal imaging of the fiber network in lyophilized samples. This work may open up avenues for in‐situ imaging analysis and evaluation of gelatin‐based biomaterials during processes such as in vivo degradation and mineralization.

Fluorogenic in‐situ Labelling of Gelatin Polymer in Aqueous Solution and Hydrogel