Covalent Anchoring of Enzyme‐Activatable Fluorescent Signals for in Situ Imaging and Longitudinal Staging of Cellular Senescence in Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease caused by the pathological accumulation of senescent cells. In this fibrotic microenvironment, senescence-associated β-galactosidase (SA-β-Gal) serves as a key biomarker. However, existing SA-β-Gal molecular probes suffer from signal diffusion and rapid clearance due to the extracellular leakage, compromising imaging fidelity. To address this issue, an enzyme-activated covalent labelling strategy is proposed, and as a design paradigm developing TCFM-Gal, a imaging tool integrating β-galactosidic fluorophore with an ortho-difluoromethyl leaving group. Upon SA-β-Gal-mediated hydrolysis, TCFM-Gal generates a quinone methide intermediate that covalently anchors to surrounding nucleophiles, confining fluorescence within lysosomes. In senescent cells, TCFM-Gal achieves extended lysosome-retained imaging, maintaining over 85% signal retention within 24 h. In the IPF model, TCFM-Gal enables longitudinal tracking of senescent cells and maintains a high signal intensity after 24 h. Rapid circulatory washout and high interstitial fluid pressure make conventional probes ineffective in the lungs. TCFM-Gal overcomes this not just by sensing, but by anchoring the signals in situ. Moreover, TCFM-Gal facilitates staging assessments of fibrosis progression by quantifying the senescence burden across disease phases. This study establishes an enzyme-activated covalent labeling paradigm for high-fidelity senescence imaging, advancing the spatiotemporal mapping of senescence in lung diseases.