The majority of human senescent cells exhibit overexpression of senescence-associated β-galactosidase (SA-β-gal), rendering this enzyme the most extensively utilized biomarker of senescence and a key prodrug target for intervening in aging. However, strategies for the authentic in situ identification of SA-β-gal with single-molecule resolution have not been established, limiting the accuracy of senescence targeting. Here we present an unprecedented molecular approach to identify SA-β-gal in a manner that avoids dissociation from the target enzyme, which integrates the atomic hybridization of SA-β-gal substrates with protein-environment-sensitive fluorescence technology. Using this design principle, we created nine fluorescent probes with distinct working modes for the single-molecule (target enzyme)-resolved identification of SA-β-gal. Five of them formed crystal complexes with the wild-type β-galactosidase by binding at the active site, clearly evidencing their single-molecule resolution capability. Our strategy, capable of both identifying and locating SA-β-gal, facilitated its application in dynamic single-molecule localization microscopic imaging, achieving the in situ tracking of SA-β-gal in living cells at the nanoscale. Significantly, the probing of SA-β-gal with high fidelity enabled the precise evaluation of the aging degree in mice. As such, our research provides a promising method for the authentic in situ identification of this senescence-associated protein with single-molecule resolution.
