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However, strategies for the authentic in situ identification of SA\u2010\u03b2\u2010gal with single\u2010molecule resolution have not been established, limiting the accuracy of senescence targeting. Here we present an unprecedented molecular approach to identify SA\u2010\u03b2\u2010gal in a manner that avoids dissociation from the target enzyme, which integrates the atomic hybridization of SA\u2010\u03b2\u2010gal substrates with protein\u2010environment\u2010sensitive fluorescence technology. Using this design principle, we created nine fluorescent probes with distinct working modes for the single\u2010molecule (target enzyme)\u2010resolved identification of SA\u2010\u03b2\u2010gal. Five of them formed crystal complexes with the wild\u2010type \u03b2\u2010galactosidase by binding at the active site, clearly evidencing their single\u2010molecule resolution capability. Our strategy, capable of both identifying and locating SA\u2010\u03b2\u2010gal, facilitated its application in dynamic single\u2010molecule localization microscopic imaging, achieving the in situ tracking of SA\u2010\u03b2\u2010gal in living cells at the nanoscale. Significantly, the probing of SA\u2010\u03b2\u2010gal 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\u2010associated protein with single\u2010molecule resolution.<\/jats:p>","DOI":"10.1002\/anie.5443283","type":"journal-article","created":{"date-parts":[[2026,8,29]],"date-time":"2026-08-29T05:45:13Z","timestamp":1787982313000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Identification of Senescence\u2010Associated \u03b2\u2010Galactosidase With Single\u2010Molecule Resolution"],"prefix":"10.1002","volume":"65","author":[{"given":"Guangwei","family":"Wang","sequence":"first","affiliation":[{"name":"Engineering Research Center of Western Resource Innovation Medicine Green Manufacturing of the Ministry of Education, School of Chemical Engineering College of Chemistry and Materials Science Northwest University  Xi'an 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