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Herein, we rationally designed four coumarin\u2010based bioorthogonal fluorescent probes by site\u2014specifically conjugating photoactivatable caging groups and HaloTag ligands to coumarin fluorophore. Fluorescence characterization revealed that probes\n                    <jats:bold>Y8<\/jats:bold>\n                    and\n                    <jats:bold>Y9<\/jats:bold>\n                    exhibited a pronounced aggregation\u2010induced emission (AIE) effect. While probes\n                    <jats:bold>Y11<\/jats:bold>\n                    and\n                    <jats:bold>Y12<\/jats:bold>\n                    displayed rapid photoresponse in aqueous environments,\n                    <jats:bold>Y12<\/jats:bold>\n                    achieved stable conjugation to HaloTag\u2010fused proteins of interest (POIs) with robust photoactivated fluorescence. We further evaluated the intracellular fluorescence labeling performance of probes\n                    <jats:bold>4<\/jats:bold>\n                    ,\n                    <jats:bold>Y9<\/jats:bold>\n                    , and\n                    <jats:bold>Y12<\/jats:bold>\n                    targeting the STING protein and confirmed that the combination of\n                    <jats:bold>Y12<\/jats:bold>\n                    with genetically encoded STING\u2010Halo enabled high\u2010efficiency photoactivated fluorescence labeling, with a 16.5\u2010fold fluorescence enhancement upon light irradiation. This ultra\u2010high signal\u2010to\u2010background ratio (S\/N) facilitated the real\u2010time monitoring of dynamic STING activation, during which\n                    <jats:bold>Y12<\/jats:bold>\n                    _STING translocated from diffuse staining to fluorescent foci in cells stimulated with the STING agonist MSA\u20102. This fluorescent dye\/protein hybrid system holds great promise for advancing the mapping of protein dynamics in cell homeostasis and medicine research.\n                  <\/jats:p>","DOI":"10.1002\/bio.70525","type":"journal-article","created":{"date-parts":[[2026,6,5]],"date-time":"2026-06-05T06:51:31Z","timestamp":1780642291000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Construction of Coumarin\u2010Conjugated Macromolecular Probe for Photoactivatable Bioimaging"],"prefix":"10.1002","volume":"41","author":[{"given":"Xuekun","family":"Zhang","sequence":"first","affiliation":[{"name":"Key Laboratory of Carbohydrate Vaccines and Drugs in Jiangsu Province, School of Life Sciences and Health Engineering Jiangnan University  Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Qin","family":"Xu","sequence":"additional","affiliation":[{"name":"Key Laboratory of Carbohydrate Vaccines and Drugs in Jiangsu Province, School of Life Sciences and Health Engineering Jiangnan University  Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ying","family":"Wang","sequence":"additional","affiliation":[{"name":"Key Laboratory of Carbohydrate Vaccines and Drugs in Jiangsu Province, School of Life Sciences and Health Engineering Jiangnan University  Wuxi China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7130-769X","authenticated-orcid":false,"given":"Chenxu","family":"Yan","sequence":"additional","affiliation":[{"name":"Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Shanghai Key Laboratory of Functional Materials Chemistry, Feringa Nobel Prize Scientist Joint Research Center, Institute of Fine Chemicals, Frontiers Science Center for Materiobiology and Dynamic Chemistry, School of Chemistry and Molecular Engineering East China University of Science and Technology  Shanghai China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0640-5901","authenticated-orcid":false,"given":"Andong","family":"Shao","sequence":"additional","affiliation":[{"name":"Key Laboratory of Carbohydrate Vaccines and Drugs in Jiangsu Province, School of Life Sciences and Health Engineering Jiangnan University  Wuxi 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