{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,15]],"date-time":"2026-06-15T14:54:26Z","timestamp":1781535266922,"version":"3.54.5"},"reference-count":56,"publisher":"Wiley","issue":"31","license":[{"start":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T00:00:00Z","timestamp":1776038400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T00:00:00Z","timestamp":1776038400000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Small"],"published-print":{"date-parts":[[2026,6]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                  <jats:p>\n                    BacFlash is a recently described transient bacterial process characterized by synchronized intracellular alkalization, membrane depolarization, and reactive oxygen species (ROS) generation, driven by regulated proton efflux. This phenomenon reveals a previously unrecognized mode of rapid physiological regulation in bacteria. Current techniques for studying BacFlash dynamics rely on intracellular fluorescent labeling with organic dyes or fluorescent proteins sensitive to pH, membrane potential, or ROS. Here, we introduce an extracellular, label\u2010free fluorescence imaging strategy for real\u2010time monitoring of BacFlash\u2010associated proton dynamics at the single\u2010cell level. By confining individual\n                    <jats:italic>E. Coli<\/jats:italic>\n                    cells within femtoliter\u2010scale microwells and minimizing extracellular buffering, we directly quantified transient proton extrusion, revealing an estimated release of \u223c10\n                    <jats:sup>5\u2013<\/jats:sup>\n                    10\n                    <jats:sup>6<\/jats:sup>\n                    protons per cell per second\u2014a parameter not previously reported. Further analysis under alkaline conditions uncovered two kinetically distinct forms of BacFlash, differing by over an order of magnitude in proton efflux rates and durations. These dynamic patterns were further modulated by ultraviolet stimulation. Our findings demonstrate that BacFlash is not a stochastic fluctuation but a tunable, metabolically linked signaling process. Moreover, our ability to quantify proton translocation at single\u2010cell resolution opens new avenues for mechanistic studies of bacterial bioenergetics and antimicrobial strategies targeting proton\u2010driven physiological processes.\n                  <\/jats:p>","DOI":"10.1002\/smll.202513944","type":"journal-article","created":{"date-parts":[[2026,4,13]],"date-time":"2026-04-13T07:14:31Z","timestamp":1776064471000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Extracellular Fluorescence Recording of BacFlash Dynamics Reveals Two Distinct Modes of Proton Extrusion"],"prefix":"10.1002","volume":"22","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0097-6305","authenticated-orcid":false,"given":"Jia","family":"Gao","sequence":"first","affiliation":[{"name":"State Key Laboratory of Analytical Chemistry for Life Science School of Chemistry and Chemical Engineering ChemBIC (Chemistry and Biomedicine Innovation Center) Nanjing University  Nanjing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yaoyao","family":"Zhang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Analytical Chemistry for Life Science School of Chemistry and Chemical Engineering ChemBIC (Chemistry and Biomedicine Innovation Center) Nanjing University  Nanjing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hua","family":"Su","sequence":"additional","affiliation":[{"name":"School of Pharmacy Nanjing University of Chinese Medicine  Nanjing China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4628-1755","authenticated-orcid":false,"given":"Wei","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Analytical Chemistry for Life Science School of Chemistry and Chemical Engineering ChemBIC (Chemistry and Biomedicine Innovation Center) Nanjing University  Nanjing 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