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Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein,\n                    <jats:italic>Bacillus<\/jats:italic>\n                    FoF\n                    <jats:sub>1<\/jats:sub>\n                    -ATP synthase \u03b5 subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT\/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.\n                  <\/jats:p>","DOI":"10.7554\/elife.57544","type":"journal-article","created":{"date-parts":[[2020,7,10]],"date-time":"2020-07-10T08:00:43Z","timestamp":1594368043000},"update-policy":"https:\/\/doi.org\/10.7554\/elife.57544","source":"Crossref","is-referenced-by-count":58,"title":["Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor"],"prefix":"10.7554","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9838-832X","authenticated-orcid":true,"given":"Nami","family":"Kitajima","sequence":"first","affiliation":[{"name":"Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kenji","family":"Takikawa","sequence":"additional","affiliation":[{"name":"Department of Pharmacology, Graduate School of Medicine, The 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