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Here, we present a mesoscopic spinning-disk confocal (MSDC) imaging platform that physically suppresses background fluorescence while preserving the throughput required for large-scale functional imaging. By systematically optimizing the mesoscopic spatial filtering configuration and hardware synchronization scheme, the MSDC system achieves a field of view of \u223c6.8 \u00d7 6.8 mm2 with uniform \u223c8 \u03bcm lateral and \u223c50 \u03bcm axial sectioning thicknesses at full-frame rates up to 100 fps. Ex vivo evaluations demonstrate that the platform successfully suppresses out-of-focus fluorescence by \u223c90%, thereby preserving structural contrast and improving imaging fidelity in the presence of strong background signals. In vivo cerebrovascular imaging shows that the MSDC platform substantially reduces background-induced dark-vessel artifacts and enables positive-contrast three-dimensional reconstruction of cerebrovascular networks across cortical depths. When integrated with extended constrained nonnegative matrix factorization analysis, the platform further supports extraction of high-signal-to-noise calcium dynamics from hundreds of active Layer 2\/3 neurons distributed across multiple functional brain regions. Together, these results establish MSDC as a robust and cost-effective analytical imaging platform for high-fidelity structural and functional interrogation of fluorescent signals in complex biological specimens.<\/jats:p>","DOI":"10.1021\/acs.analchem.6c02597","type":"journal-article","created":{"date-parts":[[2026,8,30]],"date-time":"2026-08-30T17:53:32Z","timestamp":1788112412000},"page":"25689-25699","source":"Crossref","is-referenced-by-count":0,"title":["Large-Field\nMesoscopic Spinning-Disk Confocal Microscopy\nEnhances Quantitative Fidelity for In Vivo Cerebrovascular and Calcium\nSensing"],"prefix":"10.1021","volume":"98","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6419-6813","authenticated-orcid":true,"given":"Bingchen","family":"Che","sequence":"first","affiliation":[{"name":"Institute of Physics, Chinese Academy of Sciences , , ,","place":["Beijing, China, 100190"]},{"name":"Liyang Tianmu Lake Center of Applied Medical Physics Co., Ltd. , ,","place":["Changzhou, China, 213333"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sheng","family":"Gao","sequence":"additional","affiliation":[{"name":"Institute of Physics, Chinese Academy of Sciences , , ,","place":["Beijing, China, 100190"]},{"name":"Northwestern Polytechnical University , , ,","place":["Xi\u2019an, China, 710129"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuyou","family":"Huang","sequence":"additional","affiliation":[{"name":"Xuanwu Hospital of Capital Medical University , , ,","place":["Beijing, China, 100053"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jieyu","family":"Zhang","sequence":"additional","affiliation":[{"name":"Tsinghua University , , ,","place":["Beijing, China, 100084"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jie","family":"Liu","sequence":"additional","affiliation":[{"name":"Liyang Tianmu Lake Center of Applied Medical Physics Co., Ltd. , ,","place":["Changzhou, China, 213333"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zengcai V.","family":"Guo","sequence":"additional","affiliation":[{"name":"Tsinghua University , , ,","place":["Beijing, China, 100084"]},{"name":"Tsinghua-Peking Joint Center for Life Sciences , ,","place":["Beijing, China, 100084"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7133-0558","authenticated-orcid":true,"given":"Yan","family":"He","sequence":"additional","affiliation":[{"name":"Tsinghua University , , ,","place":["Beijing, China, 100084"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hao","family":"Xie","sequence":"additional","affiliation":[{"name":"Institute of Physics, Chinese Academy of Sciences , , ,","place":["Beijing, China, 100190"]},{"name":"Liyang Tianmu Lake Center of Applied Medical Physics Co., Ltd. , ,","place":["Changzhou, China, 213333"]},{"name":"University of Chinese Academy of Sciences , ,","place":["Nanjing, China, 211135"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,24]]},"reference":[{"issue":"11","key":"2026090808073096700_cit1","doi-asserted-by":"publisher","first-page":"7106","DOI":"10.1021\/acs.chemrev.3c00776","article-title":"Fluorescent\nProbes for Disease Diagnosis","volume":"124","author":"Wang","year":"2024","journal-title":"Chem. 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Chem."},{"issue":"5","key":"2026090808073096700_cit4","doi-asserted-by":"publisher","first-page":"3294","DOI":"10.1021\/acs.analchem.8b03992","article-title":"Fluorescence\nLifetime Imaging of p-tau Protein in Single Neuron with\na Highly Selective Fluorescent Probe","volume":"91","author":"Ge","year":"2019","journal-title":"Anal.\nChem."},{"issue":"10","key":"2026090808073096700_cit5","doi-asserted-by":"publisher","first-page":"13267","DOI":"10.1021\/acsomega.8b02055","article-title":"Analytical and Quantitative in Vivo\nMonitoring of Brain\nNeurochemistry by Electrochemical and Imaging Approaches","volume":"3","author":"Wu","year":"2018","journal-title":"ACS Omega"},{"issue":"12","key":"2026090808073096700_cit6","doi-asserted-by":"publisher","first-page":"3102","DOI":"10.1021\/acssensors.9b01713","article-title":"In Vivo Electrochemical Sensors for\nNeurochemicals: Recent Update","volume":"4","author":"Xu","year":"2019","journal-title":"ACS Sens."},{"issue":"8","key":"2026090808073096700_cit7","doi-asserted-by":"publisher","first-page":"2062","DOI":"10.1021\/jacsau.3c00220","article-title":"New Opportunities of Electrochemistry for Monitoring,\nModulating, and Mimicking the Brain Signals","volume":"3","author":"Wu","year":"2023","journal-title":"JACS Au"},{"issue":"1","key":"2026090808073096700_cit8","doi-asserted-by":"publisher","first-page":"33","DOI":"10.1016\/j.neuron.2020.09.031","article-title":"Mesoscopic Imaging: Shining a Wide\nLight on Large-Scale Neural Dynamics","volume":"108","author":"Cardin","year":"2020","journal-title":"Neuron"},{"issue":"1","key":"2026090808073096700_cit9","doi-asserted-by":"publisher","first-page":"9","DOI":"10.1016\/j.cell.2021.12.007","article-title":"Fluorescence\nimaging of large-scale neural ensemble\ndynamics","volume":"185","author":"Kim","year":"2022","journal-title":"Cell"},{"key":"2026090808073096700_cit10","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.59841","article-title":"Sources of\nwidefield fluorescence from the brain","volume":"9","author":"Waters","year":"2020","journal-title":"Elife"},{"issue":"3","key":"2026090808073096700_cit11","doi-asserted-by":"publisher","first-page":"390","DOI":"10.1111\/j.1365-2818.2007.01861.x","article-title":"Evaluating\nperformance\nin three-dimensional fluorescence microscopy","volume":"228","author":"Murray","year":"2007","journal-title":"J. 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Image Process."},{"key":"2026090808073096700_cit28","first-page":"120","article-title":"Fast Normalized\nCross-Correlation","volume-title":"Proceedings of Vision\nInterface 1995","author":"Lewis"},{"issue":"1","key":"2026090808073096700_cit29","doi-asserted-by":"publisher","first-page":"72","DOI":"10.2307\/1412159","article-title":"The Proof\nand Measurement of Association between Two Things","volume":"15","author":"Spearman","year":"1904","journal-title":"Am. J. Psychol."},{"issue":"7321","key":"2026090808073096700_cit30","doi-asserted-by":"publisher","first-page":"232","DOI":"10.1038\/nature09613","article-title":"Glial and neuronal\ncontrol of brain\nblood flow","volume":"468","author":"Attwell","year":"2010","journal-title":"Nature"},{"issue":"1","key":"2026090808073096700_cit31","doi-asserted-by":"publisher","first-page":"17","DOI":"10.1016\/j.neuron.2017.07.030","article-title":"The neurovascular\nunit coming of age: A journey through neurovascular coupling in health\nand disease","volume":"96","author":"Iadecola","year":"2017","journal-title":"Neuron"},{"issue":"3","key":"2026090808073096700_cit32","doi-asserted-by":"publisher","DOI":"10.1002\/advs.202205191","article-title":"Simultaneous functional magnetic\nresonance and optoacoustic imaging of brain-wide sensory responses\nin mice","volume":"10","author":"Chen","year":"2023","journal-title":"Adv. Sci."},{"issue":"30","key":"2026090808073096700_cit33","doi-asserted-by":"crossref","DOI":"10.1126\/sciadv.adw5275","article-title":"Photoacoustic and fluorescence hybrid microscope for\ncortex-wide imaging of neurovascular dynamics with subcellular resolution","volume":"11","author":"Liu","year":"2025","journal-title":"Sci. Adv."},{"issue":"6","key":"2026090808073096700_cit34","doi-asserted-by":"publisher","DOI":"10.1126\/sciadv.aea1602","article-title":"Noninvasive photoacoustic computed mesoscopy for longitudinal brain\nimaging","volume":"12","author":"Ruan","year":"2026","journal-title":"Sci. Adv."},{"issue":"2","key":"2026090808073096700_cit35","doi-asserted-by":"publisher","first-page":"201","DOI":"10.1038\/nprot.2009.222","article-title":"Thinned-skull cranial\nwindow technique for long-term imaging of the cortex in live mice","volume":"5","author":"Yang","year":"2010","journal-title":"Nat. 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Express"},{"issue":"6","key":"2026090808073096700_cit49","doi-asserted-by":"publisher","first-page":"L7","DOI":"10.1016\/0584-8539(95)01421-P","article-title":"Absorption and fluorescence properties\nof fluorescein","volume":"51","author":"Sjo\u0308back","year":"1995","journal-title":"Spectrochim. Acta, Part A"},{"issue":"5","key":"2026090808073096700_cit50","doi-asserted-by":"publisher","first-page":"510","DOI":"10.1002\/lsm.1900120510","article-title":"Optical\nproperties of Intralipid: a phantom medium for light propagation studies","volume":"12","author":"Flock","year":"1992","journal-title":"Lasers Surg. Med."},{"issue":"4","key":"2026090808073096700_cit51","doi-asserted-by":"publisher","first-page":"041102","DOI":"10.1117\/1.2335429","article-title":"Review\nof tissue simulating phantoms for optical spectroscopy,\nimaging and dosimetry","volume":"11","author":"Pogue","year":"2006","journal-title":"J. Biomed. 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Biol."},{"issue":"5","key":"2026090808073096700_cit53","doi-asserted-by":"publisher","first-page":"942","DOI":"10.1016\/j.neuron.2015.02.022","article-title":"Transgenic mice for\nintersectional targeting of neural sensors and\neffectors with high specificity and performance","volume":"85","author":"Madisen","year":"2015","journal-title":"Neuron"}],"container-title":["Analytical Chemistry"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.analchem.6c02597","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/35\/25689\/67327996\/acs.analchem.6c02597.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/ancham\/article-pdf\/98\/35\/25689\/67327996\/acs.analchem.6c02597.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,8]],"date-time":"2026-09-08T12:31:51Z","timestamp":1788870711000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/ancham\/article\/98\/35\/25689\/5321757\/Large-Field-Mesoscopic-Spinning-Disk-Confocal"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,24]]},"references-count":53,"journal-issue":{"issue":"35","published-online":{"date-parts":[[2026,8,24]]},"published-print":{"date-parts":[[2026,9,8]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.analchem.6c02597","relation":{},"ISSN":["0003-2700","1520-6882"],"issn-type":[{"value":"0003-2700","type":"print"},{"value":"1520-6882","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,24]]}}}