Rapid Single-Cell Measurement of Transient Transmembrane Water Flow under Osmotic Gradient

Rapid Single-Cell Measurement of Transient Transmembrane Water Flow under Osmotic Gradient | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Rapid Single-Cell Measurement of Transient Transmembrane Water Flow under Osmotic Gradient Hong Jiang, Jinnawat Jongkhumkrong, Y. J. Chao, Qian Wang, Guiren Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7909902/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 25 Feb, 2026 Read the published version in The Journal of Membrane Biology → Version 1 posted 9 You are reading this latest preprint version Abstract Aquaporins (AQPs) are critical for transmembrane water transport in response to osmotic gradients, but their gating and regulatory mechanisms remain poorly understood. A central challenge is the lack of methods to measure water flow across AQPs from individual cells with the spatiotemporal resolution and sensitivity equivalent to patch-clamp recordings of ion fluxes—a limitation stemming from the electrically silent nature of water flow. Here, we present a novel optical technique—Flow-Induced Fluorescence Increase Velocimetry (FIFIV) based on Laser-Induced Fluorescence Photobleaching Anemometry (LIFPA)—that enables direct, real-time monitoring of cytoplasmic flow induced by AQP-mediated transmembrane water transport under osmotic pressure gradients. Using small molecular fluorescent dyes to label cytoplasm in single adherent MDA-MB-231 breast cancer cells, we show detection of instantaneous extremely low transmembrane water flow signals on the order of 1 µm/s following localized hypotonic stimulation. This approach circumvents the limitations of traditional volume-based osmotic permeability assays, achieving single-cell sensitivity and temporal resolution comparable to electrophysiological measurements of ion flux. FIFIV provides a paradigm-shifting tool to explore AQP function, regulation, and gating with potential applications in physiopathological studies and drug discovery. Full Text Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 25 Feb, 2026 Read the published version in The Journal of Membrane Biology → Version 1 posted Editorial decision: Revision requested 29 Nov, 2025 Reviews received at journal 28 Nov, 2025 Reviewers agreed at journal 17 Nov, 2025 Reviews received at journal 05 Nov, 2025 Reviewers agreed at journal 27 Oct, 2025 Reviewers invited by journal 22 Oct, 2025 Editor assigned by journal 22 Oct, 2025 Submission checks completed at journal 22 Oct, 2025 First submitted to journal 20 Oct, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7909902","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":538807222,"identity":"f9bde2ab-1250-45b3-b7da-a44ddd284ff8","order_by":0,"name":"Hong Jiang","email":"","orcid":"","institution":"University of South Carolina","correspondingAuthor":false,"prefix":"","firstName":"Hong","middleName":"","lastName":"Jiang","suffix":""},{"id":538807223,"identity":"54a2aa17-b585-4ea1-b539-7f9271ad6b95","order_by":1,"name":"Jinnawat Jongkhumkrong","email":"","orcid":"","institution":"University of South Carolina, United States","correspondingAuthor":false,"prefix":"","firstName":"Jinnawat","middleName":"","lastName":"Jongkhumkrong","suffix":""},{"id":538807224,"identity":"fd0a2757-84ec-421a-9af9-2a33d9dec5e2","order_by":2,"name":"Y. J. Chao","email":"","orcid":"","institution":"University of South Carolina","correspondingAuthor":false,"prefix":"","firstName":"Y.","middleName":"J.","lastName":"Chao","suffix":""},{"id":538807225,"identity":"c26dac3a-484e-475b-a2f8-1cdfb8fbe529","order_by":3,"name":"Qian Wang","email":"","orcid":"","institution":"University of South Carolina, United States","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Wang","suffix":""},{"id":538807226,"identity":"fa033ae9-c3af-4862-a3c4-5c0dadcbe9c1","order_by":4,"name":"Guiren Wang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYFACxgYog/kACpcYLWwJxGqBAx4D4rTIRyS3ffi447C8Of+azx9+MNjIbjhAQIvhjcTmmTPPHDbcOePtNskehjRjwlpmJDYz87bdZtxw4+w2ZgaGw4nEafnbdtt+w40zjz8zMPwnrEVeAqiFse124obzPQzSDAwHCGsx4HnYzNjb9j95ww02M8keg2TjmQRtaU9/zPCzLc12w/nDjz/8qLCT7SNoC1yBRAKIS0A52JYGGIufkOmjYBSMglEwYgEAD1VLYfNBCHkAAAAASUVORK5CYII=","orcid":"","institution":"University of South Carolina","correspondingAuthor":true,"prefix":"","firstName":"Guiren","middleName":"","lastName":"Wang","suffix":""}],"badges":[],"createdAt":"2025-10-21 03:08:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7909902/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7909902/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00232-026-00375-6","type":"published","date":"2026-02-25T15:59:18+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":94979123,"identity":"96b02b08-1655-46fd-a3d5-44b31b2d9a19","added_by":"auto","created_at":"2025-11-03 04:45:49","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2350585,"visible":true,"origin":"","legend":"","description":"","filename":"singlecellwaterfluxmeasurement05final.docx","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/3c9a5e821ab19c274b7d2b88.docx"},{"id":94979119,"identity":"f199b6c3-3d11-403f-a291-5bea553f65e6","added_by":"auto","created_at":"2025-11-03 04:45:49","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6620,"visible":true,"origin":"","legend":"","description":"","filename":"d58d3ab263a9425bbf690469e782f797.json","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/83c571b19de284a4de3ddbea.json"},{"id":94979122,"identity":"b31a80a6-93d9-42a0-84c5-0a08681e68f7","added_by":"auto","created_at":"2025-11-03 04:45:49","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":153508,"visible":true,"origin":"","legend":"","description":"","filename":"d58d3ab263a9425bbf690469e782f7971enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/c78d898c1c07da83a30e47a2.xml"},{"id":94979125,"identity":"77fbc365-0a96-4cfb-935c-da2a7e363f82","added_by":"auto","created_at":"2025-11-03 04:45:50","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":627036,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/681286978c6216236d020ef2.jpeg"},{"id":94979120,"identity":"835a5702-0349-4d0e-81b3-149b2e1d8bdf","added_by":"auto","created_at":"2025-11-03 04:45:49","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":139057,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/a74cb2fad8ae1e11c0d9f1ad.jpeg"},{"id":94979121,"identity":"542ee864-be9d-4560-a445-71c813636d45","added_by":"auto","created_at":"2025-11-03 04:45:49","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":293903,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/88cf25ec50885da71683eabd.jpeg"},{"id":94979128,"identity":"0e04f5c4-cd30-45e0-8b4f-ceb7087ccd69","added_by":"auto","created_at":"2025-11-03 04:45:50","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":301585,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/263f78ab6557205169db9464.jpeg"},{"id":94979124,"identity":"db6e58f7-689a-42cb-8d05-949f7fc9c656","added_by":"auto","created_at":"2025-11-03 04:45:49","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":561353,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/94cb0adc39381e73ec583315.jpeg"},{"id":94989374,"identity":"da5de3a2-c546-49c0-9d58-acdf165f2ebd","added_by":"auto","created_at":"2025-11-03 07:12:42","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":140819,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/4f14fbdd65d1076a0b77f08f.png"},{"id":94979126,"identity":"e4fef766-f18a-4aa1-a2dd-1f0bb7aaef11","added_by":"auto","created_at":"2025-11-03 04:45:50","extension":"png","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":37812,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/100ff369cc8fadaa9ae483a7.png"},{"id":94979132,"identity":"9c658f44-562c-4c16-81eb-d67c92eabf75","added_by":"auto","created_at":"2025-11-03 04:45:50","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":50020,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/500f7a0367043cabdbd7cd1f.png"},{"id":94989171,"identity":"dc6243d5-e159-46a7-a0ed-7e411ecd5daf","added_by":"auto","created_at":"2025-11-03 07:12:19","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":86400,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/17a3d353f1025dbad55bde7c.png"},{"id":94979129,"identity":"f1a8c954-7cb8-463a-a6b3-5c189d776cd9","added_by":"auto","created_at":"2025-11-03 04:45:50","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":154283,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/7e92261c742c5668db04d8f6.png"},{"id":94979131,"identity":"a31f5c3f-abbb-4b00-979e-e626d0c212d3","added_by":"auto","created_at":"2025-11-03 04:45:50","extension":"xml","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":152067,"visible":true,"origin":"","legend":"","description":"","filename":"d58d3ab263a9425bbf690469e782f7971structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/dfc5e6afadc2eb709667ac0b.xml"},{"id":94979133,"identity":"ad8382b1-5254-44d7-8bc2-e4d72195cee7","added_by":"auto","created_at":"2025-11-03 04:45:50","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":163742,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1/731409cce1d6955ed8ac953e.html"},{"id":103765680,"identity":"673b3538-768e-4ab3-a409-78e26b419ec5","added_by":"auto","created_at":"2026-03-02 16:07:24","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":693043,"visible":true,"origin":"","legend":"","description":"","filename":"singlecellwaterfluxmeasurement05final.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7909902/v1_covered_987d5e75-3432-4795-bc89-a1f239d687ff.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Rapid Single-Cell Measurement of Transient Transmembrane Water Flow under Osmotic Gradient","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-membrane-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmbi","sideBox":"Learn more about [The Journal of Membrane Biology](http://link.springer.com/journal/232)","snPcode":"232","submissionUrl":"https://submission.nature.com/new-submission/232/3","title":"The Journal of Membrane Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7909902/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7909902/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAquaporins (AQPs) are critical for transmembrane water transport in response to osmotic gradients, but their gating and regulatory mechanisms remain poorly understood. A central challenge is the lack of methods to measure water flow across AQPs from individual cells with the spatiotemporal resolution and sensitivity equivalent to patch-clamp recordings of ion fluxes\u0026mdash;a limitation stemming from the electrically silent nature of water flow. Here, we present a novel optical technique\u0026mdash;Flow-Induced Fluorescence Increase Velocimetry (FIFIV) based on Laser-Induced Fluorescence Photobleaching Anemometry (LIFPA)\u0026mdash;that enables direct, real-time monitoring of cytoplasmic flow induced by AQP-mediated transmembrane water transport under osmotic pressure gradients. Using small molecular fluorescent dyes to label cytoplasm in single adherent MDA-MB-231 breast cancer cells, we show detection of instantaneous extremely low transmembrane water flow signals on the order of 1 \u0026micro;m/s following localized hypotonic stimulation. This approach circumvents the limitations of traditional volume-based osmotic permeability assays, achieving single-cell sensitivity and temporal resolution comparable to electrophysiological measurements of ion flux. FIFIV provides a paradigm-shifting tool to explore AQP function, regulation, and gating with potential applications in physiopathological studies and drug discovery.\u003c/p\u003e","manuscriptTitle":"Rapid Single-Cell Measurement of Transient Transmembrane Water Flow under Osmotic Gradient","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-03 04:45:45","doi":"10.21203/rs.3.rs-7909902/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-29T05:42:38+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-28T07:25:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"232438707871826491532759583763500115532","date":"2025-11-17T09:17:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-05T18:16:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145983249279995633856819995422929243860","date":"2025-10-27T15:13:30+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-22T22:00:10+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-22T04:30:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-22T04:30:14+00:00","index":"","fulltext":""},{"type":"submitted","content":"The Journal of Membrane Biology","date":"2025-10-21T03:05:49+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"the-journal-of-membrane-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jmbi","sideBox":"Learn more about [The Journal of Membrane Biology](http://link.springer.com/journal/232)","snPcode":"232","submissionUrl":"https://submission.nature.com/new-submission/232/3","title":"The Journal of Membrane Biology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5ad34128-1e0b-421f-8104-43095cefaf1e","owner":[],"postedDate":"November 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-02T16:04:16+00:00","versionOfRecord":{"articleIdentity":"rs-7909902","link":"https://doi.org/10.1007/s00232-026-00375-6","journal":{"identity":"the-journal-of-membrane-biology","isVorOnly":false,"title":"The Journal of Membrane Biology"},"publishedOn":"2026-02-25 15:59:18","publishedOnDateReadable":"February 25th, 2026"},"versionCreatedAt":"2025-11-03 04:45:45","video":"","vorDoi":"10.1007/s00232-026-00375-6","vorDoiUrl":"https://doi.org/10.1007/s00232-026-00375-6","workflowStages":[]},"version":"v1","identity":"rs-7909902","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7909902","identity":"rs-7909902","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}