Three-photon holographic microscopy for deep precise optogenetics

Three-photon holographic microscopy for deep precise optogenetics | 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 Article Three-photon holographic microscopy for deep precise optogenetics Aysha Mohamed Lafirdeen, Cécile Telliez, Nataf Jérémie, Thi Phong Lien Ung, and 15 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9271774/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted You are reading this latest preprint version Abstract Precise manipulation of neurons across cortical layers requires optical approaches that maintain single-cell specificity deep within scattering brain tissue. Two-photon optogenetics provides targeted photostimulation in vivo and has expanded experimental access to defined neuronal populations beyond the limits of single-photon excitation. However, its application remains largely confined to the upper cortical layers (∼250–300 µm) in mice due to scattering. Here we introduce three-photon temporally focused computer-generated holography (3P TF-CGH) for precise optogenetic activation beyond this depth limit. We characterize 3P excitation of multiple excitatory and inhibitory opsins in organotypic slices, demonstrating cubic power dependence, efficient photocurrents and preserved temporal fidelity. We demonstrated that temporally focused holography maintains tight axial confinement in scattering tissue and minimizes superficial out-of-focus excitation under conditions required for deep targeting. In vivo, we combined 3P holographic stimulation at 1700 nm with simultaneous 3P calcium imaging at 1300 nm to achieve reliable neuronal activation across cortical layers down to 800 µm. Photostimulation remained stable across repeated trials without detectable physiological perturbation. By extending cell-resolved optogenetic control well beyond the established depth limits of 2P approaches, 3P holographic optogenetics enables non-invasive, all-optical interrogation of deep cortical circuits. Biological sciences/Biological techniques/Optogenetics Biological sciences/Biological techniques/Microscopy/Ca2+ imaging Biological sciences/Biological techniques/Imaging/Fluorescence imaging Full Text Additional Declarations There is NO Competing Interest. All animal procedures were carried out in accordance with the European Directive 2010/63/EU and approved by the local Animal Experimentation Ethics Committee (CETEA n.44) and the French Ministry of Research and Education (authorization #43746-202306061507449 v6). Supplementary Files SupplementaryVideo3.avi Supplementary Video 3 SupplementaryVideo2.avi Supplementary Video 2 3PmanuscriptSI.pdf Supplementary Information SupplementaryVideo1.avi Supplementary Video 1 Cite Share Download PDF Status: Under Review Version 1 posted 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. 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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-9271774","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":626600874,"identity":"c4b9e04e-10d6-4937-b163-35ba70178811","order_by":0,"name":"Aysha Mohamed Lafirdeen","email":"","orcid":"","institution":"Vision Institute, Sorbonne University","correspondingAuthor":false,"prefix":"","firstName":"Aysha","middleName":"Mohamed","lastName":"Lafirdeen","suffix":""},{"id":626600875,"identity":"92908f0f-ac53-459c-b036-416709ef1fac","order_by":1,"name":"Cécile Telliez","email":"","orcid":"","institution":"Vision Institute, Sorbonne 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