Oblique Line Scan Illumination Enables Expansive, Accurate and Sensitive Single Protein Measurements in Solution and in Living Cells

Oblique Line Scan Illumination Enables Expansive, Accurate and Sensitive Single Protein Measurements in Solution and in Living Cells | 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 Oblique Line Scan Illumination Enables Expansive, Accurate and Sensitive Single Protein Measurements in Solution and in Living Cells Daniel Anderson, Amine Driouchi, Mason Bretan, Brynmor Davis, and 34 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3790116/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Feb, 2025 Read the published version in Nature Methods → Version 1 posted You are reading this latest preprint version Abstract Single-molecule localization microscopy (SMLM) techniques, such as single-molecule tracking (SMT), enable in situ measurements in cells from which data-rich metrics can be extracted. SMT has been successfully applied to a variety of biological questions and model systems, aiming to unravel the spatiotemporal regulation of molecular mechanisms that govern protein function, downstream pathway effects, and cellular function. While powerful, SMLM often suffers from low throughput and illumination inhomogeneity, along with microscope and user-induced technical biases. Due to technical limitations in scaling SMLM techniques, a tradeoff between spatiotemporal resolution and throughput has been made historically, restricting broad application of these technologies. Here we address these limitations using Oblique Line Scan (OLS), a robust single-objective light-sheet based illumination and detection modality that achieves nanoscale spatial resolution and sub-millisecond temporal resolution across a 250 x 190 µm field of view. We demonstrate OLS-enabled SMT on Halo-Tagged proteins in living cells capturing protein motion up to 14 µm 2 /s. By exploiting the adaptability of the acquisition frame rate and the improved rejection of out of focus light, we extend the utility of OLS beyond cellular compartments with in-solution SMT (isSMT) for single-molecule measurement of ligand-protein interactions and disruption of protein-protein interactions (PPI). We illustrate the versatility of OLS by showcasing two-color SMT, STORM, and single molecule fluorescence recovery after photobleaching (FRAP). OLS expands the range of SMLM applications and paves the way for robust, high-throughput single-molecule investigations of protein dynamics required for drug screening and systems biology studies, both in cells and in solution. Biological sciences/Biophysics/Single-molecule biophysics Biological sciences/Biological techniques/High-throughput screening Biological sciences/Biological techniques/Microscopy/Super-resolution microscopy Biological sciences/Biological techniques/Microscopy/Light-sheet microscopy Biological sciences/Drug discovery Full Text Additional Declarations Yes there is potential Competing Interest. All authors are or have been employees or founders of Eikon Therapeutics. Cite Share Download PDF Status: Published Journal Publication published 18 Feb, 2025 Read the published version in Nature Methods → 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. 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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-3790116","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":264796277,"identity":"591d4368-78f9-4923-93ae-4fda7f81caf9","order_by":0,"name":"Daniel 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SMT has been successfully applied to a variety of biological questions and model systems, aiming to unravel the spatiotemporal regulation of molecular mechanisms that govern protein function, downstream pathway effects, and cellular function. While powerful, SMLM often suffers from low throughput and illumination inhomogeneity, along with microscope and user-induced technical biases. Due to technical limitations in scaling SMLM techniques, a tradeoff between spatiotemporal resolution and throughput has been made historically, restricting broad application of these technologies. Here we address these limitations using Oblique Line Scan (OLS), a robust single-objective light-sheet based illumination and detection modality that achieves nanoscale spatial resolution and sub-millisecond temporal resolution across a 250 x 190 µm field of view. We demonstrate OLS-enabled SMT on Halo-Tagged proteins in living cells capturing protein motion up to 14 µm\u003csup\u003e2\u003c/sup\u003e /s. By exploiting the adaptability of the acquisition frame rate and the improved rejection of out of focus light, we extend the utility of OLS beyond cellular compartments with in-solution SMT (isSMT) for single-molecule measurement of ligand-protein interactions and disruption of protein-protein interactions (PPI). We illustrate the versatility of OLS by showcasing two-color SMT, STORM, and single molecule fluorescence recovery after photobleaching (FRAP). OLS expands the range of SMLM applications and paves the way for robust, high-throughput single-molecule investigations of protein dynamics required for drug screening and systems biology studies, both in cells and in solution.\u003c/p\u003e","manuscriptTitle":"Oblique Line Scan Illumination Enables Expansive, Accurate and Sensitive Single Protein Measurements in Solution and in Living Cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-01-08 02:58:38","doi":"10.21203/rs.3.rs-3790116/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-methods","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"nmeth","sideBox":"Learn more about [Nature Methods](http://www.nature.com/nmeth)","snPcode":"","submissionUrl":"","title":"Nature Methods","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"b84eed5c-e0be-4c84-bf7b-84b5d2670802","owner":[],"postedDate":"January 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":27903689,"name":"Biological sciences/Biophysics/Single-molecule biophysics"},{"id":27903690,"name":"Biological sciences/Biological techniques/High-throughput screening"},{"id":27903691,"name":"Biological sciences/Biological techniques/Microscopy/Super-resolution microscopy"},{"id":27903692,"name":"Biological sciences/Biological techniques/Microscopy/Light-sheet microscopy"},{"id":27903693,"name":"Biological sciences/Drug discovery"}],"tags":[],"updatedAt":"2025-02-19T08:09:17+00:00","versionOfRecord":{"articleIdentity":"rs-3790116","link":"https://doi.org/10.1038/s41592-025-02594-6","journal":{"identity":"nature-methods","isVorOnly":false,"title":"Nature Methods"},"publishedOn":"2025-02-18 05:00:00","publishedOnDateReadable":"February 18th, 2025"},"versionCreatedAt":"2024-01-08 02:58:38","video":"","vorDoi":"10.1038/s41592-025-02594-6","vorDoiUrl":"https://doi.org/10.1038/s41592-025-02594-6","workflowStages":[]},"version":"v1","identity":"rs-3790116","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3790116","identity":"rs-3790116","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}