Generalized photoactivatable fluorescent probes enable background-suppressed 3D single-molecule imaging in hydrogels

Generalized photoactivatable fluorescent probes enable background-suppressed 3D single-molecule imaging in hydrogels | 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 Generalized photoactivatable fluorescent probes enable background-suppressed 3D single-molecule imaging in hydrogels Huang Tang, Shuting Liu, Xi Zhang, Xing-Hua Xia, Boyang Hua This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9070027/v2 This work is licensed under a CC BY 4.0 License Status: Posted Version 2 posted You are reading this latest preprint version Show more versions Abstract Compared with the broad range of regular fluorophores, photoactivatable (PA) fluorophores remain limited in choice, partly due to their commercial availability, complex synthesis routes, and in some case suboptimal photophysical and photochemical properties. Here, we present a type of generalized and modular PA fluorescent probes using DNA as scaffolds. These probes are readily accessible, flexible with fluorophore cores and attachment moieties, and have an improved performance than some existing PA fluorophores. With the probes, we implement 3D single-molecule imaging in hydrogels, overcoming the “concentration barrier” and observing single enzymatic reactions and DNA hybridization events in real time. These versatile PA probes could facilitate a wider adoption of many single-molecule imaging techniques. Biological sciences/Biophysics/Single-molecule biophysics Biological sciences/Biological techniques/Imaging/Fluorescence imaging photoactivatable fluorescent probes confocal photoactivation diffusion and excitation (PhADE) 3D single-molecule imaging hydrogels concentration barrier Figures Figure 1 Figure 2 Figure 3 Full Text Additional Declarations The authors declare no competing interests. Supplementary Files PAfluorescentprobesSI22.docx Supplemenatry information figures1figure1.jpg Supplementary Figure 1 figures2figure1.jpg Supplementary Figure 2 figures3figure2.jpg Supplementary Figure 3 figures4figure2.jpg Supplementary Figure 4 figures5figure2.jpg Supplementary Figure 5 figures6figure2.jpg Supplementary Figure 6 figures7figure3.jpg Supplementary Figure 7 Cite Share Download PDF Status: Posted Version 2 posted You are reading this latest preprint version Show more versions 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-9070027","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":611844462,"identity":"7ecf40a9-26d3-43f8-937e-abc5be04be41","order_by":0,"name":"Huang Tang","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Huang","middleName":"","lastName":"Tang","suffix":""},{"id":611844463,"identity":"e66a92bc-49f5-46b6-83e2-fd9e3d7dcdf8","order_by":1,"name":"Shuting Liu","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Shuting","middleName":"","lastName":"Liu","suffix":""},{"id":611844464,"identity":"18536670-20d2-4486-a613-bab3337d3249","order_by":2,"name":"Xi Zhang","email":"","orcid":"","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Xi","middleName":"","lastName":"Zhang","suffix":""},{"id":611844461,"identity":"76a1d57d-f428-4d86-8f90-7bf88b51433a","order_by":3,"name":"Xing-Hua Xia","email":"","orcid":"https://orcid.org/0000-0001-9831-4048","institution":"Nanjing University","correspondingAuthor":false,"prefix":"","firstName":"Xing-Hua","middleName":"","lastName":"Xia","suffix":""},{"id":611844460,"identity":"bdb9e886-99f9-4b9e-99ab-a30ddebd695c","order_by":4,"name":"Boyang Hua","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDCCAwwMH4AUD4ME8wEGCagIIS2MMyBa2BJI08LAIMFjALcXL+A7fvZgw8cddTIM0j3fHli2Mcjx3Uhg/FyAR4vkmbzExpln2HgYZM5uN5BsYzCWvJHALD0DjxaDAznmj3nbeIB+yd0mAdSSuOFGAhszDz4t598YNv9tkwBqyXkG0lJPWMuNHMNmxjYDkBY2kJYEA0JaJG+8MWzsbUvgYZNIM5OQOCdhOPPMw2ZpfFr4zucYNvxsq7Pnl0h+Ji1RZiPPdzz54Gd8WuCADYiZJcCRydhAjAYIYPxAvNpRMApGwSgYQQAAwStGwHPOjlYAAAAASUVORK5CYII=","orcid":"https://orcid.org/0009-0001-6488-6096","institution":"Nanjing University","correspondingAuthor":true,"prefix":"","firstName":"Boyang","middleName":"","lastName":"Hua","suffix":""}],"badges":[],"createdAt":"2026-03-09 07:57:07","currentVersionCode":2,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-9070027/v2","doiUrl":"https://doi.org/10.21203/rs.3.rs-9070027/v2","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105906039,"identity":"a12c7ed3-c4f9-4cea-8ab1-efec7bbc1745","added_by":"auto","created_at":"2026-04-01 10:16:55","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":951143,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D single-molecule imaging of Acrydite-FQD\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e594 immobilized within a hydrogel matrix.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Structures of FQD\u003csub\u003e1\u003c/sub\u003e and FQD\u003csub\u003e2\u003c/sub\u003e probes. FQD\u003csub\u003e2\u003c/sub\u003e represents the bimolecular construct annealed of two complementary oligo strands, whereas FQD\u003csub\u003e1\u003c/sub\u003e denotes the unimolecular construct made of a single oligo strand. \u003cstrong\u003eb\u003c/strong\u003e, Preparation workflow of anchoring Acrydite-FQD\u003csub\u003e2\u003c/sub\u003e594 in a polyacrylamide hydrogel during the polymerization process. \u003cstrong\u003ec-e\u003c/strong\u003e, Representative volumetric 3D confocal reconstructions of FQD\u003csub\u003e2\u003c/sub\u003e594 molecules revealing that Acrydite-Oligo A-AF594 is immobilized in the hydrogel matrix (\u003cstrong\u003ec\u003c/strong\u003e); subsequent hybridization with the PC Linker and BHQ2-modified Oligo B yields FQD\u003csub\u003e2\u003c/sub\u003e594 in the non-fluorescent “off” state (\u003cstrong\u003ed\u003c/strong\u003e); upon photoactivation, the PC Linker undergoes cleavage and BHQ2 hence dissociates, converting FQD\u003csub\u003e2\u003c/sub\u003e594 to the fluorescent \"on\" state (\u003cstrong\u003ee\u003c/strong\u003e). Images were acquired using the Z-Stack mode. The image size was 6.84 × 6.84 × 2.5 mm\u003csup\u003e3\u003c/sup\u003e, with a Z-step size of 0.5 mm covering a Z-range of 2.0–4.5 mm (relative to the coverslip surface at 0 mm). Both hydrogel polymerization and oligo hybridization were conducted in 1X PBS buffer (pH 7.4). \u003cstrong\u003ef\u003c/strong\u003e, Confocal images of FQD\u003csub\u003e2\u003c/sub\u003e594 at varying Z depths. The image size was 6.84 × 6.84 mm\u003csup\u003e2\u003c/sup\u003e. \u003cstrong\u003eg\u003c/strong\u003e, Statistics of the SNR of FQD\u003csub\u003e2\u003c/sub\u003e594 molecules across different imaging depths. 3 random imaging regions (6.84 × 6.84 mm\u003csup\u003e2\u003c/sup\u003e) with 75–98 molecules were analyzed per Z depth. \u003cstrong\u003eh\u003c/strong\u003e, Representative single-molecule images of FQD\u003csub\u003e2\u003c/sub\u003e594 before (BP) and after (AP) photoactivation, and intensity trajectories corresponding to the specific spots (I, II) annotated in the left images. The imaging depth was 2 μm, and the image size was 3.42 × 3.42 mm\u003csup\u003e2\u003c/sup\u003e. \u003cstrong\u003ei\u003c/strong\u003e, Quantification of the average spot counts per imaging area before hybridization (BH), after hybridization/before photoactivation (AH/BP), and after photoactivation (AP). 5 random imaging regions (Z depth at 2 μm, 13.76 × 13.76 mm\u003csup\u003e2\u003c/sup\u003e) were analyzed per condition. \u003cstrong\u003ej\u003c/strong\u003e, Quantification of the average spot counts of photoactivated FQD\u003csub\u003e2\u003c/sub\u003e594 per imaging area at different 405-nm laser powers. 5 random imaging regions (Z depth at 2 μm, 13.76 × 13.76 mm\u003csup\u003e2\u003c/sup\u003e) were analyzed per condition. For the above confocal experiments, a scanning speed of 97.65 ms/pixel and a pixel size of 53.84 × 53.84 nm\u003csup\u003e2\u003c/sup\u003e were used. Photoactivation of 1 frame (equivalent to a single-molecule activation time of 4.78 ms per frame) was performed using a 20% 405-nm laser power unless otherwise specified. Error bars, the standard deviation (SD). Scale bars, 1 mm. All the single-molecule images were obtained in Buffer B, and 1X Gloxy was added before imaging.\u003c/p\u003e","description":"","filename":"figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9070027/v2/f381305ff748d82efc455fec.jpg"},{"id":105905993,"identity":"0621e2c4-ecf5-48b8-a4b5-c7ae33661fcf","added_by":"auto","created_at":"2026-04-01 10:16:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":529913,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D confocal PhADE imaging.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Principle of 3D confocal PhADE imaging. Acrydite-FQD\u003csub\u003e2\u003c/sub\u003e594 is immobilized, while diffusing FQD\u003csub\u003e2\u003c/sub\u003e594 is infused into the hydrogel. Upon photoactivation (405 nm), both the immobilized Acrydite-FQD\u003csub\u003e2\u003c/sub\u003e594 and the diffusing FQD\u003csub\u003e2\u003c/sub\u003e594 near the focal spot are converted to the “on” state. During excitation (580 nm), the non-immobilized FQD\u003csub\u003e2\u003c/sub\u003e594 diffuses out of the imaging volume, leading to a time-dependent increase in the SNR of the immobilized Acrydite-FQD\u003csub\u003e2\u003c/sub\u003e594. \u003cstrong\u003eb\u003c/strong\u003e, 3D confocal PhADE imaging of Acrydite-FQD\u003csub\u003e2\u003c/sub\u003e594 immobilized in a hydrogel matrix with 1 mM diffusing FQD\u003csub\u003e2\u003c/sub\u003e594 (one PhADE cycle). The imaging depth was 4 μm, and the image size was 3.42 × 3.42 mm\u003csup\u003e2\u003c/sup\u003e. Each image represents an average of five raw frames, corresponding to the time periods (A1–A8) annotated in \u003cstrong\u003ec\u003c/strong\u003e. \u003cstrong\u003ec\u003c/strong\u003e, Time-dependent SNR for the immobilized Acrydite-FQD\u003csub\u003e2\u003c/sub\u003e594 in a single PhADE cycle. \"Point A\" denotes the specific spot marked in \u003cstrong\u003eb\u003c/strong\u003e; \"Average\" represents the mean SNR over all identified spots (n = 9). For the confocal experiments in \u003cstrong\u003ea-c\u003c/strong\u003e, a scanning speed of 97.65 ms/pixel and a pixel size of 53.84 × 53.84 nm\u003csup\u003e2\u003c/sup\u003e were used. Photoactivation of 2 frames (equivalent to a single-molecule activation time of 4.78 ms per frame) was performed using a 20% 405-nm laser power in \u003cstrong\u003ea-c\u003c/strong\u003e. \u003cstrong\u003ed\u003c/strong\u003e, Intensity decay curves of background fluorescence in continuous PhADE cycles at 1 mM FQD\u003csub\u003e2\u003c/sub\u003e594. The double-exponential decay time constants were 0.6 s and 4.0 s, respectively. For the confocal experiments in \u003cstrong\u003ed\u003c/strong\u003e, a scanning speed of 97.65 ms/pixel and a pixel size of 53.84 × 53.84 nm\u003csup\u003e2\u003c/sup\u003e were used. Photoactivation of 2 frames (equivalent to a single-molecule activation time of 4.78 ms per frame) was performed using a 20% 405-nm laser power in \u003cstrong\u003ed\u003c/strong\u003e. The duration of diffusion and excitation was 20 frames. \u003cstrong\u003ee\u003c/strong\u003e, Workflow and representative images of 3D confocal PhADE imaging (continuous PhADE cycles). \u003cstrong\u003ef\u003c/strong\u003e, Workflow of observing individual RCA reactions in the hydrogel with 3D confocal PhADE imaging. The RCA initiation complex, consisting of the Acrydite-labeled RCA primer and the circular RCA template, is copolymerized into the hydrogel network, followed by the infusion of RCA probe-FQD\u003csub\u003e2\u003c/sub\u003e594. The RCA reaction is initiated by the addition of phi29 DNA Polymerase and dNTPs. As the reaction proceeds, each amplification cycle generates a binding site for RCA probe-FQD\u003csub\u003e2\u003c/sub\u003e594, resulting in a progressive increase in fluorescence intensity. \u003cstrong\u003eg\u003c/strong\u003e, 3D confocal imaging of individual RCA reactions (without PhADE) at 2.5 mM RCA probe-AF594. The imaging depth was 10 μm, and the image size was 13.76 × 13.76 mm\u003csup\u003e2\u003c/sup\u003e. Each image represents an average of two raw frames. \u003cstrong\u003eh\u003c/strong\u003e, 3D confocal PhADE imaging of individual RCA reactions at 2.5 mM RCA probe-FQD\u003csub\u003e2\u003c/sub\u003e594 (continuous PhADE cycles). The imaging depth was 10 μm, and the image size was 13.76 × 13.76 mm\u003csup\u003e2\u003c/sup\u003e. Each image represents an average of two raw frames. \u003cstrong\u003ei\u003c/strong\u003e, Fluorescence intensity trajectories representing the growth curves of individual RCA reactions. The addition of phi29 DNA Polymerase and dNTPs was at t = 0 s. 13 consecutive movies were obtained from different regions, with each movie consisting of 25 PhADE cycles. For the confocal experiments in \u003cstrong\u003ef-i\u003c/strong\u003e, a scanning speed of 9.75 ms/pixel and a pixel size of 53.84 × 53.84 nm\u003csup\u003e2\u003c/sup\u003e were used. Photoactivation of 1 frame (equivalent to a single-molecule activation time of 4.78 ms per frame) was performed using a 20% 405-nm laser power in \u003cstrong\u003ef-i\u003c/strong\u003e. The duration of diffusion and excitation was 2 frames (approximately 2.6 s). Scale bars, 1 mm. All the single-molecule images in \u003cstrong\u003ea-e\u003c/strong\u003e were obtained in Buffer B, and 1X Gloxy was added before imaging. All the single-molecule results in \u003cstrong\u003ef-i\u003c/strong\u003e were obtained in Buffer D, and 1X Gloxy was added before imaging.\u003c/p\u003e","description":"","filename":"figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9070027/v2/6b6cbc16eca2e9ff4874fd47.jpg"},{"id":105906030,"identity":"c5935e22-5fee-44a7-993a-78b778ea0db8","added_by":"auto","created_at":"2026-04-01 10:16:33","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1031409,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e3D PA DNA-PAINT imaging.\u003c/strong\u003e \u003cstrong\u003ea\u003c/strong\u003e, Workflow of 3D PA DNA-PAINT imaging in a hydrogel matrix. The FQD\u003csub\u003e1\u003c/sub\u003e594-labeled imager strands interact with the Acrydite-AF488-labeled docking strands in the hydrogel. \u003cstrong\u003eb\u003c/strong\u003e, A representative volumetric 3D confocal reconstruction revealing that the Acrydite-AF488-docking strands are immobilized in the hydrogel matrix. Images were acquired using the Z-Stack mode. The image size was 6.84 × 6.84 × 5 mm\u003csup\u003e3\u003c/sup\u003e, with a Z-step size of 1 mm covering a Z-range of 2.0–7.0 mm (relative to the coverslip surface at 0 mm). \u003cstrong\u003ec\u003c/strong\u003e, Co-localization analysis between the AF488 channel (left) and the FQD\u003csub\u003e1\u003c/sub\u003e594 channel (middle), and the co-localized pixel positions (right). The imaging depth was 4 μm, and the image size was 2.42 × 2.42 mm\u003csup\u003e2\u003c/sup\u003e. \u003cstrong\u003ed\u003c/strong\u003e, Representative intensity trajectories with the unbound or dissociated state (t\u003csub\u003ed\u003c/sub\u003e) and the bound state (t\u003csub\u003eb\u003c/sub\u003e). Comparison between the confocal mode (top) and the confocal PhADE mode (bottom). \u003cstrong\u003ee\u003c/strong\u003e, Rate of dissociation (k\u003csub\u003eoff\u003c/sub\u003e = 1/t\u003csub\u003eb\u003c/sub\u003e) as a function of the FQD\u003csub\u003e1\u003c/sub\u003e594-imager strand concentration. \u003cstrong\u003ef\u003c/strong\u003e, Rate of binding (k\u003csub\u003eon\u003c/sub\u003e = 1/t\u003csub\u003ed\u003c/sub\u003e) as a function of the FQD\u003csub\u003e1\u003c/sub\u003e594-imager strand concentration. The mean dwelltimes were derived from 3 random groups with 92–259 and 70–194 events in \u003cstrong\u003ee,f\u003c/strong\u003e, respectively. \u003cstrong\u003eg\u003c/strong\u003e, A representative diffraction-limited single-molecule spot (left) and its corresponding super-resolution localization cluster (right). \u003cstrong\u003eh\u003c/strong\u003e, Cross-section profiles in \u003cstrong\u003eg\u003c/strong\u003e. For the above confocal experiments, a scanning speed of 102.38 ms/pixel and a pixel size of 51.53 × 51.53 nm\u003csup\u003e2\u003c/sup\u003e were used. Photoactivation of 1 frame (equivalent to a single-molecule activation time of 5.01 ms per frame) was performed using a 20% 405-nm laser power. The duration of diffusion and excitation was 1 frame (approximately 0.52 s). Error bars, the standard deviation (SD). Scale bars, 0.5 mm. All the single-molecule results were obtained in Buffer C, and 1X Gloxy was added before imaging.\u003c/p\u003e","description":"","filename":"figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9070027/v2/b202a9d72b3e07a288d1cf94.jpg"},{"id":105909833,"identity":"78198f07-4ee6-42e0-8075-0a8d00a20438","added_by":"auto","created_at":"2026-04-01 10:44:22","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3164916,"visible":true,"origin":"","legend":"","description":"","filename":"PAfluorescentprobes53.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9070027/v2_covered_824db5c6-3c99-44db-9310-5204957f4309.pdf"},{"id":105906627,"identity":"67777c32-ea8c-4f22-9066-e21683536a62","added_by":"auto","created_at":"2026-04-01 10:23:52","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":29815,"visible":true,"origin":"","legend":"Supplemenatry 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single-molecule imaging, hydrogels, concentration barrier","lastPublishedDoi":"10.21203/rs.3.rs-9070027/v2","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9070027/v2","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCompared with the broad range of regular fluorophores, photoactivatable (PA) fluorophores remain limited in choice, partly due to their commercial availability, complex synthesis routes, and in some case suboptimal photophysical and photochemical properties. Here, we present a type of generalized and modular PA fluorescent probes using DNA as scaffolds. These probes are readily accessible, flexible with fluorophore cores and attachment moieties, and have an improved performance than some existing PA fluorophores. With the probes, we implement 3D single-molecule imaging in hydrogels, overcoming the \u0026ldquo;concentration barrier\u0026rdquo; and observing single enzymatic reactions and DNA hybridization events in real time. These versatile PA probes could facilitate a wider adoption of many single-molecule imaging techniques.\u003c/p\u003e","manuscriptTitle":"Generalized photoactivatable fluorescent probes enable background-suppressed 3D single-molecule imaging in hydrogels","msid":"","msnumber":"","nonDraftVersions":[{"code":2,"date":"2026-03-31 18:18:28","doi":"10.21203/rs.3.rs-9070027/v2","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}},{"code":1,"date":"2026-03-25 08:21:52","doi":"10.21203/rs.3.rs-9070027/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"36e675f6-83b4-4672-82df-c0620d1aac65","owner":[],"postedDate":"March 31st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":65097456,"name":"Biological sciences/Biophysics/Single-molecule biophysics"},{"id":65097457,"name":"Biological sciences/Biological techniques/Imaging/Fluorescence imaging"}],"tags":[],"updatedAt":"2026-03-25T08:21:53+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-31 18:18:28","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v2","identity":"rs-9070027","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9070027","identity":"rs-9070027","version":["v2"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}