Hidden Markov model analysis of fluorescence blinking in fluorescently labeled DNA

Hidden Markov model analysis of fluorescence blinking in fluorescently labeled DNA | 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 Hidden Markov model analysis of fluorescence blinking in fluorescently labeled DNA Tatsuhiro Furuta, Shuya Fan, Tadao Takada, Yohei Kondo, Mamoru Fujitsuka, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7965396/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 27 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted 11 You are reading this latest preprint version Abstract We investigate the transition processes between the emitting (ON) and non-emitting (OFF) states of fluorescent molecules using a machine-learning approach. In fluorescently labeled DNA, continuous fluorescence is observed under irradiation; however, the system occasionally transitions to a non-emitting state, often associated with a charge-separated configuration. The resulting fluorescence trajectories exhibit characteristic blinking behavior ---alternating ON and OFF states--- which is heavily obscured by various sources of noise, making reliable state classification challenging. Because such trajectories represent typical stochastic time-series data, advanced analytical techniques are required. In this study, we apply a hidden Markov model to extract hidden ON/OFF states from noisy fluorescence trajectories using the forward-filtered backward-blocking Gibbs sampling algorithm, and construct probability density functions of the ON- and OFF-state durations to characterize the blinking dynamics. From these distributions, the characteristic relaxation times are evaluated as 17.6 ms for the ON state and 7.8 ms for the OFF state. The relatively long OFF period indicates that the charge-separated state in the DNA-ATTO655 system is fairly stable, suggesting suppressed charge recombination. In addition, during the ON state, the period of the light absorption-emission cycle is estimated to be approximately 25 $\mu$s, implying a relatively long waiting time for excitation. These results provide new insights into the fluorescence dynamics of single DNA-fluorophore systems. Finally, we discuss the detailed conditions required for reliable time-series analysis in terms of the photon-count histogram shape and the time-bin width used in the trajectories. Biological sciences/Biophysics Physical sciences/Physics Full Text Additional Declarations No competing interests reported. Supplementary Files SIHMMSRonlinesubmission.pdf Cite Share Download PDF Status: Published Journal Publication published 27 Feb, 2026 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 04 Dec, 2025 Reviews received at journal 02 Dec, 2025 Reviewers agreed at journal 23 Nov, 2025 Reviews received at journal 22 Nov, 2025 Reviewers agreed at journal 12 Nov, 2025 Reviewers agreed at journal 04 Nov, 2025 Reviewers invited by journal 03 Nov, 2025 Editor assigned by journal 03 Nov, 2025 Editor invited by journal 03 Nov, 2025 Submission checks completed at journal 31 Oct, 2025 First submitted to journal 31 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. 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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-7965396","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":543844237,"identity":"1e3b74e4-ea10-4008-ac32-a970afa2c957","order_by":0,"name":"Tatsuhiro Furuta","email":"","orcid":"","institution":"Kyushu Institute of Technology","correspondingAuthor":false,"prefix":"","firstName":"Tatsuhiro","middleName":"","lastName":"Furuta","suffix":""},{"id":543844238,"identity":"558471e7-6a93-4f4e-bdcd-f2fd562a13da","order_by":1,"name":"Shuya Fan","email":"","orcid":"","institution":"Osaka 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The relatively long OFF period indicates that the charge-separated state in the DNA-ATTO655 system is fairly stable, suggesting suppressed charge recombination. In addition, during the ON state, the period of the light absorption-emission cycle is estimated to be approximately 25 $\\mu$s, implying a relatively long waiting time for excitation. These results provide new insights into the fluorescence dynamics of single DNA-fluorophore systems. 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