Singlet oxygen phosphorescence detection in living adherent cells in vitro

Singlet oxygen phosphorescence detection in living adherent cells in vitro | 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 Singlet oxygen phosphorescence detection in living adherent cells in vitro Steffen Hackbarth, Muriel Kabus, Xiuli Zheng, Tong Mu, Ronald Sroka, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9130479/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Objective Photosensitization by singlet oxygen is a chemical reaction with cellular quenchers, mainly proteins. Therefore, optical detection of singlet oxygen in cells affects the investigated cells and potentially, falsifies the observed results of photosensitization. For this reason, the illumination intensity has to be kept low during a measurement to avoid killing and destruction of the investigated cells. Material & Methods The individual illumination dose for cells under investigation are reduced by distributing it among a large number of adherent growing cells. Adherent cell lines were investigated with regard to singlet oxygen production by means of simultaneous fiber-coupled irradiation at 635 nm and singlet oxygen phosphorescence detection using a specified photomultiplier tube in combination with the TCMPC counting system. Singlet oxygen kinetics could be measured in two different cell types incubated with different photosensitizers, by scanning a major part of the area of a confluent layer of cells in six well plates. Result In each case, singlet oxygen signals kinetics could be observed and analyzed. Incubation with increasing amounts of photosensitizer resulted in both, stronger signals and longer singlet oxygen decay times. Conclusion Focused excitation and observation in combination with scanning across the whole well results in successful determination of singlet oxygen kinetics from incubated adherent cells without detaching them from the well plate. Further development is required to reduce artifacts, likely originating from the plate bottom. Full Text Additional Declarations Competing interest reported. S.H. is co-founder and shareholder of SHB Analytics GmbH Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 14 May, 2026 Reviews received at journal 04 May, 2026 Reviewers agreed at journal 19 Apr, 2026 Reviewers agreed at journal 15 Apr, 2026 Reviewers agreed at journal 14 Apr, 2026 Reviewers invited by journal 14 Apr, 2026 Editor assigned by journal 17 Mar, 2026 Submission checks completed at journal 16 Mar, 2026 First submitted to journal 15 Mar, 2026 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-9130479","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":625673841,"identity":"22540342-efb7-4abf-92ea-b4b696c214ad","order_by":0,"name":"Steffen Hackbarth","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABFUlEQVRIie2RMUsDMRTH3/NAF2+/pehHiGSQoh/mwkFdiouLg+DBQSZrVwvFz3BT6WYk0C4Ht2YoaDm4qYubbr5XFXHIuQrmNyV/8st7LwEIBP4gAiACuIQUAAvAfJug4W3eqVQ/FfhdQc0KL74U6FCO92wj4/vVuahtIXBuKRk9m/UcemOP0r8ZyCyetRfCqSLFylKyFEZVICeeMsIMIxvPrCodFga1vS7dAIzSoErjUeoNKVNS6setAuKp/VAefIobRlmck2K4MVbc7mcVzyzCtfJoumjVhGYRSp/xdKwk8s7bWNYkm6uVuq2XTfKiT+jFFrh+06e9sWd8ZmefPuKQ70y/w8R/nsBXUg467gwEAoF/zjv6xWz9T7g0FQAAAABJRU5ErkJggg==","orcid":"","institution":"Humboldt-Universität zu Berlin","correspondingAuthor":true,"prefix":"","firstName":"Steffen","middleName":"","lastName":"Hackbarth","suffix":""},{"id":625673842,"identity":"0397d344-afd1-42c2-941b-49806be54e70","order_by":1,"name":"Muriel Kabus","email":"","orcid":"","institution":"Laser-Forschungslabor and Labor für Tumor-Immunologie, LMU University Hospital, LMU Munich","correspondingAuthor":false,"prefix":"","firstName":"Muriel","middleName":"","lastName":"Kabus","suffix":""},{"id":625673843,"identity":"f16a4a04-9986-457a-ba47-fec179fe33b0","order_by":2,"name":"Xiuli Zheng","email":"","orcid":"","institution":"Technical Institute of Physics and Chemistry","correspondingAuthor":false,"prefix":"","firstName":"Xiuli","middleName":"","lastName":"Zheng","suffix":""},{"id":625673844,"identity":"a5d555ce-51b4-478d-8b08-66cdfc198387","order_by":3,"name":"Tong Mu","email":"","orcid":"","institution":"Technical Institute of Physics and Chemistry","correspondingAuthor":false,"prefix":"","firstName":"Tong","middleName":"","lastName":"Mu","suffix":""},{"id":625673845,"identity":"fe7a0a40-d992-44b0-a1fc-e5fb73cd672d","order_by":4,"name":"Ronald Sroka","email":"","orcid":"","institution":"Laser-Forschungslabor and Labor für Tumor-Immunologie, LMU University Hospital, LMU Munich","correspondingAuthor":false,"prefix":"","firstName":"Ronald","middleName":"","lastName":"Sroka","suffix":""},{"id":625673846,"identity":"2ad8f3f2-7419-4473-b625-a627a77de68d","order_by":5,"name":"Weimin Liu","email":"","orcid":"","institution":"Technical Institute of Physics and Chemistry","correspondingAuthor":false,"prefix":"","firstName":"Weimin","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-03-15 18:08:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9130479/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9130479/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107486810,"identity":"d5d8e28a-c81c-4cb7-b0fc-4aff787b1bc4","added_by":"auto","created_at":"2026-04-22 02:39:00","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":313188,"visible":true,"origin":"","legend":"","description":"","filename":"Singletoxygenphosphorescencedetectioninlivingadherentcellsinvitrosubmit.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9130479/v1_covered_f2cbf15d-91ec-4034-9ad7-1fa76e6ea063.pdf"}],"financialInterests":"Competing interest reported. 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Therefore, optical detection of singlet oxygen in cells affects the investigated cells and potentially, falsifies the observed results of photosensitization. For this reason, the illumination intensity has to be kept low during a measurement to avoid killing and destruction of the investigated cells.\u003c/p\u003e\u003ch2\u003eMaterial \u0026amp; Methods\u003c/h2\u003e \u003cp\u003eThe individual illumination dose for cells under investigation are reduced by distributing it among a large number of adherent growing cells. Adherent cell lines were investigated with regard to singlet oxygen production by means of simultaneous fiber-coupled irradiation at 635 nm and singlet oxygen phosphorescence detection using a specified photomultiplier tube in combination with the TCMPC counting system. 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