Lifetime imaging of singlet oxygen NIR phosphorescence

Lifetime imaging of singlet oxygen NIR phosphorescence | 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 Lifetime imaging of singlet oxygen NIR phosphorescence Mauricio Baptista, Isabel Bacellar, Carlos Marques, André Schroder, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7474505/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 1 O 2 is the first excited state of molecular oxygen and the key intermediate in photosensitized oxidation reactions. It is sensed by its near-infrared (NIR) phosphorescence emission that peaks at 1270 nm. However, several intrinsic properties of the 1 O 2 emission, such as the very low yield of photon emission, the comparable rates for decay and di!usion in dense media, and the low e”ciency of the cameras available in this wavelength range, make the acquisition of microscopic images of 1 O 2 localization a real challenge. Here, we describe a 1 O 2 phosphorescence lifetime imaging microscope ( 1 O 2 -PLIM) that allows acquiring lifetime and intensity profiles of 1 O 2 phosphorescence emission with micrometer resolution. Calibration carried out with photosensitizer solutions returned the expected lifetimes for 1 O 2 generation and decay. Nanometer-sized beads allowed the reconstruction of the excitation volume and the consequent estimation of detection limit as being 1 million 1 O 2 molecules in 2 femtoliters of ethanol. Scanning samples in a confocal configuration provided reconstruction of intensity and lifetime images of 1 O 2 emission from complex systems such as micrometer-sized polymer beads bound to photosensitizers, as well as from HaCaT keratynocytes previously incubated with a photosensitizer. Raw image data was corrected for the 1 O 2 emission lifetime and for the di!usion pathway within the confocal volume, using a mathematical model specifically developed for this purpose. This work provides a proof of principle for 1 O 2 imaging that will stimulate development of novel instrumentation, allowing a better understanding of how light interacts with matter and a!ects processes relevant to biology, medicine, agriculture, food and materials science. Physical sciences/Chemistry/Analytical chemistry/Imaging studies Biological sciences/Chemical biology/Biophysical chemistry Singlet-oxygen microscopy PLIM Diffusion-corrected lifetime Full Text Additional Declarations There is NO Competing Interest. 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. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7474505","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":522083385,"identity":"7f8f816c-9687-4700-abd5-87ddb56a3fed","order_by":0,"name":"Mauricio Baptista","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2ElEQVRIiWNgGAWjYBACAxCRAMT8zMgiBLQwNiQAKclmkrSAKIMDxGoxZz97/MGDP3/kjY8zb2Dm+XOHwVz6AH4tlj15iQ2JbQaG2w6zFTDztj1jsOxLIOCwAzmGDYkNBozbDvMYMPM2HGYwOEPIL+ffGDYk/DGw39wM1MLzhxgtN4C2JLAZJAI9AtTCRoQWyxnvEmckthknzwD65eDctmc8lj0EtJjz5x74+OOPnG1//+GND978uSNnzkNACwMDQgUoag4Q1oCiBYgPEKFjFIyCUTAKRhoAAP8sRHLByRG0AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0001-7079-7666","institution":"Universidade de Sao Paulo","correspondingAuthor":true,"prefix":"","firstName":"Mauricio","middleName":"","lastName":"Baptista","suffix":""},{"id":522083386,"identity":"efca8e81-c42b-4f44-81b2-3212ce0dcd2e","order_by":1,"name":"Isabel Bacellar","email":"","orcid":"","institution":"Universidade de Sao Paulo","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"","lastName":"Bacellar","suffix":""},{"id":522083387,"identity":"561814d3-ac70-4928-bb67-6cdb0290d602","order_by":2,"name":"Carlos Marques","email":"","orcid":"https://orcid.org/0000-0002-3952-0498","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Carlos","middleName":"","lastName":"Marques","suffix":""},{"id":522083388,"identity":"949f70a2-53af-4b61-8728-e53b261a0c11","order_by":3,"name":"André Schroder","email":"","orcid":"","institution":"Laboratoire LAMCOS - 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It is sensed by its near-infrared (NIR) phosphorescence emission that peaks at 1270 nm. However, several intrinsic properties\u003c/p\u003e\n\u003cp\u003eof the \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e emission, such as the very low yield of photon emission, the comparable rates for decay and di!usion in dense media, and the low e”ciency of the cameras available in this wavelength range, make the acquisition of microscopic images of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e localization a real challenge. Here, we describe a \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e phosphorescence lifetime imaging microscope (\u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e -PLIM) that allows acquiring lifetime and intensity profiles of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e phosphorescence emission with micrometer resolution. Calibration carried out with photosensitizer solutions returned the expected lifetimes for \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e generation and decay. Nanometer-sized beads allowed the reconstruction of the excitation volume and the consequent estimation of detection limit as being 1 million \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e molecules in 2 femtoliters of ethanol. Scanning samples in a confocal configuration provided reconstruction of intensity and lifetime images of \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e emission from complex systems such as micrometer-sized polymer beads bound to photosensitizers, as well as from HaCaT keratynocytes previously incubated with a photosensitizer. Raw image data was corrected for the \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e emission lifetime and for the di!usion pathway within the confocal volume, using a mathematical model specifically developed for this purpose. This work provides a proof of principle for \u003csup\u003e1\u003c/sup\u003eO\u003csub\u003e2\u003c/sub\u003e imaging that will stimulate development of novel instrumentation, allowing a better understanding of how light interacts with matter and a!ects processes relevant to biology, medicine, agriculture, food and materials science.\u003c/p\u003e","manuscriptTitle":"Lifetime imaging of singlet oxygen NIR phosphorescence","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-10 03:32:22","doi":"10.21203/rs.3.rs-7474505/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"communications-chemistry","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"commschem","sideBox":"Learn more about [Communications Chemistry](http://www.nature.com/commschem/)","snPcode":"","submissionUrl":"","title":"Communications Chemistry","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Communications Series","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"285250ba-f8a4-4e52-b526-ab7af4e164a2","owner":[],"postedDate":"October 10th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":55486557,"name":"Physical sciences/Chemistry/Analytical chemistry/Imaging studies"},{"id":55486558,"name":"Biological sciences/Chemical biology/Biophysical chemistry"}],"tags":[],"updatedAt":"2026-04-19T06:45:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-10 03:32:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7474505","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7474505","identity":"rs-7474505","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}