A bright synthetic near-infrared luciferin enhances the capabilities of deep-tissue bioluminescence imaging using firefly luciferases

A bright synthetic near-infrared luciferin enhances the capabilities of deep-tissue bioluminescence imaging using firefly luciferases | 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 A bright synthetic near-infrared luciferin enhances the capabilities of deep-tissue bioluminescence imaging using firefly luciferases Takahiro Kuchimaru, Ryohei Saito-Moriya, Satoshi Iwano, Nobuo Kitada, and 26 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5464096/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 Synthetic bioluminescence reactions exhibiting near-infrared (NIR)-shifted spectra have been explored to improve deep-tissue imaging through the design of firefly luciferin analogues. Although the NIR bioluminescence reactions improve the tissue penetration of bioluminescence signals from deep tissues, their photon output is markedly lower compared to the natural reaction with D-luciferin and firefly luciferase (Fluc), often by an order of magnitude or more. Consequently, in most instances, the sensitivity of NIR bioluminescence imaging (NIR-BLI) has not yet substantially surpassed that of BLI with the natural firefly reaction. Here, we present a synthetic firefly luciferin, named AkaSuke, that generates intense NIR bioluminescence (λmax = 680 nm) in reaction with Fluc, greatly improving the detection sensitivity beyond that of the D-luciferin/Fluc reaction for targeting deep tissue. AkaSuke enables sensitive visualizations of ectopic hematogenesis through entire tissues of mice over time following transplantation of bone marrow stem cells labeled with Fluc. We additionally identify a Japanese firefly luciferase, DkumLuc1, that displays higher catalytic activities for bioluminescence emission of AkaSuke compared to typical Fluc, resulting in detection sensitivity comparable to that of AkaLumine/Akaluc reaction, one of the most sensitive bioluminescence systems for deep tissue imaging. We further propose the potential of the AkaSuke/DkumLuc1 reaction as an orthogonal pair with the AkaLumine/Akaluc for sensitive dual-target tracking in mice. Overall results suggest that AkaSuke enhances the capabilities of deep-tissue bioluminescence imaging using Fluc and its variant, and could serve as an emerging benchmark for the molecular design of NIR luciferin analogues. Biological sciences/Biological techniques/Imaging Biological sciences/Chemical biology/Chemical tools Near-infrared bioluminescence Synthetic firefly luciferin Deep-tissue imaging Orthogonal bioluminescence reaction Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Full Text Additional Declarations Yes there is potential Competing Interest. R.S.-M., N.K., R.I., H.Ao. and S.A.M. are co-inventers on patents JP7255828, US11807612 and CN115996914A submitted by Tokyo University of Pharmacy & Life Sciences and The University of Electro-Communications that covers of use and creation of AkaSuke and 1b. S.I. and A.M. are inventers on patents US10815462B2, JP7356749B2 and EP3505625B1 submitted by RIKEN that covers of use and creation of Akaluc. K.O. and T.Su are co-inventors on patent JP5860651 by OLYMPUS that covers of the use and creation of DkumLuc1. Supplementary Files SupplementaryVideo1AkaSuke.mp4 Supplementary Video 1 SupplementaryVideo2AkaLumine.mp4 Supplementary Video 2 SuplFigSaitoMoriyaetal.pdf SuplMethSaitoMoriyaetal.pdf 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-5464096","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":398418499,"identity":"26845844-e731-4445-be09-edc7b65cbbdb","order_by":0,"name":"Takahiro 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Miyawaki","email":"","orcid":"https://orcid.org/0000-0002-0671-4376","institution":"RIKEN","correspondingAuthor":false,"prefix":"","firstName":"Atsushi","middleName":"","lastName":"Miyawaki","suffix":""},{"id":398418528,"identity":"a7c49196-2b67-481d-9afd-49349c2fe6ec","order_by":29,"name":"Shojiro A Maki","email":"","orcid":"","institution":"The University of Electro-Communications, Tokyo","correspondingAuthor":false,"prefix":"","firstName":"Shojiro","middleName":"A","lastName":"Maki","suffix":""}],"badges":[],"createdAt":"2024-11-16 06:05:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5464096/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5464096/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":74516700,"identity":"8f84c69c-797b-45e9-8f22-650c139218c8","added_by":"auto","created_at":"2025-01-23 05:07:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":110847,"visible":true,"origin":"","legend":"\u003cp\u003eSynthetic firefly luciferin analogues with an extended π conjugation and a 5-membered ring. a Molecular structure of the synthetic luciferin AkaSuke, 1b and 1c. The structure of D-luciferin, AkaLumine and seMpai is also shown. b Bioluminescence spectrum of the substrates in reaction with natural Fluc. Source data are provided as a Source Data file.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-5464096/v1/289d0425c01ea590d60728c5.png"},{"id":74516701,"identity":"55238954-acee-4842-a1ba-31dc4f16e0cc","added_by":"auto","created_at":"2025-01-23 05:07:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":178196,"visible":true,"origin":"","legend":"\u003cp\u003eCharacterization of AkaSuke in living cells. a Bioluminescence emission from living cells expressing Fluc (E0771/Venus-Fluc) in reaction with the substrate at indicated concentrations. Bioluminescence emissions were measured using an open filter. Data were statistically analyzed with two-tailed Student’s t-test (n = 3 biologically independent samples). Data are presented as mean values ± SEM. Source data are provided as a Source Data file. b Bioluminescence emission from E0771/Venus-Fluc was measured using a near-infrared (Cy5.5) filter. Data were statistically analyzed with two tailed Student’s t-test (n = 3 biologically independent samples). Data are presented as mean values ± SEM. Source data are provided as a Source Data file. c The correlation between Fluc-expressing cell number and bioluminescence emission of AkaSuke. E0771/Venus-Fluc cells were treated with 250 μM AkaSuke. Data are presented as mean values (n = 3 biologically independent samples). Source data are provided as a Source Data file. d The bioluminescence kinetics of substrates in living cells expressing Fluc. E0771/Venus-Fluc cells were treated with the substrate concentration of 25 μM for D luciferin and AkaSuke, 1 μM for AkaLumine. Thick lines represent the mean values, which are calculated from individual thin lines (n = 3 independent biological samples). Source data are provided as a Source Data file. All experiments were repeated at least twice, and similar results were reproducible.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-5464096/v1/222db2daa92fbccbf72a4745.png"},{"id":74516706,"identity":"b6123bdb-89af-4545-bdb4-e2ae149283cc","added_by":"auto","created_at":"2025-01-23 05:07:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":699801,"visible":true,"origin":"","legend":"\u003cp\u003eNIR-BLI of living mice using the AkaSuke/Fluc rection. a Representative bioluminescence images of mice harboring E0771/Venus-Fluc lung metastasis obtained by D-luciferin and AkaSuke in same mice. The mice were intraperitoneally injected with D-luciferin (10 μmol/body) and AkaSuke (3 μmol/body). b Quantitative analysis of bioluminescence emission from lung metastasis generated with D-luciferin and AkaSuke (n = 8 independent metastatic lesions in 5 mice, p-value for D-luciferin vs AkaSuke, two tailed Student’s t test). Source data are provided as a Source Data file. c BLI of hematopoietic expansion process using AkaSuke. The experimental diagram (left) and representative time-course bioluminescence images obtained by repeatedly injecting AkaSuke (3 μmol/body) (right panel). Bone marrow cells (BM cells) were isolated from transgenic mice expressing Fluc (ffluc Tg mice) and transplanted into wild-type NOD SCID mice. d Representative bioluminescence images of hematopoiesis obtained by D luciferin and AkaSuke in same mice. The mice were intraperitoneally injected with i.p. injection of D-luciferin (10 μmol/body) and AkaSuke (3 μmol/body) after transplanting BM cells isolated from ffluc Tg mice. e Quantitative analysis of bioluminescence emission from lymph nodes (LN) and hind limbs (HL) obtained with D-luciferin and AkaSuke (n = 12 independent LN and HL in 6 mice, p-value for D-luciferin vs AkaSuke, two-tailed Student’s t test). Source data are provided as a Source Data file. All experiments were repeated at least twice, and similar results were reproducible.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-5464096/v1/ab62ed698c15a04e63e79a73.png"},{"id":74516703,"identity":"9b5a48b4-1ab9-424b-8457-81fce60ac7a2","added_by":"auto","created_at":"2025-01-23 05:07:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":295053,"visible":true,"origin":"","legend":"\u003cp\u003eEnhanced deep tissue BLI using AkaSuke/DkumLuc1. a Bioluminescence spectrum of DkumLuc1 in reaction with D-luciferin and AkaSuke. b Bioluminescence emission from living HEK293T cells transiently expressing DkumLuc1 or Akaluc treated with AkaSuke or AkaLumine at indicated concentrations (n = 3, p-value for 1000 or 4000 μM AkaSuke vs 250 μM AkaLumine, one-way ANOVA followed by Bonferroni’s multiple comparison). c Representative BL images of cells trapped in the lung obtained with the AkaSuke/DkumLuc1 or AkaLumine/Akaluc reaction. HEK293T cells expressing DkumLuc1 or Akaluc were intravenously injected from the tail vein 15 minutes before i.p. administration of AkaSuke (3 μmol/body) or AkaLumine (1.5 μmol/body). d Quantitative analysis of BL emission from the lung using the AkaSuke/DkumLuc1 or AkaLumine/Akaluc reaction (n = 8 independent lung tissue in 4 mice, p-value, two-tailed Student’s t test).\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-5464096/v1/a00f1f784744ff2f2c20a484.png"},{"id":74516705,"identity":"595e08aa-84a5-4b5b-849b-452b1673f521","added_by":"auto","created_at":"2025-01-23 05:07:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":695632,"visible":true,"origin":"","legend":"\u003cp\u003eDual target detection with NIR-BLI by combining AkaSuke/DkumLuc1 and AkaLumine/AkaLuc. a Bioluminescence emission from living HEK293T cells expressing DkumLuc1 or Akaluc treated with AkaLumine at indicated concentrations. b Bioluminescence emission from living HEK293T cells expressing DkumLuc1 or Akaluc treated with AkaSuke at indicated concentrations. c Representative dual NIR bioluminescence images of T cell migration (AkaLumine/Akaluc) and xenogeneic transplanted cell proliferation (AkaSuke/DkumLuc1). The bioluminescence images for xenotransplant proliferation were acquired by i.p. injection of AkaSuke (3 μmol/body) 6 hours after i.p. injection of AkaLumine (1.5 μmol/body). d NIR bioluminescence monitoring of T cell accumulation at xenotransplantation sites (left panel) and xenogeneic grafted cells (right panel). Thick lines represent the mean values, which are calculated from individual thin lines (n = 8 independent xenotransplantation sites in 4 mice). Source data are provided as a Source Data file. All experiments were repeated at least twice, and similar results were reproducible.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-5464096/v1/2e523ac9961cf3ceba740902.png"},{"id":74518769,"identity":"62fb3566-5dd2-4108-979c-54ab38ac2784","added_by":"auto","created_at":"2025-01-23 05:31:42","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1477184,"visible":true,"origin":"","legend":"","description":"","filename":"ManuscripttextSaitoMoriyaetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5464096/v1_covered_d4bf30d9-ab7d-4da9-8745-d766a72fb84a.pdf"},{"id":74516702,"identity":"b7de9370-32a3-4d2a-8822-3db200df9722","added_by":"auto","created_at":"2025-01-23 05:07:38","extension":"mp4","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":26359373,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Video 1\u003c/p\u003e","description":"","filename":"SupplementaryVideo1AkaSuke.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5464096/v1/0c87d5232f0f79821002318f.mp4"},{"id":74516704,"identity":"f5a9da98-a4a5-490a-89bd-152510771a09","added_by":"auto","created_at":"2025-01-23 05:07:38","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":28467285,"visible":true,"origin":"","legend":"\u003cp\u003eSupplementary Video 2\u003c/p\u003e","description":"","filename":"SupplementaryVideo2AkaLumine.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5464096/v1/432d04020f1c431a4f1e30d3.mp4"},{"id":74516707,"identity":"93ea6968-0c70-4b75-a15e-37b1f404aed4","added_by":"auto","created_at":"2025-01-23 05:07:39","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":1738038,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cbr\u003e\u003c/p\u003e","description":"","filename":"SuplFigSaitoMoriyaetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5464096/v1/f178b26815e1c35a315bc74c.pdf"},{"id":74516708,"identity":"4df14aa2-0bca-49ba-8a8d-d37608800f52","added_by":"auto","created_at":"2025-01-23 05:07:39","extension":"pdf","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":16530294,"visible":true,"origin":"","legend":"","description":"","filename":"SuplMethSaitoMoriyaetal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5464096/v1/6eb66b432c1d293cdaf5af0e.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\nR.S.-M., N.K., R.I., H.Ao. and S.A.M. are co-inventers on patents JP7255828, US11807612 and CN115996914A submitted by Tokyo University of Pharmacy \u0026 Life Sciences and The University of Electro-Communications that covers of use and creation of AkaSuke and 1b. S.I. and A.M. are inventers on patents US10815462B2, JP7356749B2 and EP3505625B1 submitted by RIKEN that covers of use and creation of Akaluc. K.O. and T.Su are co-inventors on patent JP5860651 by OLYMPUS that covers of the use and creation of DkumLuc1.","formattedTitle":"A bright synthetic near-infrared luciferin enhances the capabilities of deep-tissue bioluminescence imaging using firefly luciferases","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Near-infrared bioluminescence, Synthetic firefly luciferin, Deep-tissue imaging, Orthogonal bioluminescence reaction","lastPublishedDoi":"10.21203/rs.3.rs-5464096/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5464096/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Synthetic bioluminescence reactions exhibiting near-infrared (NIR)-shifted spectra have been explored to improve deep-tissue imaging through the design of firefly luciferin analogues. Although the NIR bioluminescence reactions improve the tissue penetration of bioluminescence signals from deep tissues, their photon output is markedly lower compared to the natural reaction with D-luciferin and firefly luciferase (Fluc), often by an order of magnitude or more. Consequently, in most instances, the sensitivity of NIR bioluminescence imaging (NIR-BLI) has not yet substantially surpassed that of BLI with the natural firefly reaction. Here, we present a synthetic firefly luciferin, named AkaSuke, that generates intense NIR bioluminescence (λmax = 680 nm) in reaction with Fluc, greatly improving the detection sensitivity beyond that of the D-luciferin/Fluc reaction for targeting deep tissue. AkaSuke enables sensitive visualizations of ectopic hematogenesis through entire tissues of mice over time following transplantation of bone marrow stem cells labeled with Fluc. We additionally identify a Japanese firefly luciferase, DkumLuc1, that displays higher catalytic activities for bioluminescence emission of AkaSuke compared to typical Fluc, resulting in detection sensitivity comparable to that of AkaLumine/Akaluc reaction, one of the most sensitive bioluminescence systems for deep tissue imaging. We further propose the potential of the AkaSuke/DkumLuc1 reaction as an orthogonal pair with the AkaLumine/Akaluc for sensitive dual-target tracking in mice. Overall results suggest that AkaSuke enhances the capabilities of deep-tissue bioluminescence imaging using Fluc and its variant, and could serve as an emerging benchmark for the molecular design of NIR luciferin analogues.","manuscriptTitle":"A bright synthetic near-infrared luciferin enhances the capabilities of deep-tissue bioluminescence imaging using firefly luciferases","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-23 05:07:33","doi":"10.21203/rs.3.rs-5464096/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"6a36d333-aa7d-46cf-8dfe-09c13e840200","owner":[],"postedDate":"January 23rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":42444002,"name":"Biological sciences/Biological techniques/Imaging"},{"id":42444003,"name":"Biological sciences/Chemical biology/Chemical tools"}],"tags":[],"updatedAt":"2025-01-23T05:07:33+00:00","versionOfRecord":[],"versionCreatedAt":"2025-01-23 05:07:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5464096","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5464096","identity":"rs-5464096","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}