{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,13]],"date-time":"2026-10-13T00:46:22Z","timestamp":1791852382183,"version":"4.3.4"},"reference-count":35,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2022,1,11]],"date-time":"2022-01-11T00:00:00Z","timestamp":1641859200000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2022,1,11]],"date-time":"2022-01-11T00:00:00Z","timestamp":1641859200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["EXC 2067\/1\u2013390729940"],"award-info":[{"award-number":["EXC 2067\/1\u2013390729940"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Commun Biol"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>DNA point accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful super-resolution technique highly suitable for multi-target (multiplexing) bio-imaging. However, multiplexed imaging of cells is still challenging due to the dense and sticky environment inside a cell. Here, we combine fluorescence lifetime imaging microscopy (FLIM) with DNA-PAINT and use the lifetime information as a multiplexing parameter for targets identification. In contrast to Exchange-PAINT, fluorescence lifetime PAINT (FL-PAINT) can image multiple targets simultaneously and does not require any fluid exchange, thus leaving the sample undisturbed and making the use of flow chambers\/microfluidic systems unnecessary. We demonstrate the potential of FL-PAINT by simultaneous imaging of up to three targets in a cell using both wide-field FLIM and 3D time-resolved confocal laser scanning microscopy (CLSM). FL-PAINT can be readily combined with other existing techniques of multiplexed imaging and is therefore a perfect candidate for high-throughput multi-target bio-imaging.<\/jats:p>","DOI":"10.1038\/s42003-021-02976-4","type":"journal-article","created":{"date-parts":[[2022,1,11]],"date-time":"2022-01-11T06:03:04Z","timestamp":1641880984000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":63,"title":["Fluorescence lifetime DNA-PAINT for multiplexed super-resolution imaging of cells"],"prefix":"10.1038","volume":"5","author":[{"given":"Nazar","family":"Oleksiievets","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6749-7555","authenticated-orcid":false,"given":"Yelena","family":"Sargsyan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6638-6298","authenticated-orcid":false,"given":"Jan Christoph","family":"Thiele","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nikolaos","family":"Mougios","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4271-7897","authenticated-orcid":false,"given":"Shama","family":"Sograte-Idrissi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2893-481X","authenticated-orcid":false,"given":"Oleksii","family":"Nevskyi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1775-2159","authenticated-orcid":false,"given":"Ingo","family":"Gregor","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4968-9713","authenticated-orcid":false,"given":"Felipe","family":"Opazo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sven","family":"Thoms","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5091-7157","authenticated-orcid":false,"given":"J\u00f6rg","family":"Enderlein","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3967-1755","authenticated-orcid":false,"given":"Roman","family":"Tsukanov","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,1,11]]},"reference":[{"key":"2976_CR1","doi-asserted-by":"publisher","first-page":"C12015","DOI":"10.1088\/1748-0221\/9\/12\/C12015","volume":"9","author":"Y Prokazov","year":"2014","unstructured":"Prokazov, Y., Turbin, E., Weber, A., Hartig, R. & Zuschratter, W. Position sensitive detector for fluorescence lifetime imaging. J. Instrum. 9, C12015\u2013C12015 (2014).","journal-title":"J. Instrum."},{"key":"2976_CR2","doi-asserted-by":"publisher","first-page":"162365","DOI":"10.1016\/j.nima.2019.162365","volume":"942","author":"K Suhling","year":"2019","unstructured":"Suhling, K. et al. Wide-field time-correlated single photon counting-based fluorescence lifetime imaging microscopy. Nucl. Instrum. Methods Phys. Res. Sect. A: Accelerators, Spectrometers, Detect. Associated Equip. 942, 162365 (2019).","journal-title":"Nucl. Instrum. Methods Phys. Res. Sect. A: Accelerators, Spectrometers, Detect. Associated Equip."},{"key":"2976_CR3","doi-asserted-by":"publisher","first-page":"3494","DOI":"10.1021\/acs.jpca.0c01513","volume":"124","author":"N Oleksiievets","year":"2020","unstructured":"Oleksiievets, N. et al. Wide-field fluorescence lifetime imaging of single molecules. J. Phys. Chem. A 124, 3494\u20133500 (2020).","journal-title":"J. Phys. Chem. A"},{"key":"2976_CR4","doi-asserted-by":"publisher","first-page":"14190","DOI":"10.1021\/acsnano.0c07322","volume":"14","author":"JC Thiele","year":"2020","unstructured":"Thiele, J. C. et al. Confocal fluorescence-lifetime single-molecule localization microscopy. ACS Nano 14, 14190\u201314200 (2020).","journal-title":"ACS Nano"},{"key":"2976_CR5","doi-asserted-by":"publisher","first-page":"4756","DOI":"10.1021\/nl103427w","volume":"10","author":"R Jungmann","year":"2010","unstructured":"Jungmann, R. et al. Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami. Nano Lett. 10, 4756\u20134761 (2010).","journal-title":"Nano Lett."},{"key":"2976_CR6","doi-asserted-by":"publisher","first-page":"1198","DOI":"10.1038\/nprot.2017.024","volume":"12","author":"J Schnitzbauer","year":"2017","unstructured":"Schnitzbauer, J., Strauss, M. T., Schlichthaerle, T., Schueder, F. & Jungmann, R. Super-resolution microscopy with DNA-PAINT. Nat. Protoc. 12, 1198\u20131228 (2017).","journal-title":"Nat. Protoc."},{"key":"2976_CR7","doi-asserted-by":"publisher","first-page":"2431","DOI":"10.1002\/cphc.201402179","volume":"15","author":"M Raab","year":"2014","unstructured":"Raab, M., Schmied, J. J., Jusuk, I., Forthmann, C. & Tinnefeld, P. Fluorescence microscopy with 6 nm resolution on DNA origami. ChemPhysChem 15, 2431\u20132435 (2014).","journal-title":"ChemPhysChem"},{"key":"2976_CR8","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-017-02028-8","volume":"8","author":"F Schueder","year":"2017","unstructured":"Schueder, F. et al. Multiplexed 3D super-resolution imaging of whole cells using spinning disk confocal microscopy and DNA-PAINT. Nat. Commun. 8, 2090 (2017).","journal-title":"Nat. Commun."},{"key":"2976_CR9","doi-asserted-by":"publisher","first-page":"313","DOI":"10.1038\/nmeth.2835","volume":"11","author":"R Jungmann","year":"2014","unstructured":"Jungmann, R. et al. Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat. Methods 11, 313\u2013318 (2014).","journal-title":"Nat. Methods"},{"key":"2976_CR10","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-33052-3","volume":"8","author":"CK Spahn","year":"2018","unstructured":"Spahn, C. K. et al. A toolbox for multiplexed super-resolution imaging of the E. coli nucleoid and membrane using novel PAINT labels. Sci. Rep. 8, 14768 (2018).","journal-title":"Sci. Rep."},{"key":"2976_CR11","doi-asserted-by":"publisher","unstructured":"Sograte-Idrissi, S. et al. Nanobody detection of standard fluorescent proteins enables multi-target DNA-PAINT with high resolution and minimal displacement errors. Cells https:\/\/doi.org\/10.3390\/cells8010048 (2019).","DOI":"10.3390\/cells8010048"},{"key":"2976_CR12","doi-asserted-by":"publisher","first-page":"162","DOI":"10.1038\/nature22369","volume":"546","author":"AM Valm","year":"2017","unstructured":"Valm, A. M. et al. Applying systems-level spectral imaging and analysis to reveal the organelle interactome. Nature 546, 162\u2013167 (2017).","journal-title":"Nature"},{"key":"2976_CR13","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-020-15362-1","volume":"11","author":"M Klevanski","year":"2020","unstructured":"Klevanski, M. et al. Automated highly multiplexed super-resolution imaging of protein nano-architecture in cells and tissues. Nat. Commun. 11, 1552 (2020).","journal-title":"Nat. Commun."},{"key":"2976_CR14","doi-asserted-by":"publisher","first-page":"12991","DOI":"10.1073\/pnas.1804725115","volume":"115","author":"PA G\u00f3mez-Garc\u00eda","year":"2018","unstructured":"G\u00f3mez-Garc\u00eda, P. A., Garbacik, E. T., Otterstrom, J. J., Garcia-Parajo, M. F. & Lakadamyali, M. Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT. Proc. Natl Acad. Sci. USA 115, 12991 (2018).","journal-title":"Proc. Natl Acad. Sci. USA"},{"key":"2976_CR15","doi-asserted-by":"publisher","first-page":"1749","DOI":"10.1126\/science.1146598","volume":"317","author":"M Bates","year":"2007","unstructured":"Bates, M., Huang, B., Dempsey, G. T. & Zhuang, X. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749 (2007).","journal-title":"Science"},{"key":"2976_CR16","doi-asserted-by":"publisher","first-page":"1047","DOI":"10.1038\/nmeth.1274","volume":"5","author":"B Huang","year":"2008","unstructured":"Huang, B., Jones, S. A., Brandenburg, B. & Zhuang, X. Whole-cell 3D STORM reveals interactions between cellular structures with nanometer-scale resolution. Nat. Methods 5, 1047\u20131052 (2008).","journal-title":"Nat. Methods"},{"key":"2976_CR17","doi-asserted-by":"publisher","first-page":"606","DOI":"10.1126\/science.aak9913","volume":"355","author":"F Balzarotti","year":"2017","unstructured":"Balzarotti, F. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606\u2013612 (2017).","journal-title":"Science"},{"key":"2976_CR18","doi-asserted-by":"publisher","first-page":"257","DOI":"10.1038\/nmeth.3740","volume":"13","author":"T Nieh\u00f6rster","year":"2016","unstructured":"Nieh\u00f6rster, T. et al. Multi-target spectrally resolved fluorescence lifetime imaging microscopy. Nat. Methods 13, 257\u2013262 (2016).","journal-title":"Nat. Methods"},{"key":"2976_CR19","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-12301-7","volume":"10","author":"H G\u00f6tzke","year":"2019","unstructured":"G\u00f6tzke, H. et al. The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications. Nat. Commun. 10, 4403 (2019).","journal-title":"Nat. Commun."},{"key":"2976_CR20","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-019-09231-9","volume":"10","author":"P Almada","year":"2019","unstructured":"Almada, P. et al. Automating multimodal microscopy with NanoJ-Fluidics. Nat. Commun. 10, 1223 (2019).","journal-title":"Nat. Commun."},{"key":"2976_CR21","doi-asserted-by":"publisher","unstructured":"Tomov, T. E. et al. DNA bipedal motor achieves a large number of steps due to operation using microfluidics-based interface. ACS Nano 11, 4002\u20134008, https:\/\/doi.org\/10.1021\/acsnano.7b00547 (2017).","DOI":"10.1021\/acsnano.7b00547"},{"key":"2976_CR22","doi-asserted-by":"publisher","first-page":"3080","DOI":"10.1039\/C6SC05420J","volume":"8","author":"SS Agasti","year":"2017","unstructured":"Agasti, S. S. et al. DNA-barcoded labeling probes for highly multiplexed Exchange-PAINT imaging. Chem. Sci. 8, 3080\u20133091 (2017).","journal-title":"Chem. Sci."},{"key":"2976_CR23","doi-asserted-by":"publisher","first-page":"2641","DOI":"10.1021\/acs.nanolett.9b00508","volume":"19","author":"OK Wade","year":"2019","unstructured":"Wade, O. K. et al. 124-color super-resolution imaging by engineering DNA-PAINT blinking kinetics. Nano Lett. 19, 2641\u20132646 (2019).","journal-title":"Nano Lett."},{"key":"2976_CR24","doi-asserted-by":"publisher","first-page":"071101","DOI":"10.1063\/1.4961054","volume":"109","author":"LM Hirvonen","year":"2016","unstructured":"Hirvonen, L. M. et al. Picosecond wide-field time-correlated single photon counting fluorescence microscopy with a delay line anode detector. Appl. Phys. Lett. 109, 071101 (2016).","journal-title":"Appl. Phys. Lett."},{"key":"2976_CR25","doi-asserted-by":"crossref","unstructured":"Hartig, R., Prokazov, Y., Turbin, E. & Zuschratter, W. in Fluorescence Spectroscopy and Microscopy: Methods and Protocols (eds Yves Engelborghs & A. J. W. G. Visser) 457\u2212480 (Humana Press, 2014).","DOI":"10.1007\/978-1-62703-649-8_20"},{"key":"2976_CR26","doi-asserted-by":"publisher","first-page":"474005","DOI":"10.1088\/1361-6463\/aae0e2","volume":"51","author":"V Fabricius","year":"2018","unstructured":"Fabricius, V., Lef\u00e8bre, J., Geertsema, H., Marino, S. F. & Ewers, H. Rapid and efficient C-terminal labeling of nanobodies for DNA-PAINT. J. Phys. D: Appl. Phys. 51, 474005 (2018).","journal-title":"J. Phys. D: Appl. Phys."},{"key":"2976_CR27","unstructured":"O\u2019Connor, D. V. Time-Correlated Single Photon Counting (Academic Press, 2012)."},{"key":"2976_CR28","doi-asserted-by":"publisher","first-page":"10226","DOI":"10.1039\/D0NR00227E","volume":"12","author":"S Sograte-Idrissi","year":"2020","unstructured":"Sograte-Idrissi, S. et al. Circumvention of common labelling artefacts using secondary nanobodies. Nanoscale 12, 10226\u201310239 (2020).","journal-title":"Nanoscale"},{"key":"2976_CR29","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-018-24119-2","volume":"8","author":"K Soliman","year":"2018","unstructured":"Soliman, K., G\u00f6ttfert, F., Rosewich, H., Thoms, S. & G\u00e4rtner, J. Super-resolution imaging reveals the sub-diffraction phenotype of Zellweger Syndrome ghosts and wild-type peroxisomes. Sci. Rep. 8, 7809 (2018).","journal-title":"Sci. Rep."},{"key":"2976_CR30","doi-asserted-by":"publisher","DOI":"10.1038\/srep37947","volume":"6","author":"SC Stein","year":"2016","unstructured":"Stein, S. C. & Thiart, J. TrackNTrace: A simple and extendable open-source framework for developing single-molecule localization and tracking algorithms. Sci. Rep. 6, 37947 (2016).","journal-title":"Sci. Rep."},{"key":"2976_CR31","doi-asserted-by":"publisher","first-page":"6264","DOI":"10.1073\/pnas.93.13.6264","volume":"93","author":"T Ha","year":"1996","unstructured":"Ha, T. et al. Probing the interaction between two single molecules: Fluorescence resonance energy transfer between a single donor and a single acceptor. Proc. Natl Acad. Sci. USA 93, 6264 (1996).","journal-title":"Proc. Natl Acad. Sci. USA"},{"key":"2976_CR32","doi-asserted-by":"publisher","first-page":"78","DOI":"10.1006\/meth.2001.1217","volume":"25","author":"T Ha","year":"2001","unstructured":"Ha, T. Single-molecule fluorescence resonance energy transfer. Methods 25, 78\u201386 (2001).","journal-title":"Methods"},{"key":"2976_CR33","doi-asserted-by":"publisher","first-page":"1789","DOI":"10.1021\/ar500027d","volume":"47","author":"R Tsukanov","year":"2014","unstructured":"Tsukanov, R., Tomov, T. E., Liber, M., Berger, Y. & Nir, E. Developing DNA nanotechnology using single-molecule fluorescence. Acc. Chem. Res. 47, 1789\u20131798 (2014).","journal-title":"Acc. Chem. Res."},{"key":"2976_CR34","doi-asserted-by":"publisher","first-page":"124","DOI":"10.1038\/nphoton.2013.345","volume":"8","author":"AI Chizhik","year":"2014","unstructured":"Chizhik, A. I., Rother, J., Gregor, I., Janshoff, A. & Enderlein, J. Metal-induced energy transfer for live cell nanoscopy. Nat. Photonics 8, 124\u2013127 (2014).","journal-title":"Nat. Photonics"},{"key":"2976_CR35","doi-asserted-by":"publisher","first-page":"2616","DOI":"10.1021\/acs.nanolett.8b00425","volume":"18","author":"S Isbaner","year":"2018","unstructured":"Isbaner, S. et al. Axial colocalization of single molecules with nanometer accuracy using metal-induced energy transfer. Nano Lett. 18, 2616\u20132622 (2018).","journal-title":"Nano Lett."}],"container-title":["Communications Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s42003-021-02976-4.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s42003-021-02976-4","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s42003-021-02976-4.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,11,25]],"date-time":"2022-11-25T03:12:59Z","timestamp":1669345979000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s42003-021-02976-4"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,1,11]]},"references-count":35,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2022,12]]}},"alternative-id":["2976"],"URL":"https:\/\/doi.org\/10.1038\/s42003-021-02976-4","relation":{},"ISSN":["2399-3642"],"issn-type":[{"value":"2399-3642","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,1,11]]},"assertion":[{"value":"25 May 2021","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"8 December 2021","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"11 January 2022","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing non-financial interests but the following competing financial interests: F.O. is a shareholder of NanoTag Biotechnologies GmbH. All other authors declare no competing interest.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"38"}}