{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T05:57:17Z","timestamp":1790747837803,"version":"4.1.0"},"reference-count":32,"publisher":"National Academy of Sciences","issue":"51","license":[{"start":{"date-parts":[[2019,6,3]],"date-time":"2019-06-03T00:00:00Z","timestamp":1559520000000},"content-version":"vor","delay-in-days":182,"URL":"https:\/\/www.pnas.org\/site\/aboutpnas\/licenses.xhtml"}],"content-domain":{"domain":["www.pnas.org"],"crossmark-restriction":true},"short-container-title":["Proc. Natl. Acad. Sci. U.S.A."],"published-print":{"date-parts":[[2018,12,18]]},"abstract":"<jats:p>Recent advancements in single-molecule-based superresolution microscopy have made it possible to visualize biological structures with unprecedented spatial resolution. Determining the spatial coorganization of these structures within cells under physiological and pathological conditions is an important biological goal. This goal has been stymied by the current limitations of carrying out superresolution microscopy in multiple colors. Here, we develop an approach for simultaneous multicolor superresolution imaging which relies solely on fluorophore excitation, rather than fluorescence emission properties. By modulating the intensity of the excitation lasers at different frequencies, we show that the color channel can be determined based on the fluorophore\u2019s response to the modulated excitation. We use this frequency multiplexing to reduce the image acquisition time of multicolor superresolution DNA-PAINT while maintaining all its advantages: minimal color cross-talk, minimal photobleaching, maximal signal throughput, ability to maintain the fluorophore density per imaged color, and ability to use the full camera field of view. We refer to this imaging modality as \u201cfrequency multiplexed DNA-PAINT,\u201d or fm-DNA-PAINT for short. We also show that frequency multiplexing is fully compatible with STORM superresolution imaging, which we term fm-STORM. Unlike fm-DNA-PAINT, fm-STORM is prone to color cross-talk. To overcome this caveat, we further develop a machine-learning algorithm to correct for color cross-talk with more than 95% accuracy, without the need for prior information about the imaged structure.<\/jats:p>","DOI":"10.1073\/pnas.1804725115","type":"journal-article","created":{"date-parts":[[2018,12,3]],"date-time":"2018-12-03T20:15:27Z","timestamp":1543868127000},"page":"12991-12996","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":75,"title":["Excitation-multiplexed multicolor superresolution imaging with fm-STORM and fm-DNA-PAINT"],"prefix":"10.1073","volume":"115","author":[{"given":"Pablo A.","family":"G\u00f3mez-Garc\u00eda","sequence":"first","affiliation":[{"name":"ICFO \u2013 Institut de Ciencies Fotoniques, Barcelona Institute of Science and Technology, 08860 Castelldefels, Barcelona, Spain;"},{"name":"Centre for Genomic Regulation (CRG), The Barcelona Institute of Science and Technology, 08003 Barcelona, Spain;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Erik T.","family":"Garbacik","sequence":"additional","affiliation":[{"name":"ICFO \u2013 Institut de Ciencies Fotoniques, Barcelona Institute of Science and Technology, 08860 Castelldefels, Barcelona, Spain;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0176-9976","authenticated-orcid":false,"given":"Jason J.","family":"Otterstrom","sequence":"additional","affiliation":[{"name":"ICFO \u2013 Institut de Ciencies Fotoniques, Barcelona Institute of Science and Technology, 08860 Castelldefels, Barcelona, Spain;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6618-3944","authenticated-orcid":false,"given":"Maria F.","family":"Garcia-Parajo","sequence":"additional","affiliation":[{"name":"ICFO \u2013 Institut de Ciencies Fotoniques, Barcelona Institute of Science and Technology, 08860 Castelldefels, Barcelona, Spain;"},{"name":"Instituci\u00f3 Catalana de Recerca i Estudis Avan\u00e7ats, 08010 Barcelona, Spain;"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Melike","family":"Lakadamyali","sequence":"additional","affiliation":[{"name":"ICFO \u2013 Institut de Ciencies Fotoniques, Barcelona Institute of Science and Technology, 08860 Castelldefels, Barcelona, Spain;"},{"name":"Department of Physiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2018,12,3]]},"reference":[{"key":"e_1_3_4_1_2","doi-asserted-by":"crossref","first-page":"1749","DOI":"10.1126\/science.1146598","article-title":"Multicolor super-resolution imaging with photo-switchable fluorescent probes","volume":"317","author":"Bates M","year":"2007","unstructured":"M Bates, B Huang, GT Dempsey, X Zhuang, Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749\u20131753 (2007).","journal-title":"Science"},{"key":"e_1_3_4_2_2","doi-asserted-by":"crossref","first-page":"99","DOI":"10.1002\/cphc.201100735","article-title":"Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection","volume":"13","author":"Bates M","year":"2012","unstructured":"M Bates, GT Dempsey, KH Chen, X Zhuang, Multicolor super-resolution fluorescence imaging via multi-parameter fluorophore detection. ChemPhysChem 13, 99\u2013107 (2012).","journal-title":"ChemPhysChem"},{"key":"e_1_3_4_3_2","doi-asserted-by":"crossref","first-page":"e101772","DOI":"10.1371\/journal.pone.0101772","article-title":"Cross-talk-free multi-color STORM imaging using a single fluorophore","volume":"9","author":"Tam J","year":"2014","unstructured":"J Tam, GA Cordier, JS Borbely, A Sandoval \u00c1lvarez, M Lakadamyali, Cross-talk-free multi-color STORM imaging using a single fluorophore. PLoS One 9, e101772 (2014).","journal-title":"PLoS One"},{"key":"e_1_3_4_4_2","doi-asserted-by":"crossref","first-page":"e0123941","DOI":"10.1371\/journal.pone.0123941","article-title":"Sequential superresolution imaging of multiple targets using a single fluorophore","volume":"10","author":"Valley CC","year":"2015","unstructured":"CC Valley, S Liu, DS Lidke, KA Lidke, Sequential superresolution imaging of multiple targets using a single fluorophore. PLoS One 10, e0123941 (2015).","journal-title":"PLoS One"},{"key":"e_1_3_4_5_2","doi-asserted-by":"crossref","first-page":"1027","DOI":"10.1038\/nmeth.1768","article-title":"Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging","volume":"8","author":"Dempsey GT","year":"2011","unstructured":"GT Dempsey, JC Vaughan, KH Chen, M Bates, X Zhuang, Evaluation of fluorophores for optimal performance in localization-based super-resolution imaging. Nat Methods 8, 1027\u20131036 (2011).","journal-title":"Nat Methods"},{"key":"e_1_3_4_6_2","doi-asserted-by":"crossref","first-page":"3106","DOI":"10.3390\/molecules16043106","article-title":"Chemically induced photoswitching of fluorescent probes\u2013A general concept for super-resolution microscopy","volume":"16","author":"Endesfelder U","year":"2011","unstructured":"U Endesfelder, , Chemically induced photoswitching of fluorescent probes\u2013A general concept for super-resolution microscopy. Molecules 16, 3106\u20133118 (2011).","journal-title":"Molecules"},{"key":"e_1_3_4_7_2","doi-asserted-by":"crossref","first-page":"229","DOI":"10.1111\/boc.201100011","article-title":"Multi-colour direct STORM with red emitting carbocyanines","volume":"104","author":"Lampe A","year":"2012","unstructured":"A Lampe, V Haucke, SJ Sigrist, M Heilemann, J Schmoranzer, Multi-colour direct STORM with red emitting carbocyanines. Biol Cell 104, 229\u2013237 (2012).","journal-title":"Biol Cell"},{"key":"e_1_3_4_8_2","doi-asserted-by":"crossref","first-page":"991","DOI":"10.1038\/nprot.2011.336","article-title":"Direct stochastic optical reconstruction microscopy with standard fluorescent probes","volume":"6","author":"van de Linde S","year":"2011","unstructured":"S van de Linde, , Direct stochastic optical reconstruction microscopy with standard fluorescent probes. Nat Protoc 6, 991\u20131009 (2011).","journal-title":"Nat Protoc"},{"key":"e_1_3_4_9_2","doi-asserted-by":"crossref","first-page":"590","DOI":"10.1038\/nphoton.2016.137","article-title":"Multicolour localization microscopy by point-spread-function engineering","volume":"10","author":"Shechtman Y","year":"2016","unstructured":"Y Shechtman, LE Weiss, AS Backer, MY Lee, WE Moerner, Multicolour localization microscopy by point-spread-function engineering. Nat Photonics 10, 590\u2013594 (2016).","journal-title":"Nat Photonics"},{"key":"e_1_3_4_10_2","doi-asserted-by":"crossref","first-page":"935","DOI":"10.1038\/nmeth.3528","article-title":"Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy","volume":"12","author":"Zhang Z","year":"2015","unstructured":"Z Zhang, SJ Kenny, M Hauser, W Li, K Xu, Ultrahigh-throughput single-molecule spectroscopy and spectrally resolved super-resolution microscopy. Nat Methods 12, 935\u2013938 (2015).","journal-title":"Nat Methods"},{"key":"e_1_3_4_11_2","doi-asserted-by":"crossref","first-page":"10411","DOI":"10.1038\/ncomms10411","article-title":"Broadband single-molecule excitation spectroscopy","volume":"7","author":"Piatkowski L","year":"2016","unstructured":"L Piatkowski, E Gellings, NF van Hulst, Broadband single-molecule excitation spectroscopy. Nat Commun 7, 10411 (2016).","journal-title":"Nat Commun"},{"key":"e_1_3_4_12_2","doi-asserted-by":"crossref","first-page":"313","DOI":"10.1038\/nmeth.2835","article-title":"Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT","volume":"11","author":"Jungmann R","year":"2014","unstructured":"R Jungmann, , Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT. Nat Methods 11, 313\u2013318 (2014).","journal-title":"Nat Methods"},{"key":"e_1_3_4_13_2","doi-asserted-by":"crossref","first-page":"1198","DOI":"10.1038\/nprot.2017.024","article-title":"Super-resolution microscopy with DNA-PAINT","volume":"12","author":"Schnitzbauer J","year":"2017","unstructured":"J Schnitzbauer, MT Strauss, T Schlichthaerle, F Schueder, R Jungmann, Super-resolution microscopy with DNA-PAINT. Nat Protoc 12, 1198\u20131228 (2017).","journal-title":"Nat Protoc"},{"key":"e_1_3_4_14_2","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1038\/nmeth.3804","article-title":"Quantitative super-resolution imaging with qPAINT","volume":"13","author":"Jungmann R","year":"2016","unstructured":"R Jungmann, , Quantitative super-resolution imaging with qPAINT. Nat Methods 13, 439\u2013442 (2016).","journal-title":"Nat Methods"},{"key":"e_1_3_4_15_2","doi-asserted-by":"crossref","first-page":"6428","DOI":"10.1021\/acs.nanolett.7b03425","article-title":"Fast, background-free DNA-PAINT imaging using FRET-based probes","volume":"17","author":"Auer A","year":"2017","unstructured":"A Auer, MT Strauss, T Schlichthaerle, R Jungmann, Fast, background-free DNA-PAINT imaging using FRET-based probes. Nano Lett 17, 6428\u20136434 (2017).","journal-title":"Nano Lett"},{"key":"e_1_3_4_16_2","doi-asserted-by":"crossref","first-page":"5346","DOI":"10.1073\/pnas.0501606102","article-title":"Color-blind fluorescence detection for four-color DNA sequencing","volume":"102","author":"Lewis EK","year":"2005","unstructured":"EK Lewis, , Color-blind fluorescence detection for four-color DNA sequencing. Proc Natl Acad Sci USA 102, 5346\u20135351 (2005).","journal-title":"Proc Natl Acad Sci USA"},{"key":"e_1_3_4_17_2","doi-asserted-by":"crossref","first-page":"603","DOI":"10.1016\/0968-4328(93)90038-3","article-title":"Confocal scanning microfluorometry of dual-labelled specimens using two excitation wavelengths and lock-in detection technique","volume":"24","author":"\u00c5slund N","year":"1993","unstructured":"N \u00c5slund, K Carlsson, Confocal scanning microfluorometry of dual-labelled specimens using two excitation wavelengths and lock-in detection technique. Micron 24, 603\u2013609 (1993).","journal-title":"Micron"},{"key":"e_1_3_4_18_2","doi-asserted-by":"crossref","first-page":"5809","DOI":"10.1364\/OE.23.005809","article-title":"Frequency- and spectrally-encoded confocal microscopy","volume":"23","author":"Hwang J","year":"2015","unstructured":"J Hwang, , Frequency- and spectrally-encoded confocal microscopy. Opt Express 23, 5809\u20135821 (2015).","journal-title":"Opt Express"},{"key":"e_1_3_4_19_2","doi-asserted-by":"crossref","first-page":"10221","DOI":"10.1364\/OE.22.010221","article-title":"Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging","volume":"22","author":"Zhao M","year":"2014","unstructured":"M Zhao, Y Li, L Peng, Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging. Opt Express 22, 10221\u201310232 (2014).","journal-title":"Opt Express"},{"key":"e_1_3_4_20_2","doi-asserted-by":"crossref","first-page":"725","DOI":"10.1016\/j.bpj.2018.07.008","article-title":"Frequency-encoded multicolor fluorescence imaging with single-photon-counting color-blind detection","volume":"115","author":"Garbacik ET","year":"2018","unstructured":"ET Garbacik, M Sanz-Paz, KJE Borgman, F Campelo, MF Garcia-Parajo, Frequency-encoded multicolor fluorescence imaging with single-photon-counting color-blind detection. Biophys J 115, 725\u2013736 (2018).","journal-title":"Biophys J"},{"key":"e_1_3_4_21_2","doi-asserted-by":"crossref","first-page":"793","DOI":"10.1038\/nmeth929","article-title":"Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM)","volume":"3","author":"Rust MJ","year":"2006","unstructured":"MJ Rust, M Bates, X Zhuang, Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat Methods 3, 793\u2013795 (2006).","journal-title":"Nat Methods"},{"key":"e_1_3_4_22_2","doi-asserted-by":"crossref","first-page":"810","DOI":"10.1126\/science.1153529","article-title":"Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy","volume":"319","author":"Huang B","year":"2008","unstructured":"B Huang, W Wang, M Bates, X Zhuang, Three-dimensional super-resolution imaging by stochastic optical reconstruction microscopy. Science 319, 810\u2013813 (2008).","journal-title":"Science"},{"key":"e_1_3_4_23_2","doi-asserted-by":"crossref","first-page":"373","DOI":"10.1038\/nmeth.1449","article-title":"Fast, single-molecule localization that achieves theoretically minimum uncertainty","volume":"7","author":"Smith CS","year":"2010","unstructured":"CS Smith, N Joseph, B Rieger, KA Lidke, Fast, single-molecule localization that achieves theoretically minimum uncertainty. Nat Methods 7, 373\u2013375 (2010).","journal-title":"Nat Methods"},{"key":"e_1_3_4_24_2","doi-asserted-by":"crossref","first-page":"e0128135","DOI":"10.1371\/journal.pone.0128135","article-title":"Quantifying and optimizing single-molecule switching nanoscopy at high speeds","volume":"10","author":"Lin Y","year":"2015","unstructured":"Y Lin, , Quantifying and optimizing single-molecule switching nanoscopy at high speeds. PLoS One 10, e0128135 (2015).","journal-title":"PLoS One"},{"key":"e_1_3_4_25_2","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1038\/nmeth.1296","article-title":"A bright and photostable photoconvertible fluorescent protein","volume":"6","author":"McKinney SA","year":"2009","unstructured":"SA McKinney, CS Murphy, KL Hazelwood, MW Davidson, LL Looger, A bright and photostable photoconvertible fluorescent protein. Nat Methods 6, 131\u2013133 (2009).","journal-title":"Nat Methods"},{"key":"e_1_3_4_26_2","doi-asserted-by":"crossref","first-page":"993","DOI":"10.1146\/annurev.biochem.77.061906.092014","article-title":"Super-resolution fluorescence microscopy","volume":"78","author":"Huang B","year":"2009","unstructured":"B Huang, M Bates, X Zhuang, Super-resolution fluorescence microscopy. Annu Rev Biochem 78, 993\u20131016 (2009).","journal-title":"Annu Rev Biochem"},{"key":"e_1_3_4_27_2","doi-asserted-by":"crossref","first-page":"843","DOI":"10.1016\/j.neuron.2010.11.021","article-title":"Superresolution imaging of chemical synapses in the brain","volume":"68","author":"Dani A","year":"2010","unstructured":"A Dani, B Huang, J Bergan, C Dulac, X Zhuang, Superresolution imaging of chemical synapses in the brain. Neuron 68, 843\u2013856 (2010).","journal-title":"Neuron"},{"key":"e_1_3_4_28_2","doi-asserted-by":"crossref","first-page":"1184","DOI":"10.1038\/nmeth.4486","article-title":"Localization-based super-resolution imaging meets high-content screening","volume":"14","author":"Beghin A","year":"2017","unstructured":"A Beghin, , Localization-based super-resolution imaging meets high-content screening. Nat Methods 14, 1184\u20131190 (2017).","journal-title":"Nat Methods"},{"key":"e_1_3_4_29_2","doi-asserted-by":"crossref","first-page":"aaa6090","DOI":"10.1126\/science.aaa6090","article-title":"Spatially resolved, highly multiplexed RNA profiling in single cells","volume":"348","author":"Chen KH","year":"2015","unstructured":"KH Chen, AN Boettiger, JR Moffitt, S Wang, X Zhuang, Spatially resolved, highly multiplexed RNA profiling in single cells. Science 348, aaa6090 (2015).","journal-title":"Science"},{"key":"e_1_3_4_30_2","doi-asserted-by":"crossref","first-page":"21301","DOI":"10.1073\/pnas.1213818110","article-title":"Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes","volume":"109","author":"Beliveau BJ","year":"2012","unstructured":"BJ Beliveau, , Versatile design and synthesis platform for visualizing genomes with Oligopaint FISH probes. Proc Natl Acad Sci USA 109, 21301\u201321306 (2012).","journal-title":"Proc Natl Acad Sci USA"},{"key":"e_1_3_4_31_2","doi-asserted-by":"crossref","first-page":"1522","DOI":"10.1016\/j.bpj.2011.07.049","article-title":"Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells","volume":"101","author":"Gunewardene MS","year":"2011","unstructured":"MS Gunewardene, , Superresolution imaging of multiple fluorescent proteins with highly overlapping emission spectra in living cells. Biophys J 101, 1522\u20131528 (2011).","journal-title":"Biophys J"},{"key":"e_1_3_4_32_2","doi-asserted-by":"crossref","first-page":"3","DOI":"10.1016\/j.bpj.2015.05.026","article-title":"Dual-color 3D superresolution microscopy by combined spectral-demixing and biplane imaging","volume":"109","author":"Winterflood CM","year":"2015","unstructured":"CM Winterflood, E Platonova, D Albrecht, H Ewers, Dual-color 3D superresolution microscopy by combined spectral-demixing and biplane imaging. Biophys J 109, 3\u20136 (2015).","journal-title":"Biophys J"}],"container-title":["Proceedings of the National Academy of Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pnas.org\/doi\/pdf\/10.1073\/pnas.1804725115","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,6,7]],"date-time":"2022-06-07T14:30:46Z","timestamp":1654612246000},"score":1,"resource":{"primary":{"URL":"https:\/\/pnas.org\/doi\/full\/10.1073\/pnas.1804725115"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2018,12,3]]},"references-count":32,"journal-issue":{"issue":"51","published-print":{"date-parts":[[2018,12,18]]}},"alternative-id":["10.1073\/pnas.1804725115"],"URL":"https:\/\/doi.org\/10.1073\/pnas.1804725115","relation":{},"ISSN":["0027-8424","1091-6490"],"issn-type":[{"value":"0027-8424","type":"print"},{"value":"1091-6490","type":"electronic"}],"subject":[],"published":{"date-parts":[[2018,12,3]]},"assertion":[{"value":"2018-12-03","order":2,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}