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Here, we introduce spectroscopic DNA-PAINT (sDNA-PAINT), a framework that integrates DNA-PAINT with spectroscopic SMLM (sSMLM) to enable simultaneous multiplexed imaging with high spatial and spectral fidelity. Using DNA origami nanorulers, in vitro and fixed-cell imaging, we show that sDNA-PAINT conditions significantly reduce spectral uncertainty and enhance photon budgets compared to conventional glass and antibody-conjugated conditions in sSMLM imaging. Across representative dyes from three fluorophore families (rhodamine, cyanine, and oxazine), narrow spectral centroid distributions are observed to enable reliable statistical discrimination at the single-molecule level, even for dyes with heavily overlapping ensemble spectra. In four-color subcellular imaging, sDNA-PAINT achieves accurate spatial reconstruction and high classification accuracy within a single acquisition. sDNA-PAINT provides a pathway toward high-fidelity simultaneous multiplexed super-resolution interaction imaging.<\/jats:p>","DOI":"10.1021\/acs.nanolett.6c01802","type":"journal-article","created":{"date-parts":[[2026,8,10]],"date-time":"2026-08-10T14:02:36Z","timestamp":1786370556000},"page":"10651-10659","source":"Crossref","is-referenced-by-count":1,"title":["Spectroscopic DNA-PAINT for Simultaneous Multiplexed\nSuper-Resolution Microscopy"],"prefix":"10.1021","volume":"26","author":[{"given":"Md Abul","family":"Shahid","sequence":"first","affiliation":[{"name":"North Carolina State University , , , ,","place":["Raleigh, North Carolina, United States, 27606"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shohag Chandra","family":"Das","sequence":"additional","affiliation":[{"name":"North Carolina State University , , , ,","place":["Raleigh, North Carolina, United States, 27606"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kripa","family":"Patel","sequence":"additional","affiliation":[{"name":"North Carolina State University , , , ,","place":["Raleigh, North Carolina, United States, 27606"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiayi","family":"Gong","sequence":"additional","affiliation":[{"name":"North Carolina State University , , , ,","place":["Raleigh, North Carolina, United States, 27606"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongjing","family":"Mao","sequence":"additional","affiliation":[{"name":"North Carolina State University , , , ,","place":["Raleigh, North Carolina, United States, 27606"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David A.","family":"Miller","sequence":"additional","affiliation":[{"name":"Duke University , , , ,","place":["Durham, North Carolina, United States, 27708"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1011-3001","authenticated-orcid":true,"given":"Yang","family":"Zhang","sequence":"additional","affiliation":[{"name":"North Carolina State University , , , ,","place":["Raleigh, North Carolina, United States, 27606"]},{"name":"North Carolina State University and University of North Carolina at Chapel Hill , , , ,","place":["Raleigh, North Carolina, United States, 27695"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,10]]},"reference":[{"issue":"1","key":"2026081910183783300_cit1","doi-asserted-by":"publisher","first-page":"1","DOI":"10.1038\/s43586-021-00038-x","article-title":"Single-Molecule Localization\nMicroscopy","volume":"1","author":"Lelek","year":"2021","journal-title":"Nat. Rev. Methods Primer"},{"issue":"18","key":"2026081910183783300_cit2","doi-asserted-by":"publisher","first-page":"9412","DOI":"10.1021\/acs.chemrev.7b00767","article-title":"Switchable\nFluorophores for Single-Molecule Localization\nMicroscopy","volume":"118","author":"Li","year":"2018","journal-title":"Chem. Rev."},{"issue":"5793","key":"2026081910183783300_cit3","doi-asserted-by":"publisher","first-page":"1642","DOI":"10.1126\/science.1127344","article-title":"Imaging Intracellular\nFluorescent Proteins at Nanometer\nResolution","volume":"313","author":"Betzig","year":"2006","journal-title":"Science"},{"issue":"43","key":"2026081910183783300_cit4","doi-asserted-by":"publisher","first-page":"17436","DOI":"10.1073\/pnas.1215175109","article-title":"Counting Single\nPhotoactivatable Fluorescent Molecules by Photoactivated Localization\nMicroscopy (PALM)","volume":"109","author":"Lee","year":"2012","journal-title":"Proc. Natl. Acad. Sci. U\nA"},{"issue":"6","key":"2026081910183783300_cit5","doi-asserted-by":"publisher","first-page":"2447","DOI":"10.3390\/molecules28062447","article-title":"Photoactivatable\nBODIPYs for Live-Cell PALM","volume":"28","author":"Zhang","year":"2023","journal-title":"Molecules"},{"issue":"2","key":"2026081910183783300_cit6","doi-asserted-by":"publisher","first-page":"155","DOI":"10.1038\/nmeth.1176","article-title":"High-Density\nMapping of Single-Molecule\nTrajectories with Photoactivated Localization Microscopy","volume":"5","author":"Manley","year":"2008","journal-title":"Nat. Methods"},{"issue":"5845","key":"2026081910183783300_cit7","doi-asserted-by":"publisher","first-page":"1749","DOI":"10.1126\/science.1146598","article-title":"Multicolor Super-Resolution\nImaging with Photo-Switchable Fluorescent Probes","volume":"317","author":"Bates","year":"2007","journal-title":"Science"},{"issue":"12","key":"2026081910183783300_cit8","doi-asserted-by":"publisher","first-page":"1027","DOI":"10.1038\/nmeth.1768","article-title":"Evaluation\nof Fluorophores for Optimal Performance in Localization-Based Super-Resolution\nImaging","volume":"8","author":"Dempsey","year":"2011","journal-title":"Nat. Meth"},{"key":"2026081910183783300_cit9","doi-asserted-by":"publisher","first-page":"1047","DOI":"10.1038\/nmeth.1274","article-title":"Whole-Cell 3D STORM\nReveals Interactions between Cellular Structures with Nanometer-Scale\nResolution","volume":"5","author":"Huang","year":"2008","journal-title":"Nat. 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Imaging"},{"issue":"12","key":"2026081910183783300_cit28","doi-asserted-by":"publisher","DOI":"10.1063\/1.5054144","article-title":"Theoretical Analysis\nof Spectral Precision in Spectroscopic Single-Molecule Localization\nMicroscopy","volume":"89","author":"Song","year":"2018","journal-title":"Rev. Sci. Instrum."},{"key":"2026081910183783300_cit29","doi-asserted-by":"publisher","first-page":"3914","DOI":"10.1021\/acs.jpclett.1c00192","article-title":"Investigating Single-Molecule\nFluorescence\nSpectral Heterogeneity of Rhodamines Using High-Throughput Single-Molecule\nSpectroscopy","volume":"12","author":"Zhang","year":"2021","journal-title":"J. Phys. Chem. Lett."},{"issue":"10","key":"2026081910183783300_cit30","doi-asserted-by":"publisher","first-page":"6428","DOI":"10.1021\/acs.nanolett.7b03425","article-title":"Fast, Background-Free\nDNA-PAINT Imaging Using FRET-Based Probes","volume":"17","author":"Auer","year":"2017","journal-title":"Nano\nLett."},{"issue":"6179","key":"2026081910183783300_cit31","doi-asserted-by":"publisher","first-page":"65","DOI":"10.1126\/science.1250944","article-title":"Polyhedra Self-Assembled from DNA\nTripods and Characterized with\n3D DNA-PAINT","volume":"344","author":"Iinuma","year":"2014","journal-title":"Science"},{"issue":"3","key":"2026081910183783300_cit32","doi-asserted-by":"publisher","first-page":"313","DOI":"10.1038\/nmeth.2835","article-title":"Multiplexed 3D Cellular Super-Resolution\nImaging with DNA-PAINT and\nExchange-PAINT","volume":"11","author":"Jungmann","year":"2014","journal-title":"Nat. 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Engl."},{"issue":"7","key":"2026081910183783300_cit43","doi-asserted-by":"publisher","first-page":"2682","DOI":"10.1021\/acs.nanolett.1c04520","article-title":"Simultaneous\nMulticolor DNA-PAINT without Sequential Fluid Exchange Using Spectral\nDemixing","volume":"22","author":"Gimber","year":"2022","journal-title":"Nano Lett."},{"issue":"51","key":"2026081910183783300_cit44","doi-asserted-by":"publisher","first-page":"12991","DOI":"10.1073\/pnas.1804725115","article-title":"Excitation-Multiplexed\nMulticolor Superresolution Imaging\nwith Fm-STORM and Fm-DNA-PAINT","volume":"115","author":"Go\u0301mez-Garci\u0301a","year":"2018","journal-title":"Proc. Natl.\nAcad. Sci. U. S. A."},{"issue":"9","key":"2026081910183783300_cit45","doi-asserted-by":"publisher","first-page":"1755","DOI":"10.1038\/s41592-024-02374-8","article-title":"The DNA-PAINT Palette:\nA Comprehensive Performance\nAnalysis of Fluorescent Dyes","volume":"21","author":"Steen","year":"2024","journal-title":"Nat. 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B"},{"issue":"30","key":"2026081910183783300_cit48","doi-asserted-by":"publisher","first-page":"9374","DOI":"10.1021\/jacs.8b05098","article-title":"Site-Specific\nProtein Labeling with N-Hydroxysuccinimide-Esters and the Analysis\nof Ubiquitin Ligase Mechanisms","volume":"140","author":"Dempsey","year":"2018","journal-title":"J. Am. Chem.\nSoc."},{"issue":"86","key":"2026081910183783300_cit49","doi-asserted-by":"publisher","DOI":"10.3791\/50549","article-title":"Surface Passivation for Single-Molecule Protein Studies","author":"Chandradoss","year":"2014","journal-title":"J. Vis. Exp. JoVE"},{"key":"2026081910183783300_cit50","doi-asserted-by":"publisher","first-page":"16250","DOI":"10.1021\/acs.analchem.5c01486","article-title":"Framework for Accurate Single-Molecule\nSpectroscopic Imaging Analyses Using Monte Carlo Simulation and Deep\nLearning","volume":"97","author":"Mao","year":"2025","journal-title":"Anal. Chem."},{"issue":"8","key":"2026081910183783300_cit51","doi-asserted-by":"publisher","first-page":"1527","DOI":"10.1515\/nanoph-2021-0541","article-title":"Monolithic Dual-Wedge\nPrism-Based Spectroscopic Single-Molecule\nLocalization Microscopy","volume":"11","author":"Song","year":"2022","journal-title":"Nanophotonics"},{"issue":"11","key":"2026081910183783300_cit52","doi-asserted-by":"publisher","first-page":"4462","DOI":"10.1021\/acs.jpclett.9b03833","article-title":"Self-Healing Dyes\u2500Keeping\nthe Promise?","volume":"11","author":"Isselstein","year":"2020","journal-title":"J. Phys. Chem. Lett."},{"issue":"11","key":"2026081910183783300_cit53","doi-asserted-by":"publisher","first-page":"5729","DOI":"10.1039\/D3NR05870K","article-title":"Deep-Learning-Assisted\nSpectroscopic Single-Molecule Localization Microscopy Based on Spectrum-to-Spectrum\nDenoising","volume":"16","author":"Xu","year":"2024","journal-title":"Nanoscale"},{"issue":"33","key":"2026081910183783300_cit54","doi-asserted-by":"publisher","DOI":"10.1002\/smll.202300728","article-title":"Advancing Spectrally-Resolved Single\nMolecule Localization Microscopy with Deep Learning","volume":"19","author":"Manko","year":"2023","journal-title":"Small"}],"container-title":["Nano Letters"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/pubs.acs.org\/doi\/pdf\/10.1021\/acs.nanolett.6c01802","content-type":"application\/pdf","content-version":"vor","intended-application":"unspecified"},{"URL":"https:\/\/pubs.acs.org\/nalefd\/article-pdf\/26\/32\/10651\/66475943\/acs.nanolett.6c01802.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/pubs.acs.org\/nalefd\/article-pdf\/26\/32\/10651\/66475943\/acs.nanolett.6c01802.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,19]],"date-time":"2026-08-19T14:39:59Z","timestamp":1787150399000},"score":1,"resource":{"primary":{"URL":"https:\/\/pubs.acs.org\/nalefd\/article\/26\/32\/10651\/5251383\/Spectroscopic-DNA-PAINT-for-Simultaneous"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,10]]},"references-count":54,"journal-issue":{"issue":"32","published-online":{"date-parts":[[2026,8,10]]},"published-print":{"date-parts":[[2026,8,19]]}},"URL":"https:\/\/doi.org\/10.1021\/acs.nanolett.6c01802","relation":{},"ISSN":["1530-6984","1530-6992"],"issn-type":[{"value":"1530-6984","type":"print"},{"value":"1530-6992","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,10]]}}}