{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T11:19:00Z","timestamp":1785410340441,"version":"3.56.0"},"posted":{"date-parts":[[2026,4,16]]},"group-title":"Bioengineering","reference-count":49,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,4,16]],"date-time":"2026-04-16T00:00:00Z","timestamp":1776297600000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"funder":[{"award":["CHE-2246548"],"award-info":[{"award-number":["CHE-2246548"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"award":["CHE-2441081"],"award-info":[{"award-number":["CHE-2441081"]}],"id":[{"id":"https:\/\/ror.org\/021nxhr62","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R21GM141675"],"award-info":[{"award-number":["R21GM141675"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]},{"award":["R35GM15524"],"award-info":[{"award-number":["R35GM15524"]}],"id":[{"id":"https:\/\/ror.org\/04q48ey07","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,4,16]]},"abstract":"<jats:title>ABSTRACT<\/jats:title>\n                <jats:p>\n                  Simultaneous multiplexed super-resolution imaging remains a central challenge in single-molecule localization microscopy (SMLM), due to the limited number of spectrally distinguishable fluorophores and the trade-off between spatial and spectral precision. 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,\n                  <jats:italic>in vitro<\/jats:italic>\n                  and fixed-cell imaging, we show that sDNA-PAINT conditions significantly improve spectral precision and 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 dual-target cellular imaging, sDNA-PAINT achieves accurate spatial reconstruction and high classification accuracy, demonstrating high-fidelity simultaneous multiplexing within a single acquisition. sDNA-PAINT provides a pathway toward high-throughput simultaneous multiplexed super-resolution interaction imaging.\n                <\/jats:p>","DOI":"10.64898\/2026.04.14.718276","type":"posted-content","created":{"date-parts":[[2026,4,17]],"date-time":"2026-04-17T03:40:19Z","timestamp":1776397219000},"source":"Crossref","is-referenced-by-count":0,"title":["Spectroscopic DNA-PAINT for simultaneous multiplexed super-resolution microscopy"],"prefix":"10.64898","author":[{"given":"Md Abul","family":"Shahid","sequence":"first","affiliation":[{"name":"Molecular Analytics and Photonics (MAP) Lab, Department of Textile Engineering, Chemistry and Science, North Carolina State University, Raleigh, NC 27606"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kripa","family":"Patel","sequence":"additional","affiliation":[{"name":"Molecular Analytics and Photonics (MAP) Lab, Department of Textile Engineering, Chemistry and Science, North Carolina State University, Raleigh, NC 27606"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David A.","family":"Miller","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering, Duke University, Durham, NC 27708"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1011-3001","authenticated-orcid":false,"given":"Yang","family":"Zhang","sequence":"additional","affiliation":[{"name":"Molecular Analytics and Photonics (MAP) Lab, Department of Textile Engineering, Chemistry and Science, North Carolina State University, Raleigh, NC 27606"},{"name":"Lampe Joint Department of Biomedical Engineering, North Carolina State University and University of North 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