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To acquire three dimensional (3D) super-resolution images of DNA, we combined binding activated localization microscopy (BALM) using fluorescent double-stranded DNA intercalators and optical astigmatism. We quantitatively establish the advantage of bis- over mono-intercalators before demonstrating the\u00a0approach by visualizing single DNA\u00a0molecules stretched between microspheres\u00a0at various heights. Finally, the approach is applied to the more complex environment of intact and damaged metaphase chromosomes, unravelling\u00a0their\u00a0structural features.<\/jats:p>","DOI":"10.1038\/s41598-020-68892-5","type":"journal-article","created":{"date-parts":[[2020,7,27]],"date-time":"2020-07-27T06:02:20Z","timestamp":1595829740000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":15,"title":["Three-dimensional super-resolution fluorescence imaging of DNA"],"prefix":"10.1038","volume":"10","author":[{"given":"Sevim","family":"Yardimci","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3017-8964","authenticated-orcid":false,"given":"Daniel R.","family":"Burnham","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6497-6681","authenticated-orcid":false,"given":"Samantha Y. 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