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Chromatin exhibits a hierarchical organization with structural compartments and domains at different length scales, from nanometers to micrometers. Single molecule localization microscopy (SMLM) methods, such as STORM, are essential for studying chromatin at the supra-nucleosome level due to their ability to target epigenetic marks that determine chromatin organization. Multi-label imaging of chromatin is necessary to unpack its structural complexity. However, these efforts are challenged by the high-density nuclear environment, which can affect antibody binding affinities, diffusivity and non-specific interactions. Optimizing buffer conditions, fluorophore stability, and antibody specificity is crucial for achieving effective antibody conjugates. Here, we demonstrate a sequential immunolabeling protocol that reliably enables three-color studies within the dense nuclear environment. This protocol couples multiplexed localization datasets with a robust analysis algorithm, which utilizes localizations from one target as seed points for distance, density and multi-label joint affinity measurements to explore complex organization of all three targets. Applying this multiplexed algorithm to analyze distance and joint density reveals that heterochromatin and euchromatin are not-distinct territories, but that localization of transcription and euchromatin couple with the periphery of heterochromatic clusters. This work is a crucial step in molecular imaging of the dense nuclear environment as multi-label capacity enables for investigation of complex multi-component systems like chromatin with enhanced accuracy.<\/jats:p>","DOI":"10.1038\/s41377-025-01786-1","type":"journal-article","created":{"date-parts":[[2025,3,16]],"date-time":"2025-03-16T22:45:31Z","timestamp":1742165131000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":17,"title":["Three-color single-molecule localization microscopy in chromatin"],"prefix":"10.1038","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-9278-7491","authenticated-orcid":false,"given":"Nicolas","family":"Acosta","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ruyi","family":"Gong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1110-2822","authenticated-orcid":false,"given":"Yuanzhe","family":"Su","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3007-9197","authenticated-orcid":false,"given":"Jane","family":"Frederick","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Karla I.","family":"Medina","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wing Shun","family":"Li","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0006-2034-3870","authenticated-orcid":false,"given":"Kiana","family":"Mohammadian","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Luay","family":"Almassalha","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Geng","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1981-1818","authenticated-orcid":false,"given":"Vadim","family":"Backman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,3,17]]},"reference":[{"key":"1786_CR1","doi-asserted-by":"publisher","unstructured":"Bond, C. et al. 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