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By simultaneously capturing the spatial positions and spectroscopic signatures of individual fluorescent molecules, srSMLM opens up the possibility of investigating an additional dimension in super\u2010resolution imaging. However, appropriate and dedicated tools are required to fully capitalize on the spectral dimension. Here, we propose the application of the spectral phasor analysis as an effective method for summarizing and analyzing the spectral information obtained from srSMLM experiments. The spectral phasor condenses the complete spectrum of a single emitter into a two\u2010dimensional space, preserving key spectral characteristics for single\u2010molecule spectral exploration. We demonstrate the effectiveness of spectral phasor in efficiently classifying single Nile Red fluorescence emissions from largely overlapping cyanine fluorescence signals in dual\u2010color PAINT experiments. Additionally, we employed spectral phasor with srSMLM to reveal subtle alterations occurring in the membrane of Gram\u2010positive <jats:italic>Enterococcus hirae<\/jats:italic> in response to gramicidin exposure, a membrane\u2010perturbing antibiotic treatment. Spectral phasor provides a robust, model\u2010free analytic tool for the detailed analysis of the spectral component of srSMLM, enhancing the capabilities of multi\u2010color spectrally\u2010resolved single\u2010molecule imaging.<\/jats:p>","DOI":"10.1002\/cphc.202400101","type":"journal-article","created":{"date-parts":[[2024,4,2]],"date-time":"2024-04-02T11:50:11Z","timestamp":1712058611000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Spectral Phasor Applied to Spectrally\u2010Resolved Single Molecule Localization Microscopy"],"prefix":"10.1002","volume":"25","author":[{"given":"Hanna","family":"Manko","sequence":"first","affiliation":[{"name":"Laboratoire de BioImagerie et Pathologies UMR CNRS 7021 ITI InnoVec Universit\u00e9 de Strasbourg  Illkirch France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew G.","family":"Burton","sequence":"additional","affiliation":[{"name":"Laboratoire de BioImagerie et Pathologies UMR CNRS 7021 ITI InnoVec Universit\u00e9 de Strasbourg  Illkirch France"},{"name":"Faculty of Pharmacy and Pharmaceutical Sciences Monash University  Parkville Victoria Australia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yves","family":"M\u00e9ly","sequence":"additional","affiliation":[{"name":"Laboratoire de BioImagerie et Pathologies UMR CNRS 7021 ITI InnoVec Universit\u00e9 de Strasbourg  Illkirch France"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8470-0092","authenticated-orcid":false,"given":"Julien","family":"Godet","sequence":"additional","affiliation":[{"name":"Laboratoire iCube UMR CNRS 7357 Equipe IMAGeS Universit\u00e9 de Strasbourg  Strasbourg France"},{"name":"Groupe M\u00e9thodes Recherche Clinique H\u00f4pitaux Universitaires de trasbourg  France"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2024,5,18]]},"reference":[{"key":"e_1_2_8_1_1","doi-asserted-by":"publisher","DOI":"10.1126\/science.1127344"},{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth929"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.chemrev.6b00729"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3528"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms12290"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0147506"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1038\/ncomms13544"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bpj.2017.11.013"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.biocel.2018.06.002"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1021\/acs.accounts.7b00545"},{"key":"e_1_2_8_11_1","volume":"94","author":"Song K.-H.","year":"2023","journal-title":"Rev. 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