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This offers the possibility to separate fluorophores particularly beneficial in case of similar excitation spectra. Here, we demonstrate the distinction of fluorescent molecules based on FLIM phasor analysis, called lifetime separation, in live\u2010cell imaging using open\u2010source software for analysis. We showcase two applications using\n                    <jats:italic>Caenorhabditis elegans<\/jats:italic>\n                    as a model system. First, we separated the highly spectrally overlapping fluorophores mCherry and mKate2 to distinctively track tagged proteins in six\u2010dimensional datasets to investigate cell division in the developing early embryo. Second, we separated fluorescence of tagged proteins of interest from masking natural autofluorescence in adult hermaphrodites. For FLIM data handling and workflow implementation, we developed the open\u2010source plugin napari\u2010flim\u2010phasor\u2010plotter to implement conversion, visualisation, analysis and reuse of FLIM data of different formats. Our work thus advances technical applications and bioimage data management and analysis in FLIM microscopy for life science research.\n                  <\/jats:p>","DOI":"10.1111\/jmi.70036","type":"journal-article","created":{"date-parts":[[2025,9,30]],"date-time":"2025-09-30T10:25:18Z","timestamp":1759227918000},"page":"91-106","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["A fluorescence lifetime separation approach for FLIM live\u2010cell imaging"],"prefix":"10.1111","volume":"301","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-8367-5163","authenticated-orcid":false,"given":"Cornelia","family":"Wetzker","sequence":"first","affiliation":[{"name":"B CUBE \u2013 Center for Molecular Bioengineering Technische Universit\u00e4t Dresden  Dresden Germany"},{"name":"Light Microscopy Facility Technology Platform Center for Molecular and Cellular Bioengineering Technische Universit\u00e4t Dresden  Dresden Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6165-4679","authenticated-orcid":false,"given":"Marcelo Leomil","family":"Zoccoler","sequence":"additional","affiliation":[{"name":"DFG Cluster of Excellence \u2018Physics of Life\u2019 (PoL) Technische Universit\u00e4t Dresden  Dresden Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5759-6496","authenticated-orcid":false,"given":"Svetlana","family":"Iarovenko","sequence":"additional","affiliation":[{"name":"Light Microscopy Facility Technology Platform Center for Molecular and Cellular Bioengineering Technische Universit\u00e4t Dresden  Dresden Germany"},{"name":"DFG Cluster of Excellence \u2018Physics of Life\u2019 (PoL) Technische Universit\u00e4t Dresden  Dresden Germany"},{"name":"Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA) Vienna BioCenter (VBC)  Vienna 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Bioengineering Technische Universit\u00e4t Dresden  Dresden Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0203-1436","authenticated-orcid":false,"given":"Thomas","family":"M\u00fcller\u2010Reichert","sequence":"additional","affiliation":[{"name":"Experimental Center Faculty of Medicine Carl Gustav Carus Technische Universit\u00e4t Dresden  Dresden Germany"},{"name":"Core Facility Cellular Imaging (CFCI) Faculty of Medicine Carl Gustav Carus Technische Universit\u00e4t Dresden  Dresden Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5949-2327","authenticated-orcid":false,"given":"Robert","family":"Haase","sequence":"additional","affiliation":[{"name":"DFG Cluster of Excellence \u2018Physics of Life\u2019 (PoL) Technische Universit\u00e4t Dresden  Dresden Germany"},{"name":"Center for Scalable Data Analytics and Artificial Intelligence (ScaDS.AI)  Dresden\/Leipzig Germany"},{"name":"Faculty of Mathematics and Computer Science Leipzig University  Leipzig Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3017-0978","authenticated-orcid":false,"given":"Gunar","family":"Fabig","sequence":"additional","affiliation":[{"name":"Experimental Center Faculty of Medicine Carl Gustav Carus Technische Universit\u00e4t Dresden  Dresden Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,9,30]]},"reference":[{"key":"e_1_2_6_2_1","first-page":"129","volume-title":"Methods in Molecular Biology","author":"Zimmermann 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