{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T20:36:51Z","timestamp":1790714211818,"version":"4.1.0"},"reference-count":63,"publisher":"American Chemical Society (ACS)","issue":"35","license":[{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-029"},{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-037"},{"start":{"date-parts":[[2026,8,26]],"date-time":"2026-08-26T00:00:00Z","timestamp":1787702400000},"content-version":"stm-asf","delay-in-days":0,"URL":"https:\/\/doi.org\/10.15223\/policy-045"}],"funder":[{"DOI":"10.13039\/100000002","name":"National Institutes of Health (NIH)","doi-asserted-by":"crossref","award":["1R35GM134963"],"award-info":[{"award-number":["1R35GM134963"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000002","name":"National Institutes of Health (NIH)","doi-asserted-by":"crossref","award":["S10OD024998"],"award-info":[{"award-number":["S10OD024998"]}],"id":[{"id":"10.13039\/100000002","id-type":"DOI","asserted-by":"crossref"}]},{"DOI":"10.13039\/100000001","name":"National Science Foundation (NSF)","doi-asserted-by":"crossref","award":["DGE2146752"],"award-info":[{"award-number":["DGE2146752"]}],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,9]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Fluorescence microscopy is a foundational tool in cell biology research. Its power shines brightest when used to visualize multiple independent species simultaneously, a strategy known as multiplexing. However, the compressed nature of the visible spectrum effectively limits the number of independent species that can be multiplexed, especially in live cells and using long wavelength light to limit phototoxicity. Fluorescence lifetime imaging microscopy (FLIM) can overcome this limitation, allowing the simultaneous visualization of multiple independent species in a single spectral window. Most multiplexing strategies that rely on FLIM make use of self-labeling tags to localize a fluorophore to a specific organelle or cell structure. Although simple to execute, this strategy restricts experiments to cell types that can be transfected or engineered and does not easily support long time-lapse imaging. Here we show that cell-permeant small molecule probes\u2500which are well-suited for imaging applications due to high specificity, low toxicity, and the elimination of transfection requirements\u2500can be fine-tuned via bioorthogonal chemistry to exhibit fluorescence lifetimes that are distinct for each partner and each organelle. The lifetime differences are sufficiently separable to support FLIM multiplexing. Using this strategy, we selectively image three cell structures in live cells without genetic manipulations using a single FLIM-active dye and one spectral channel.<\/jats:p>","DOI":"10.1021\/jacs.5c22980","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T11:14:51Z","timestamp":1788261291000},"page":"37642-37650","source":"Crossref","is-referenced-by-count":2,"title":["Single-Dye, Transfection-Free\nFLIM Multiplexing via\nBioorthogonal Chemistry"],"prefix":"10.1021","volume":"148","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7118-4666","authenticated-orcid":true,"given":"Neville","family":"Dadina","sequence":"first","affiliation":[{"name":"University of California , , , ,","place":["Berkeley, California, United States, 94720"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Justin H.","family":"Kwon","sequence":"additional","affiliation":[{"name":"University of California , , , ,","place":["Berkeley, California, United States, 94720"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6254-7085","authenticated-orcid":true,"given":"Lauren","family":"Lesiak","sequence":"additional","affiliation":[{"name":"University of California , , , ,","place":["Berkeley, California, United States, 94720"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shuai","family":"Zheng","sequence":"additional","affiliation":[{"name":"University of California , , , ,","place":["Berkeley, California, United States, 94720"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tvisha","family":"Singh","sequence":"additional","affiliation":[{"name":"University of California , , , ,","place":["Berkeley, California, United States, 94720"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7988-7764","authenticated-orcid":true,"given":"Madeline","family":"Zoltek","sequence":"additional","affiliation":[{"name":"University of California , , , ,","place":["Berkeley, California, United States, 94720"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6974-3476","authenticated-orcid":true,"given":"Daniel","family":"Brauer","sequence":"additional","affiliation":[{"name":"University of California , , , ,","place":["Berkeley, California, United States, 94720"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2127-3932","authenticated-orcid":true,"given":"Alanna","family":"Schepartz","sequence":"additional","affiliation":[{"name":"University of California , , , ,","place":["Berkeley, California, United States, 94720"]},{"name":"University of California , , , ,","place":["Berkeley, California, United States, 94720"]},{"name":"University of California , , , ,","place":["Berkeley, California, United States, 94720"]},{"name":"Chan Zuckerberg Biohub , , ,","place":["San Francisco, California, United States, 94158"]},{"name":"ARC Institute , , ,","place":["Palo Alto, California, United States, 94304"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,26]]},"reference":[{"issue":"12","key":"2026090910084323700_cit1","doi-asserted-by":"publisher","first-page":"910","DOI":"10.1038\/nmeth817","article-title":"Fluorescence Microscopy","volume":"2","author":"Lichtman","year":"2005","journal-title":"Nat.\nMethods"},{"key":"2026090910084323700_cit2","doi-asserted-by":"publisher","first-page":"993","DOI":"10.1146\/annurev.biochem.77.061906.092014","article-title":"Super-Resolution Fluorescence Microscopy","volume":"78","author":"Huang","year":"2009","journal-title":"Annu. Rev. Biochem."},{"issue":"9","key":"2026090910084323700_cit3","doi-asserted-by":"publisher","first-page":"767","DOI":"10.1002\/cyto.a.22295","article-title":"Fluorescence\nMicroscopy\u2500A Historical and Technical Perspective","volume":"83","author":"Renz","year":"2013","journal-title":"Cytometry, Part A"},{"issue":"1","key":"2026090910084323700_cit4","doi-asserted-by":"publisher","first-page":"86","DOI":"10.1038\/nbt765","article-title":"A General\nMethod for the Covalent Labeling of Fusion\nProteins with Small Molecules in Vivo","volume":"21","author":"Keppler","year":"2003","journal-title":"Nat.\nBiotechnol."},{"issue":"2","key":"2026090910084323700_cit5","doi-asserted-by":"publisher","first-page":"128","DOI":"10.1016\/j.chembiol.2008.01.007","article-title":"An Engineered Protein\nTag for Multiprotein Labeling in Living Cells","volume":"15","author":"Gautier","year":"2008","journal-title":"Chem. Biol."},{"issue":"6","key":"2026090910084323700_cit6","doi-asserted-by":"publisher","first-page":"373","DOI":"10.1021\/cb800025k","article-title":"HaloTag:\nA Novel\nProtein Labeling Technology for Cell Imaging and Protein Analysis","volume":"3","author":"Los","year":"2008","journal-title":"ACS Chem. Biol."},{"key":"2026090910084323700_cit7","doi-asserted-by":"publisher","first-page":"93","DOI":"10.1146\/annurev-physiol-022516-034055","article-title":"A Critical and Comparative Review\nof Fluorescent Tools for Live-Cell Imaging","volume":"79","author":"Specht","year":"2017","journal-title":"Annu. Rev. Physiol."},{"issue":"38","key":"2026090910084323700_cit8","doi-asserted-by":"publisher","first-page":"10242","DOI":"10.1002\/anie.201403349","article-title":"Super-Resolution\nImaging of the Golgi in Live Cells with a Bioorthogonal Ceramide Probe","volume":"53","author":"Erdmann","year":"2014","journal-title":"Angew. Chem., Int. 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Drug Delivery Rev."},{"issue":"1","key":"2026090910084323700_cit31","doi-asserted-by":"publisher","DOI":"10.1038\/s43586-024-00358-8","article-title":"Fluorescence Lifetime\nImaging Microscopy","volume":"4","author":"Torrado","year":"2024","journal-title":"Nat. Rev. Methods Primer"},{"issue":"12","key":"2026090910084323700_cit32","doi-asserted-by":"publisher","DOI":"10.1002\/jbio.202400450","article-title":"Fluorescence\nLifetime Imaging Techniques\u2500A\nReview on Principles, Applications and Clinical Relevance","volume":"18","author":"Shcheslavskiy","year":"2025","journal-title":"J. Biophotonics"},{"issue":"1","key":"2026090910084323700_cit33","doi-asserted-by":"publisher","first-page":"65","DOI":"10.1038\/s41592-021-01341-x","article-title":"Engineered\nHaloTag Variants for Fluorescence Lifetime\nMultiplexing","volume":"19","author":"Frei","year":"2022","journal-title":"Nat. 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Biol."},{"issue":"32","key":"2026090910084323700_cit36","doi-asserted-by":"publisher","DOI":"10.1002\/advs.202404354","article-title":"Multiplexed In Vivo Imaging with\nFluorescence Lifetime-Modulating Tags","volume":"11","author":"El Hajji","year":"2024","journal-title":"Adv.\nSci."},{"issue":"24","key":"2026090910084323700_cit37","doi-asserted-by":"publisher","first-page":"6987","DOI":"10.1016\/j.cell.2025.08.035","article-title":"Time-Resolved Fluorescent Proteins\nExpand Fluorescent Microscopy in Temporal and Spectral Domains","volume":"188","author":"Tan","year":"2025","journal-title":"Cell"},{"issue":"1","key":"2026090910084323700_cit38","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-022-17825-5","article-title":"Stimulated Emission Depletion Microscopy\nwith a Single Depletion Laser Using Five Fluorochromes and Fluorescence\nLifetime Phasor Separation","volume":"12","author":"Pisfil","year":"2022","journal-title":"Sci. Rep."},{"issue":"12","key":"2026090910084323700_cit39","doi-asserted-by":"publisher","DOI":"10.1002\/cbic.202500174","article-title":"Fluorescence\nLifetime Multiplexing\nwith Environment-Sensitive Chemigenetic Probes","volume":"26","author":"Emmert","year":"2025","journal-title":"ChemBioChem"},{"issue":"3","key":"2026090910084323700_cit40","doi-asserted-by":"publisher","first-page":"257","DOI":"10.1038\/nmeth.3740","article-title":"Multi-Target Spectrally Resolved\nFluorescence Lifetime Imaging Microscopy","volume":"13","author":"Nieho\u0308rster","year":"2016","journal-title":"Nat.\nMethods"},{"issue":"9","key":"2026090910084323700_cit41","doi-asserted-by":"publisher","first-page":"1561","DOI":"10.1021\/acscentsci.1c00703","article-title":"Bio-Orthogonal\nRed and Far-Red Fluorogenic Probes for Wash-Free Live-Cell and Super-Resolution\nMicroscopy","volume":"7","author":"Werther","year":"2021","journal-title":"ACS Cent. Sci."},{"issue":"49","key":"2026090910084323700_cit42","doi-asserted-by":"publisher","first-page":"22140","DOI":"10.1002\/anie.202008757","article-title":"Fluorescence\nQuenching Effects of Tetrazines and Their Diels\u2013Alder Products:\nMechanistic Insight Toward Fluorogenic Efficiency","volume":"59","author":"Pinto-Pacheco","year":"2020","journal-title":"Angew. Chem., Int. Ed."},{"issue":"18","key":"2026090910084323700_cit43","doi-asserted-by":"publisher","first-page":"7012","DOI":"10.1039\/D1QM00852H","article-title":"A Unified Fluorescence Quenching\nMechanism of Tetrazine-Based Fluorogenic\nDyes: Energy Transfer to a Dark State","volume":"5","author":"Chi","year":"2021","journal-title":"Mater.\nChem. Front."},{"issue":"9","key":"2026090910084323700_cit44","doi-asserted-by":"publisher","first-page":"796","DOI":"10.1021\/cbmi.3c00091","article-title":"Chemical Regulation of Fluorescence Lifetime","volume":"1","author":"Dai","year":"2023","journal-title":"Chem. Biomed. 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Soc."},{"issue":"49","key":"2026090910084323700_cit47","doi-asserted-by":"publisher","first-page":"22140","DOI":"10.1002\/anie.202008757","article-title":"Fluorescence\nQuenching Effects of Tetrazines and Their Diels\u2013Alder Products:\nMechanistic Insight Toward Fluorogenic Efficiency","volume":"59","author":"Pinto-Pacheco","year":"2020","journal-title":"Angew. Chem., Int. Ed."},{"issue":"42","key":"2026090910084323700_cit48","doi-asserted-by":"publisher","first-page":"8043","DOI":"10.1039\/c0cc03078c","article-title":"Tetrazine\u2013Trans-Cyclooctene\nLigation for the\nRapid Construction of 18F Labeled Probes","volume":"46","author":"Li","year":"2010","journal-title":"Chem.\nCommun."},{"issue":"5","key":"2026090910084323700_cit49","doi-asserted-by":"publisher","first-page":"1334","DOI":"10.1002\/anie.201610491","article-title":"Mechanism-Based Fluorogenic\nTrans-Cyclooctene\u2013Tetrazine Cycloaddition","volume":"56","author":"Va\u0301zquez","year":"2017","journal-title":"Angew. 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