{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,23]],"date-time":"2026-10-23T19:46:59Z","timestamp":1792784819111,"version":"4.5.0"},"reference-count":24,"publisher":"American Chemical Society (ACS)","issue":"37","license":[{"start":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T00:00:00Z","timestamp":1788393600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Deutsche Forschungsgemeinschaft (DFG) SFB TRR 274\/2","id":[{"id":"https:\/\/ror.org\/018mejw64","id-type":"ROR","asserted-by":"crossref"}]},{"name":"European Commission under the European Union?s Horizon 2020 research and innovation programme","id":[{"id":"https:\/\/ror.org\/00k4n6c32","id-type":"ROR","asserted-by":"crossref"}]},{"name":"European Research Council (ERC)","id":[{"id":"https:\/\/ror.org\/0472cxd90","id-type":"ROR","asserted-by":"crossref"}]},{"name":"Deutsche Forschungsgemeinschaft (DFG) SFB 1286","id":[{"id":"https:\/\/ror.org\/018mejw64","id-type":"ROR","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2026,9,22]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Fluorescence lifetime (FL) is a robust strategy for multiplexed imaging; however, variability in fluorophores\u2019 local microenvironment when used on affinity probes broadens lifetime distributions, limiting its practical applicability. We create a modular platform for FL encoding with nanobody\u2013HaloTag variants, enabling universal FL multiplexing. By characterizing dozens of fluorophores across four HaloTag variants, we generate an FL landscape that reveals tunable, consistent FL signatures within a single spectrum. Leveraging this physicochemically defined encoding, we achieve multiplexed imaging of up to eight targets in a single staining step while preserving high spatial fidelity across cellular and tissue samples. Unlike conventional antibody labeling, our approach decouples probe identity from species constraints and eliminates batch-to-batch variability in FL. These results confirm nanobody-guided FL engineering as a scalable strategy for multiplexed imaging (NanoFLex), offering a standard and straightforward framework for FL multiplexing and expanding the accessible dimensions of optical readout.<\/jats:p>","DOI":"10.1021\/acsnano.6c08378","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T22:01:50Z","timestamp":1788472910000},"page":"25442-25451","source":"Crossref","is-referenced-by-count":0,"title":["Nanobodies Equipped\nwith HaloTag Enable Scalable Fluorescence-Lifetime\nMultiplexing"],"prefix":"10.1021","volume":"20","author":[{"given":"La\u0301szlo\u0301","family":"Albert","sequence":"first","affiliation":[{"name":"University Medical Center Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37073"]},{"name":"University Medical Center Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37075"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1609-8642","authenticated-orcid":true,"given":"Samrat","family":"Basak","sequence":"additional","affiliation":[{"name":"Georg August University Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37077"]},{"name":"Ludwig-Maximilians-Universita\u0308t , , ,","place":["Mu\u0308nchen, Germany, 81377"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Henrike","family":"Ko\u0308rner","sequence":"additional","affiliation":[{"name":"University Medical Center Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37075"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nazar","family":"Oleksiievets","sequence":"additional","affiliation":[{"name":"Georg August University Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37077"]},{"name":"Max Planck Institute of Molecular Cell Biology and Genetics , ,","place":["Dresden, Germany, 01307"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nikolaos","family":"Mougios","sequence":"additional","affiliation":[{"name":"VIB Bioimaging Core Leuven, VIB Technologies , ,","place":["Leuven, Belgium, 3000"]},{"name":"VIB-KU Leuven Centre for Brain Disease Research, KU Leuven , ,","place":["Leuven, Belgium, 3000"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elena R.","family":"Cotroneo","sequence":"additional","affiliation":[{"name":"University of Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37077"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4799-4554","authenticated-orcid":true,"given":"Michelle S.","family":"Frei","sequence":"additional","affiliation":[{"name":"ETH Zu\u0308rich , , ,","place":["Zu\u0308rich, Switzerland, 8093"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5091-7157","authenticated-orcid":true,"given":"Jo\u0308rg","family":"Enderlein","sequence":"additional","affiliation":[{"name":"Georg August University Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37077"]},{"name":"University of Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37075"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3084-6595","authenticated-orcid":true,"given":"Johannes","family":"Broichhagen","sequence":"additional","affiliation":[{"name":"Leibniz-Forschungsinstitut fu\u0308r Molekulare Pharmakologie (FMP) , ,","place":["Berlin, Germany, 13125"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8130-883X","authenticated-orcid":true,"given":"Nadja A.","family":"Simeth","sequence":"additional","affiliation":[{"name":"University of Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37077"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3967-1755","authenticated-orcid":true,"given":"Roman","family":"Tsukanov","sequence":"additional","affiliation":[{"name":"Georg August University Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37077"]},{"name":"Georg-August-University Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37077"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4968-9713","authenticated-orcid":true,"given":"Felipe","family":"Opazo","sequence":"additional","affiliation":[{"name":"University Medical Center Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37073"]},{"name":"University Medical Center Go\u0308ttingen , , ,","place":["Go\u0308ttingen, Germany, 37075"]},{"name":"NanoTag Biotechnologies GmbH , ,","place":["Go\u0308ttingen, Germany, 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Biol."},{"key":"2026092210062157300_cit2","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":"2021","journal-title":"Nat. Methods"},{"issue":"3","key":"2026092210062157300_cit3","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":"25","key":"2026092210062157300_cit4","doi-asserted-by":"publisher","DOI":"10.1002\/anie.202403029","article-title":"Chemical Control of\nFluorescence Lifetime towards Multiplexing Imaging","volume":"63","author":"Ma","year":"2024","journal-title":"Angew. Chem., Int. 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