{
  "abstract": "Fluorescence lifetime (FL) is a robust strategy for multiplexed imaging; however, variability in fluorophores' local microenvironment when used on affinity probes broadens lifetime distributions, limiting its practical applicability. We create a modular platform for FL encoding with nanobody-HaloTag 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.",
  "abstract_shown": true,
  "affiliations": [
    "Institute of Neuro-And Sensory Physiology, University Medical Center Göttingen, Göttingen37073, Germany.",
    "Center for Biostructural Imaging of Neurodegeneration (BIN), University Medical Center Göttingen, Göttingen37075, Germany.",
    "III. Institute of Physics - Biophysics, Georg August University Göttingen, Göttingen37077, Germany.",
    "Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität, München81377, Germany.",
    "Institute for Neuroimmunology and Multiple Sclerosis Research, University Medical Center Göttingen, Göttingen37075, Germany.",
    "Max Planck Institute of Molecular Cell Biology and Genetics, Dresden01307, Germany.",
    "VIB Bioimaging Core Leuven, VIB Technologies , Leuven3000, Belgium.",
    "Department of Neurosciences, VIB-KU Leuven Centre for Brain Disease Research, KU Leuven, Leuven3000Belgium.",
    "Institute of Organic and Biomolecular Chemistry, University of Göttingen, Göttingen37077, Germany.",
    "Department of Chemistry and Applied Biosciences, ETH Zürich, Zürich8093, Switzerland.",
    "Cluster of Excellence \"Multiscale Bioimaging: From Molecular Machines to Networks of Excitable Cells\" (MBExC), University of Göttingen, Göttingen37075, Germany.",
    "Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) , Berlin13125, Germany.",
    "Multiscale Biology, Faculty of Biology and Psychology, Georg-August-University Göttingen, Göttingen37077, Germany.",
    "NanoTag Biotechnologies GmbH, Göttingen37079, Germany."
  ],
  "article_type": "research article",
  "arxiv": null,
  "author_total": 12,
  "authors": [
    {
      "affs": [
        0,
        1
      ],
      "name": "László Albert"
    },
    {
      "affs": [
        2,
        3
      ],
      "name": "Samrat Basak",
      "orcid": "0000-0002-1609-8642"
    },
    {
      "affs": [
        4
      ],
      "name": "Henrike Körner"
    },
    {
      "affs": [
        2,
        5
      ],
      "name": "Nazar Oleksiievets"
    },
    {
      "affs": [
        6,
        7
      ],
      "name": "Nikolaos Mougios"
    },
    {
      "affs": [
        8
      ],
      "name": "Elena R Cotroneo"
    },
    {
      "affs": [
        9
      ],
      "name": "Michelle S Frei",
      "orcid": "0000-0002-4799-4554"
    },
    {
      "affs": [
        2,
        10
      ],
      "name": "Jörg Enderlein",
      "orcid": "0000-0001-5091-7157"
    },
    {
      "affs": [
        11
      ],
      "name": "Johannes Broichhagen",
      "orcid": "0000-0003-3084-6595"
    },
    {
      "affs": [
        8
      ],
      "name": "Nadja A Simeth",
      "orcid": "0000-0001-8130-883X"
    },
    {
      "affs": [
        2,
        12
      ],
      "name": "Roman Tsukanov",
      "orcid": "0000-0002-3967-1755"
    },
    {
      "affs": [
        0,
        1,
        13
      ],
      "name": "Felipe Opazo",
      "orcid": "0000-0002-4968-9713"
    }
  ],
  "bibtex": "https://research.draconitic.com/paper/8nr0wd0txc0t6.bib",
  "cited_by": 0,
  "doi": "10.1021/acsnano.6c08378",
  "fulltext_url": "https://europepmc.org/articles/PMC13603004",
  "id": "8nr0wd0txc0t6",
  "issns": [
    "1936-0851",
    "1936-086X"
  ],
  "landing_url": "https://europepmc.org/article/MED/42916070",
  "language": "eng",
  "licence": "CC-BY",
  "licence_label": "CC BY",
  "licence_source": "article",
  "licence_url": "https://creativecommons.org/licenses/by/4.0/",
  "metadata_licence": null,
  "native_subjects": [
    [
      "openalex",
      "1304|Advanced Fluorescence Microscopy Techniques"
    ]
  ],
  "openalex": "W7207523556",
  "pdf_url": "https://europepmc.org/articles/PMC13603004?pdf=render",
  "pmcid": "PMC13603004",
  "pmid": "42916070",
  "published": "2026-09-01",
  "publisher": null,
  "ris": "https://research.draconitic.com/paper/8nr0wd0txc0t6.ris",
  "sources": [
    {
      "first_seen": "2026-10-30T12:21:02Z",
      "native_id": "MED:42916070",
      "source": "europepmc",
      "url": "https://europepmc.org/article/MED/42916070"
    }
  ],
  "subjects": [
    {
      "field": "biochemistry",
      "origin": "openalex:1304|Advanced Fluorescence Microscopy Techniques",
      "subfield": "biophysics"
    }
  ],
  "title": "Nanobodies Equipped with HaloTag Enable Scalable Fluorescence-Lifetime Multiplexing.",
  "title_text": "Nanobodies Equipped with HaloTag Enable Scalable Fluorescence-Lifetime Multiplexing.",
  "type": "peer-reviewed",
  "updated_at": "2026-10-30T12:21:02Z",
  "url": "https://research.draconitic.com/paper/8nr0wd0txc0t6",
  "venue": "ACS nano",
  "versions": []
}