Nanobodies Equipped with HaloTag Enable Scalable Fluorescence-Lifetime Multiplexing.
14 affiliations
- 1Institute of Neuro-And Sensory Physiology, University Medical Center Göttingen, Göttingen37073, Germany.
- 2Center for Biostructural Imaging of Neurodegeneration (BIN), University Medical Center Göttingen, Göttingen37075, Germany.
- 3III. Institute of Physics - Biophysics, Georg August University Göttingen, Göttingen37077, Germany.
- 4Department of Chemistry and Center for NanoScience, Ludwig-Maximilians-Universität, München81377, Germany.
- 5Institute for Neuroimmunology and Multiple Sclerosis Research, University Medical Center Göttingen, Göttingen37075, Germany.
- 6Max Planck Institute of Molecular Cell Biology and Genetics, Dresden01307, Germany.
- 7VIB Bioimaging Core Leuven, VIB Technologies , Leuven3000, Belgium.
- 8Department of Neurosciences, VIB-KU Leuven Centre for Brain Disease Research, KU Leuven, Leuven3000Belgium.
- 9Institute of Organic and Biomolecular Chemistry, University of Göttingen, Göttingen37077, Germany.
- 10Department of Chemistry and Applied Biosciences, ETH Zürich, Zürich8093, Switzerland.
- 11Cluster of Excellence "Multiscale Bioimaging: From Molecular Machines to Networks of Excitable Cells" (MBExC), University of Göttingen, Göttingen37075, Germany.
- 12Leibniz-Forschungsinstitut für Molekulare Pharmakologie (FMP) , Berlin13125, Germany.
- 13Multiscale Biology, Faculty of Biology and Psychology, Georg-August-University Göttingen, Göttingen37077, Germany.
- 14NanoTag Biotechnologies GmbH, Göttingen37079, Germany.
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.
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BibTeX
@article{albert2026nanobodies,
title = {{Nanobodies Equipped with HaloTag Enable Scalable Fluorescence-Lifetime Multiplexing.}},
author = {László Albert and Samrat Basak and Henrike Körner and Nazar Oleksiievets and Nikolaos Mougios and Elena R Cotroneo and Michelle S Frei and Jörg Enderlein and Johannes Broichhagen and Nadja A Simeth and Roman Tsukanov and Felipe Opazo},
journal = {ACS nano},
year = {2026},
month = sep,
doi = {10.1021/acsnano.6c08378},
url = {https://europepmc.org/article/MED/42916070},
note = {CC BY},
}
RIS
TY - JOUR
TI - Nanobodies Equipped with HaloTag Enable Scalable Fluorescence-Lifetime Multiplexing.
AU - László Albert
AU - Samrat Basak
AU - Henrike Körner
AU - Nazar Oleksiievets
AU - Nikolaos Mougios
AU - Elena R Cotroneo
AU - Michelle S Frei
AU - Jörg Enderlein
AU - Johannes Broichhagen
AU - Nadja A Simeth
AU - Roman Tsukanov
AU - Felipe Opazo
PY - 2026
DA - 2026/09/01
JO - ACS nano
DO - 10.1021/acsnano.6c08378
UR - https://europepmc.org/article/MED/42916070
AB - 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.
SN - 1936-0851
SN - 1936-086X
ER -