Biocompatible TADF probes for highly multiplexed fluorescence lifetime imaging

Pilar Suárez de Cepeda; Ferran Nadal-Bufi; Janine Haug; Tomas Matulaitis; Yanzi Zhou; Charles Lochenie; Quan Qi; André Jung; Dongyang Chen; Concepción González-Bello; Stefan Bräse; Eli Zysman-Colman; Marc Vendrell

doi:10.1002/anie.202521482

Biocompatible TADF probes for highly multiplexed fluorescence lifetime imaging

Pilar Suárez de Cepeda, Ferran Nadal-Bufi, Janine Haug, Tomas Matulaitis, Yanzi Zhou, Charles Lochenie, Quan Qi, André Jung, Dongyang Chen, Concepción González-Bello, Stefan Bräse^*, Eli Zysman-Colman^*, Marc Vendrell^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › peer-review

Abstract

Fluorescence lifetime imaging microscopy (FLIM) is an optical imaging modality that can provide multiplexed readouts with remarkable sensitivity to cellular microenvironments. Even though fluorescence lifetimes can distinguish fluorophores having overlapping spectral profiles, conventional fluorophores possess a narrow range of emission lifetimes (typically shorter than 5 ns) that limits their potential for multiplexed imaging. In this work, we have systematically designed and evaluated a combination of thermally activated delayed fluorescence (TADF) nanoprobes for multiplexed FLIM. We have synthesized a collection of 36 TADF biocompatible nanoprobes with long and diverse fluorescence lifetimes in aqueous media (up to 15 ns) and employed selected probes for live-cell imaging of bacterial cells under physiological conditions. By leveraging the exceptionally broad range of fluorescence lifetimes of these TADF emitters, we have achieved unprecedented simultaneous imaging of five nanoprobes within a single spectral window using a FLIM-phasor strategy. These findings demonstrate that TADF emitters are excellent scaffolds to unlock the capabilities of fluorescence lifetime imaging for multi-color biological studies.

Original language	English
Article number	e21482
Number of pages	11
Journal	Angewandte Chemie International Edition
Volume	Early View
Early online date	15 Jan 2026
DOIs	https://doi.org/10.1002/anie.202521482
Publication status	E-pub ahead of print - 15 Jan 2026

Keywords

Bacteria
Fluorescence lifetime imaging microscopy (FLIM)
Fluorophores
Nanoprobes

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10.1002/anie.202521482

Suarez-de-Cepeda_2026_ACIE_Biocompatible-TADF-probes_AAM_CCBY
Copyright © 2025 the Authors. This work has been made available online in accordance with the University of St Andrews Open Access policy. This accepted manuscript is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The final published version of this work is available at https://doi.org/10.1002/anie.202521482
Accepted author manuscript, 3.19 MBLicence: CC BY

1 Active
2 Finished

Allan Watson Programme Grant: Boron: Beyond the Reagent
Watson, A. (PI), Morris, R. (CoI), Smith, A. (CoI) & Zysman-Colman, E. (CoI)
UK Research and Innovation
1/05/23 → 30/04/28
Project: Standard
Commercialization of TADF Dendrimer: Commercialization of TADF dendrimer materials and assessment of their use in time-resolved and confocal fluorescence imaging in living cells
Zysman-Colman, E. (PI)
EPSRC
1/09/22 → 30/11/22
Project: Standard
MR TADF: Multi-resonance TADF materials for highly efficient and stable OLEDs
Zysman-Colman, E. (PI)
EPSRC
1/07/22 → 30/06/25
Project: Standard

Biocompatible TADF probes for highly multiplexed fluorescence lifetime imaging (dataset)
Suárez de Cepeda, P. (Creator), Nadal-Bufi, F. (Creator), Haug, J. J. (Creator), Matulaitis, T. (Creator), Zhou, Y. (Creator), Lochenie, C. (Creator), Qi, Q. (Creator), Jung, A. (Creator), Chen, D. (Creator), González-Bello, C. (Creator), Bräse, S. (Creator), Zysman-Colman, E. (Creator) & Vendrell, M. (Creator), University of St Andrews, 30 Jan 2026
DOI: 10.17630/ac09a45f-f5d8-4b91-9282-9bd357e8192e
Dataset

File

Cite this

Suárez de Cepeda, P., Nadal-Bufi, F., Haug, J., Matulaitis, T., Zhou, Y., Lochenie, C., Qi, Q., Jung, A., Chen, D., González-Bello, C., Bräse, S., Zysman-Colman, E., & Vendrell, M. (2026). Biocompatible TADF probes for highly multiplexed fluorescence lifetime imaging. Angewandte Chemie International Edition, Early View, Article e21482. Advance online publication. https://doi.org/10.1002/anie.202521482

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title = "Biocompatible TADF probes for highly multiplexed fluorescence lifetime imaging",

abstract = "Fluorescence lifetime imaging microscopy (FLIM) is an optical imaging modality that can provide multiplexed readouts with remarkable sensitivity to cellular microenvironments. Even though fluorescence lifetimes can distinguish fluorophores having overlapping spectral profiles, conventional fluorophores possess a narrow range of emission lifetimes (typically shorter than 5 ns) that limits their potential for multiplexed imaging. In this work, we have systematically designed and evaluated a combination of thermally activated delayed fluorescence (TADF) nanoprobes for multiplexed FLIM. We have synthesized a collection of 36 TADF biocompatible nanoprobes with long and diverse fluorescence lifetimes in aqueous media (up to 15 ns) and employed selected probes for live-cell imaging of bacterial cells under physiological conditions. By leveraging the exceptionally broad range of fluorescence lifetimes of these TADF emitters, we have achieved unprecedented simultaneous imaging of five nanoprobes within a single spectral window using a FLIM-phasor strategy. These findings demonstrate that TADF emitters are excellent scaffolds to unlock the capabilities of fluorescence lifetime imaging for multi-color biological studies.",

keywords = "Bacteria, Fluorescence lifetime imaging microscopy (FLIM), Fluorophores, Nanoprobes",

author = "\{Su{\'a}rez de Cepeda\}, Pilar and Ferran Nadal-Bufi and Janine Haug and Tomas Matulaitis and Yanzi Zhou and Charles Lochenie and Quan Qi and Andr{\'e} Jung and Dongyang Chen and Concepci{\'o}n Gonz{\'a}lez-Bello and Stefan Br{\"a}se and Eli Zysman-Colman and Marc Vendrell",

note = "Funding: The authors thank the EPSRC (EP/X525819/1, EP/Z535291/1, EP/W007517/1, EP/W015137/1) for financial support. P. S. C thanks the Xunta de Galicia for her predoctoral contract (ED481A-2023-140). J. H. acknowledges funding from the Studienstiftung des deutschen Volkes. C. G. B. acknowledges the financial support from the Axencia Galega de Innovaci{\'o}n (GAIN) [ED431C 2025/05 and Centro Singular de Investigaci{\'o}n de Galicia accreditation 2024–2027 (ED431G 2023/03)] and the European Regional Development Fund. M. V. acknowledges funding from an ERC Consolidator Grant (DYNAFLUORS, 771443).",

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T1 - Biocompatible TADF probes for highly multiplexed fluorescence lifetime imaging

AU - Suárez de Cepeda, Pilar

AU - Nadal-Bufi, Ferran

AU - Haug, Janine

AU - Matulaitis, Tomas

AU - Zhou, Yanzi

AU - Lochenie, Charles

AU - Qi, Quan

AU - Jung, André

AU - Chen, Dongyang

AU - González-Bello, Concepción

AU - Bräse, Stefan

AU - Zysman-Colman, Eli

AU - Vendrell, Marc

N1 - Funding: The authors thank the EPSRC (EP/X525819/1, EP/Z535291/1, EP/W007517/1, EP/W015137/1) for financial support. P. S. C thanks the Xunta de Galicia for her predoctoral contract (ED481A-2023-140). J. H. acknowledges funding from the Studienstiftung des deutschen Volkes. C. G. B. acknowledges the financial support from the Axencia Galega de Innovación (GAIN) [ED431C 2025/05 and Centro Singular de Investigación de Galicia accreditation 2024–2027 (ED431G 2023/03)] and the European Regional Development Fund. M. V. acknowledges funding from an ERC Consolidator Grant (DYNAFLUORS, 771443).

PY - 2026/1/15

Y1 - 2026/1/15

N2 - Fluorescence lifetime imaging microscopy (FLIM) is an optical imaging modality that can provide multiplexed readouts with remarkable sensitivity to cellular microenvironments. Even though fluorescence lifetimes can distinguish fluorophores having overlapping spectral profiles, conventional fluorophores possess a narrow range of emission lifetimes (typically shorter than 5 ns) that limits their potential for multiplexed imaging. In this work, we have systematically designed and evaluated a combination of thermally activated delayed fluorescence (TADF) nanoprobes for multiplexed FLIM. We have synthesized a collection of 36 TADF biocompatible nanoprobes with long and diverse fluorescence lifetimes in aqueous media (up to 15 ns) and employed selected probes for live-cell imaging of bacterial cells under physiological conditions. By leveraging the exceptionally broad range of fluorescence lifetimes of these TADF emitters, we have achieved unprecedented simultaneous imaging of five nanoprobes within a single spectral window using a FLIM-phasor strategy. These findings demonstrate that TADF emitters are excellent scaffolds to unlock the capabilities of fluorescence lifetime imaging for multi-color biological studies.

AB - Fluorescence lifetime imaging microscopy (FLIM) is an optical imaging modality that can provide multiplexed readouts with remarkable sensitivity to cellular microenvironments. Even though fluorescence lifetimes can distinguish fluorophores having overlapping spectral profiles, conventional fluorophores possess a narrow range of emission lifetimes (typically shorter than 5 ns) that limits their potential for multiplexed imaging. In this work, we have systematically designed and evaluated a combination of thermally activated delayed fluorescence (TADF) nanoprobes for multiplexed FLIM. We have synthesized a collection of 36 TADF biocompatible nanoprobes with long and diverse fluorescence lifetimes in aqueous media (up to 15 ns) and employed selected probes for live-cell imaging of bacterial cells under physiological conditions. By leveraging the exceptionally broad range of fluorescence lifetimes of these TADF emitters, we have achieved unprecedented simultaneous imaging of five nanoprobes within a single spectral window using a FLIM-phasor strategy. These findings demonstrate that TADF emitters are excellent scaffolds to unlock the capabilities of fluorescence lifetime imaging for multi-color biological studies.

KW - Bacteria

KW - Fluorescence lifetime imaging microscopy (FLIM)

KW - Fluorophores

KW - Nanoprobes

U2 - 10.1002/anie.202521482

DO - 10.1002/anie.202521482

M3 - Article

SN - 1433-7851

VL - Early View

JO - Angewandte Chemie International Edition

JF - Angewandte Chemie International Edition

M1 - e21482

ER -

Biocompatible TADF probes for highly multiplexed fluorescence lifetime imaging

Abstract

Keywords

Access to Document

Fingerprint

Projects

Allan Watson Programme Grant: Boron: Beyond the Reagent

Commercialization of TADF Dendrimer: Commercialization of TADF dendrimer materials and assessment of their use in time-resolved and confocal fluorescence imaging in living cells

MR TADF: Multi-resonance TADF materials for highly efficient and stable OLEDs

Datasets

Biocompatible TADF probes for highly multiplexed fluorescence lifetime imaging (dataset)

Cite this