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A palette of bridged bicycle-strengthened fluorophores

Nature Methods volume 22, pages 1276–1287 (2025)Cite this article

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Abstract

Organic fluorophores are the keystone of advanced biological imaging. The vast chemical space of fluorophores has been extensively explored in search of molecules with ideal properties. However, within the current molecular constraints, there appears to be a trade-off between high brightness, robust photostability, and tunable biochemical properties. Herein we report a general strategy to systematically boost the performance of donor-acceptor-type fluorophores, such as rhodamines, by leveraging SO2 and O-substituted azabicyclo[3.2.1] octane auxochromes. These bicyclic heterocycles give rise to a collection of ‘bridged’ dyes (BD) spanning the ultraviolet and visible range with top-notch quantum efficiencies, enhanced water solubility, and tunable cell-permeability. Notably, these azabicyclic fluorophores showed remarkable photostability compared to their tetramethyl or azetidine analogs while being completely resistant to oxidative photoblueing. Functionalized BD dyes are tailored for applications in single-molecule imaging, super-resolution imaging (STED and SIM) in fixed or live mammalian and plant cells, and live zebrafish imaging and chemogenetic voltage imaging.

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Fig. 1: Development of fluorophores with bridged bicyclic auxochromes.
Fig. 2: Photostability and photoblueing properties of BD and benchmarking dyes.
Fig. 3: BD derivatives for antibody bioconjugation and immunofluorescence imaging.
Fig. 4: BD derivatives are exceptional HaloTag ligands for the labeling and imaging of cellular targets.
Fig. 5: BDHTLs are versatile tools for general imaging in cells, plants, and animals.

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Data availability

All data associated with this study are presented in the main text or Supplementary Information. The raw high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy data are provided in the Supplementary Information. The structure of HaloTag7 bound to BD626HTL has been deposited in the Protein Data Bank (PDB 6Y7B).

The raw imaging data for Figures 4d, and 5c,f,h are provided as Supplementary Videos 1–4. All chemical probes reported in this work are available from the corresponding author upon reasonable request. Selected BDHTL dyes are commercially available from Genvivo Biotech and Spirochrome. Source data are provided with this paper.

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Acknowledgements

This project was supported by funds from National Key R&D Program of China (2021YFF0502904 to Z.C.; 2020YFA0509502 to W.D.), the Beijing Municipal Science & Technology Commission (project Z221100003422013 to Z.C.), the Beijing National Laboratory for Molecular Sciences (BNLMS202407 to Z.C.), and the Natural Science Foundation of China (32170566 to W.D.). We thank L. Chen at Peking University for the guidance in analyzing crystal structures. We thank J. Lin for the assistance with plant-cell imaging. We thank P. Xi for assistance with SIM imaging. We thank the Tsinghua University Branch of China National Center for Protein Sciences (Beijing) and Tsinghua University Technology Center for Protein Research for the X-ray crystallography facility support. We thank S. Fan, L. Wang, and M. Li at the X-ray crystallography platform of National Protein Science Facility, Tsinghua University, Beijing, for their technical help. We thank the Metabolic Mass Spectrometry Platform at the IMM, the analytical instrumentation center of Peking University, the NMR facility of the National Center for Protein Sciences at Peking University, and the National Center for Protein Sciences at Peking University.

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Authors and Affiliations

  1. College of Future Technology, Institute of Molecular Medicine, National Biomedical Imaging Center, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, State Key Laboratory of Membrane Biology, Peking University, Beijing, China

    Junwei Zhang, Kecheng Zhang, Mengling Zhang, Tianyan Liu & Zhixing Chen

  2. Institute of Neuroscience, State Key Laboratory of Neuroscience, Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences, Shanghai, China

    Kui Wang, Shilin Fang, Xinxin Zhang & Yu Mu

  3. Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, China

    Bo Wang, Siyan Zhu, Yuan Shen, Peng Zou, Wulan Deng & Zhixing Chen

  4. Biomedical Pioneering Innovation Center (BIOPIC), Peking University, Beijing, China

    Bo Wang & Wulan Deng

  5. College of Chemistry and Molecular Engineering, Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, PKU-IDG/McGovern Institute for Brain Research, Peking University, Beijing, China

    Siyan Zhu & Peng Zou

  6. College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing, China

    Hongping Qian

  7. BSJ Institute, Beijing, China

    Yumiao Ma

  8. PKU-Nanjing Institute of Translational Medicine, Nanjing, China

    Peng Chen & Zhixing Chen

  9. Genvivo Biotech, Nanjing, China

    Peng Chen

  10. Department of Biomedical Engineering, College of Future Technology, Peking University, Beijing, China

    Yunzhe Fu

  11. ShanghaiTech University, Shanghai, China

    Xinxin Zhang

  12. Chinese Institute for Brain Research (CIBR), Beijing, China

    Peng Zou

  13. Beijing Advanced Innovation Center for Genomics (ICG), Peking University, Beijing, China

    Wulan Deng

  14. University of Chinese Academy of Sciences, Beijing, China

    Yu Mu

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Contributions

Z.C. conceived the study. J.Z. and P.C. performed the chemical synthesis and characterizations. J.Z. performed the photophysical tests. K.Z. solved the crystal structures of HaloTag7–BD626. J.Z., M.Z., and T.L. performed confocal and STED imaging. K.W., S.F., and X.Z. performed in vivo imaging. B.W. performed single-molecule imaging. S.Z. performed voltage imaging. H.Q. performed SIM imaging on plant cells. Y.M. performed chemical calculations. Y.S. performed photo-crosslinking experiments. Y.F. performed SIM imaging on mammalian cells. P.Z., W.D., Y.M., and Z. C. supervised the project. J.Z. and Z.C. wrote the paper.

Corresponding author

Correspondence to Zhixing Chen.

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Competing interests

Z.C., J.Z., and P. C. are the inventors of a patent on the bridged-bicycle strengthened fluorophores (CN2023108090626, patent pending), whose value could be affected by this paper. The other authors declare no competing interests.

Peer review

Peer review information

Nature Methods thanks Lei Wang, Youjun Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Rita Strack, in collaboration with the Nature Methods team.

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Extended data

Extended Data Fig. 1 A crystal structure of BD626HTL at the HaloTag7 shows additional interactions between dye and protein.

(a)Structural comparison between HT7-CPY (cyan, PDB 6Y7B) and HT7-BD626 (yellow, PDB 9JHA); (b-c) Close-ups of the substrate binding sites. Proteins are represented as pink or gray cartoons. Fluorophores and residues are shown as sticks. A polar interaction is identified between the bridged auxochrome and Q165 and shown as dashed lines with annotated distances; (d)A overlaid comparison of CPYHTL and BD626HTL in the pocket of HT7.

Extended Data Fig. 2 BD566HTL enables time-lapse SIM imaging.

(a)Time-lapse SIM images of live COS-7 cells stably expressing TOMM20-HT7 labeled with BD566HTL (500 nM, 1 h, 37 °C, one wash), scale bar=2 μm; (b) Normalized fluorescence decay curves of samples in (a), n = 4 in 2 independent experiments; error bar show ± s.d.

Extended Data Fig. 3 BD626HTL can offer 25 z-frames for 3D reconstruction, enabling the construction of the distribution of the cytoskeleton (Cep41) throughout the entire cell.

Orthogonal views of 3D-STED images of fixed HeLa cells expressing Cpe41-HT7 labeled with BD626HTL (500 nM, 30 min, 37 °C, one wash). An area of 52.5 × 51.0 μm (x–y) was recorded in 65 nm z-stacks over 25 times; scale bar= 1 μm.

Extended Data Table 1 The major advantages BriDyes bring out in the manuscript by head-to-head comparisons
Full size table

Supplementary information

Supplementary Information

Supplementary Figures 1–12, Supplementary Tables 1 and 2, Supplementary Schemes 1–3, Synthesis and characterization of new compounds, NMR spectra, and Supplementary References.

Reporting Summary

Supplementary Video 1

Time-lapse single-molecule images of fixed U-2 OS cells stably expressing H2B-HaloTag7 labeled with TMRHTL, JF549HTL and BD566HTL; scale bar, 5 μm.

Supplementary Video 2

Time-lapse STED images of live HeLa cells expressing TOMM20-HaloTag7 labeled with BD626HTL, SiRHTL or JF646HTL; scale bar, 1 μm.

Supplementary Video 3

Time-lapse TIRF-SIM images of live Arabidopsis thalianas cells expressing HIR1-HaloTag7 labeled with BD626HTL or JF646HTL; scale bar, 1 μm.

Supplementary Video 4

Z-stack confocal images and 3D reconstruction of a live zebrafish expressing elavl3:H2B-HT7 and labeled with BD566HTL.

Source data

Source Data Fig. 2

Statistical source data.

Source Data Fig. 2

Unprocessed gels.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

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Zhang, J., Zhang, K., Wang, K. et al. A palette of bridged bicycle-strengthened fluorophores. Nat Methods 22, 1276–1287 (2025). https://doi.org/10.1038/s41592-025-02693-4

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  • DOI: https://doi.org/10.1038/s41592-025-02693-4

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