Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Single-chain ultrasmall fluorescent polymer dots enable nanometre-resolution cellular imaging and single protein tracking

Nature Photonics volume 19, pages 1336–1344 (2025)Cite this article

Subjects

Abstract

Fluorescent nanoparticles offer superior brightness and photostability compared with conventional dyes and proteins. However, their relatively large size and complex surface chemistry limit their utility for imaging nanoscale biostructures and tracking individual proteins in living cells. Here we develop single-chain ultrasmall fluorescent polymer dots (suPdots) with size below 5 nm, comparable to fluorescent proteins. Fabricated via vitrification of conjugated polymer solutions, suPdots enable tunable fluorescence as well as high-density, specific labelling of multiple subcellular organelles. We demonstrate nanoscopic imaging of continuous ring structures in clathrin-coated pits as well as multi-target stimulated emission depletion imaging. Thanks to their high brightness, suPdots enable tracking the individual steps of the kinesin-1 motor protein in living cells using standard spinning-disk fluorescence microscopy, with a 16-nm step size and 50-Hz temporal resolution. These demonstrations establish suPdots as powerful, versatile fluorescent probes for nanoscale-resolution biomolecular imaging with increased accessibility and efficiency for diverse bio-applications.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Vitreously frozen fluorescent polymer solution leads to suPdots.
Fig. 2: suPdots with well-defined composition.
Fig. 3: Single-particle photophysical properties of suPdots.
Fig. 4: High-density specific biolabelling for high-resolution imaging with suPdots.
Fig. 5: Nanometre-resolution tracking of single kinesin in living cells.

Similar content being viewed by others

Data availability

The data that support the findings of this study are available from the corresponding authors upon request. Source data are provided with this paper.

References

  1. Francisco, B. et al. Nanometer resolution imaging and tracking of fluorescent molecules with minimal photon fluxes. Science 355, 606–612 (2016).

    Google Scholar 

  2. Li, D. et al. Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics. Science 349, aab3500 (2015).

    Article  Google Scholar 

  3. Ouyang, Z. et al. Elucidating subcellular architecture and dynamics at isotropic 100-nm resolution with 4Pi-SIM. Nat. Methods 22, 335–347 (2024).

    Article  Google Scholar 

  4. Nehme, E. et al. DeepSTORM3D: dense 3D localization microscopy and PSF design by deep learning. Nat. Methods 17, 734–740 (2020).

    Article  Google Scholar 

  5. Huang, X. et al. Fast, long-term, super-resolution imaging with Hessian structured illumination microscopy. Nat. Biotechnol.36, 451–459 (2018).

    Article  Google Scholar 

  6. Gwosch, K. C. et al. MINFLUX nanoscopy delivers 3D multicolor nanometer resolution in cells. Nat. Methods 17, 217–224 (2020).

    Article  Google Scholar 

  7. Day, R. N. & Davidson, M. W. The fluorescent protein palette: tools for cellular imaging. Chem. Soc. Rev.38, 2887–2921 (2009).

    Article  Google Scholar 

  8. Zeng, S. et al. Fluorescent dyes based on rhodamine derivatives for bioimaging and therapeutics: recent progress, challenges, and prospects. Chem. Soc. Rev.52, 5607–5651 (2023).

    Article  Google Scholar 

  9. Jin, D. et al. Nanoparticles for super-resolution microscopy and single-molecule tracking. Nat. Methods 15, 415–423 (2018).

    Article  Google Scholar 

  10. Ashoka, A. H. et al. Brightness of fluorescent organic nanomaterials. Chem. Soc. Rev.52, 4525–4548 (2023).

    Article  Google Scholar 

  11. Xu, Y. et al. Recent advances in luminescent materials for super-resolution imaging via stimulated emission depletion nanoscopy. Chem. Soc. Rev. 50, 667–690 (2021).

    Article  Google Scholar 

  12. Wegner, K. D. et al. Quantum dots: bright and versatile in vitro and in vivo fluorescence imaging biosensors. Chem. Soc. Rev. 44, 4792–4834 (2015).

    Article  ADS  Google Scholar 

  13. Li, W. et al. Fluorescent nanoparticles for super-resolution imaging. Chem. Rev. 122, 12495–12543 (2022).

    Article  Google Scholar 

  14. Hoffman, D. P. et al. Correlative three-dimensional super-resolution and block-face electron microscopy of whole vitreously frozen cells. Science 367, eaaz5357 (2020).

    Article  Google Scholar 

  15. Silvester, E., et al. DNA origami signposts for identifying proteins on cell membranes by electron cryotomography. Cell 184, 1110–1121 (2021).

    Article  Google Scholar 

  16. Tao, H., et al. Nanoparticle synthesis assisted by machine learning. Nat. Rev. Mater. 6, 701–716 (2021).

    Article  ADS  Google Scholar 

  17. Li, K., et al. Polymer-encapsulated organic nanoparticles for fluorescence and photoacoustic imaging. Chem. Soc. Rev. 43, 6570–6597 (2014).

    Article  ADS  Google Scholar 

  18. Takahiro, D., et al. Direct observation of motor protein stepping in living cells using MINFLUX. Science 379, 1010–1015 (2023).

    Article  Google Scholar 

  19. Jan, O. W., et al. MINFLUX dissects the unimpeded walking of kinesin-1. Science 379, 1004–1010 (2023).

    Article  Google Scholar 

  20. Fan, Q., et al. Precise control over kinetics of molecular assembly: production of particles with tunable sizes and crystalline forms. Angew. Chem. Int. Ed. 59, 15141–15146 (2020).

    Article  Google Scholar 

  21. Yan, Z., et al. Preparation of ultrasmall AIE nanoparticles with tunable molecular packing via freeze assembly. Nano Lett. 23, 1030–1035 (2023).

    Article  ADS  Google Scholar 

  22. Mao, J., et al. Templated freezing assembly precisely regulates molecular assembly for free-standing centimeter-scale microtextured nanofilms. Sci. China Chem. 66, 878–886 (2023).

    Google Scholar 

  23. Carrie, L., et al. Effects of packing structure on the optoelectronic and charge transport properties in poly(9-9-di-n-octylfluorene-alt-benzothiadiazole). J. Am. Chem. Soc. 127, 12890–12899 (2005).

    Article  Google Scholar 

  24. Wang, D., et al. From single chains to aggregates, how conjugated polymers behave in dilute solutions. Macromolecules 46, 6217–6224 (2013).

    Article  ADS  Google Scholar 

  25. Wu, C., et al. Highly fluorescent semiconducting polymer dots for biology and medicine. Angew. Chem. Int. Ed. 52, 3086–3109 (2013).

    Article  Google Scholar 

  26. Osamu Mishima, H. E. S. The relationship between liquid, supercooled and glassy water. Nature 396, 329–335 (1998).

    Article  ADS  Google Scholar 

  27. Guan, W., et al. Measurement of solubilization location in micelles using anchored aggregation-induced emission donors. Angew. Chem. Int. Ed. 59, 12800–12805 (2020).

    Article  Google Scholar 

  28. Zhang, J., et al. Ultrabright Pdots with a large absorbance cross section and high quantum yield. ACS Appl. Mater. Interfaces 14, 13631–13637 (2022).

    Article  Google Scholar 

  29. Wang, S., et al. NIR-II excitable conjugated polymer dots with bright NIR-I emission for deep in vivo two-photon brain imaging through intact skull. Adv. Funct. Mater. 29, 1808365 (2019).

    Article  Google Scholar 

  30. Chen, Y., et al. Surface modification of gold nanoparticles with small molecules for biochemical analysis. Acc. Chem. Res. 50, 310–319 (2017).

    Article  Google Scholar 

  31. Gajdos, T., et al. Hot-band anti-Stokes fluorescence properties of Alexa Fluor 568. J. Fluoresc. 30, 437–443 (2020).

    Article  Google Scholar 

  32. Xu, J., et al. Automated stoichiometry analysis of single-molecule fluorescence imaging traces via deep learning. J. Am. Chem. Soc. 141, 6976–6985 (2019).

    Article  ADS  Google Scholar 

  33. Tang, D., et al. Topological DNA tetrahedron encapsulated gold nanoparticle enables precise ligand engineering for targeted cell imaging. Anal. Chem. 93, 17036–17042 (2021).

    Article  ADS  Google Scholar 

  34. Paul, F. B., et al. Hole-induced quenching of triplet and singlet excitons in conjugated polymers. J. Am. Chem. Soc. 127, 9556–9560 (2005).

    Article  ADS  Google Scholar 

  35. Paul, F. B., et al. Discrete intensity jumps and intramolecular electronic energy transfer in the spectroscopy of single conjugated polymer molecules. Science 277, 1074–1077 (1997).

    Article  Google Scholar 

  36. Paul, F. B. et al. Size-dependent spectroscopic properties of conjugated polymer nanoparticles. J. Phys. Chem. B 110, 25568–25572 (2006).

    Article  Google Scholar 

  37. Chen, X. et al. Small photoblinking semiconductor polymer dots for fluorescence nanoscopy. Adv. Mater. 29, 1604850 (2017).

    Article  ADS  Google Scholar 

  38. Chen, X. et al. Multicolor super-resolution fluorescence microscopy with blue and carmine small photoblinking polymer dots. ACS Nano 11, 8084–8091 (2017).

    Article  Google Scholar 

  39. Paul, F. B. et al. Collapse of stiff conjugated polymers with chemical defects into ordered, cylindrical conformations. Nature 405, 1030–1033 (2000).

    Article  Google Scholar 

  40. Joshua, C. B. et al. Conformation and energy transfer in single conjugated polymers. Acc. Chem. Res. 45, 1992–2001 (2012).

    Article  Google Scholar 

  41. Jiang, Y., et al. Light-harvesting and amplified energy transfer in conjugated polymer nanoparticles. Chem. Rev. 117, 838–859 (2017).

    Article  Google Scholar 

  42. Xu, X. et al. Influence of side-chain length and relative rigidities of backbone and side chains on glass formation of branched polymers. Macromolecules 54, 6327–6341 (2021).

    Article  ADS  Google Scholar 

  43. Wu, C. et al. Bioconjugation of ultrabright semiconducting polymer dots for specific cellular targeting. J. Am. Chem. Soc.132, 15410–15417 (2010).

    Article  ADS  Google Scholar 

  44. Wu, Y. et al. Fluorescent polymer dot-based multicolor stimulated emission depletion nanoscopy with a single laser beam pair for cellular tracking. Anal. Chem. 92, 12088–12096 (2020).

    Article  ADS  Google Scholar 

  45. Yu, J., et al. Recent advances in the development of highly luminescent semiconducting polymer dots and nanoparticles for biological imaging and medicine. Anal. Chem. 89, 42–56 (2016).

    Article  ADS  Google Scholar 

  46. Rittweger, E., et al. STED microscopy reveals crystal colour centres with nanometric resolution. Nat. Photon. 3, 144–147 (2009).

    Article  ADS  Google Scholar 

  47. Bates, M. et al. Multicolor super-resolution imaging with photo-switchable fluorescent probes. Science 317, 1749–1753 (2007).

    Article  ADS  Google Scholar 

  48. Anderson, J., et al. Hypertonic media inhibit receptor-mediated endocytosis by blocking clathrin-coated pit formation. J. Cell Biol. 108, 389–400 (1989).

    Article  Google Scholar 

  49. Liang, L., et al. Continuous-wave near-infrared stimulated-emission depletion microscopy using downshifting lanthanide nanoparticles. Nat. Nanotechnol. 16, 975–980 (2021).

    Article  ADS  Google Scholar 

  50. Zhan, Q., et al. Achieving high-efficiency emission depletion nanoscopy by employing cross relaxation in upconversion nanoparticles. Nat. Commun. 8, 1058 (2017).

    Article  ADS  Google Scholar 

  51. Hanne, J. et al. STED nanoscopy with fluorescent quantum dots. Nat. Commun.6, 7127 (2015).

    Article  ADS  Google Scholar 

  52. Pisfil, M. G. et al. Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation. Sci. Rep.12, 14027–14042 (2022).

    Article  ADS  Google Scholar 

  53. Blom, H. et al. Stimulated emission depletion microscopy. Chem. Rev. 117, 7377–7427 (2017).

    Article  Google Scholar 

  54. Peng, C. S. et al. Nanometer-resolution tracking of single cargo reveals dynein motor mechanisms. Nat. Chem. Biol. 21, 648–656 (2024).

    Article  Google Scholar 

  55. Ahmet, Y. et al. Kinesin walks hand-over-hand. Science 303, 676–678 (2004).

    Article  Google Scholar 

  56. Niekamp, S. et al. A 6-nm ultra-photostable DNA FluoroCube for fluorescence imaging. Nat. Methods17, 437–441 (2020).

    Article  Google Scholar 

  57. Hiroyasu, H., et al. Live-cell single-molecule labeling and analysis of myosin motors with quantum dots. Mol. Biol. Cell 28, 173–181 (2016).

    Google Scholar 

  58. Sun, C. et al. Intracellular tracking of single native molecules with electroporation-delivered quantum dots. Anal. Chem. 86, 11403–11409 (2014).

    Article  ADS  Google Scholar 

  59. Wichner, S. M., et al. Covalent protein labeling and improved single-molecule optical properties of aqueous CdSe/CdS quantum dots. ACS Nano 11, 6773–6781 (2017).

    Article  Google Scholar 

  60. Tinevez, J.-Y. et al. TrackMate: an open and extensible platform for single-particle tracking. Methods 115, 80–90 (2017).

    Article  Google Scholar 

  61. Loeff, L., et al. AutoStepfinder: a fast and automated step detection method for single-molecule analysis. Patterns 2, 100256 (2021).

    Article  Google Scholar 

  62. Steffen, J. S. et al. Direct optical measurement of intramolecular distances with angstrom precision. Science 386, 180–187 (2024).

    Article  ADS  Google Scholar 

  63. Zhang, X., et al. Importance of having low-density functional groups for generating high-performance semiconducting polymer dots. ACS Nano 6, 5429–5439 (2012).

    Article  Google Scholar 

  64. Ershov, D., et al. TrackMate 7: integrating state-of-the-art segmentation algorithms into tracking pipelines. Nat. Methods 19, 829–932 (2022).

    Article  Google Scholar 

Download references

Acknowledgements

This work was supported in part by the National Natural Science Foundation of China (grant numbers T2293760 and T2293762 to J.W. and 52573355 to Q.F.), the Strategic Priority Research Program of the Chinese Academy of Sciences (grant numbers XDB1030000 to J.W. and Q.F., and XDB1020000 to X.F.) and the National Key Research and Development Program of China (2022YFC2703004 to J.W. and 2022YFA1304500 to X.F.). "Pioneer" and "Leading Goose" R&D program of Zhejiang (2023SDYXS0001 to X.F. and Y.J.) and TIPC Director’s Fund to Q.F. We thank G. Qin for the moral support and H. Zhang for the polymer characterization. We also thank L. Chen and K. Wang for their help with Hessian-SIM imaging. Supplementary Fig. 4a is created with BioRender.com.

Author information

Author notes

  1. These authors contributed equally: Hongwei Yang, Zequan Yan.

Authors and Affiliations

  1. Interdisciplinary Research Center for Advanced Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, China

    Hongwei Yang, Zequan Yan, Panru Lin, Han Xue, Qingrui Fan & Jianjun Wang

  2. Hangzhou Institute of Medicine (HIM), Chinese Academy of Sciences, Hangzhou, China

    Hongwei Yang, Yifei Jiang & Xiaohong Fang

  3. Institute of Chemistry, Chinese Academy of Sciences, Beijing, China

    Hongwei Yang, Xiaolong Liu, Weifeng Liu, Yayun Wu & Xiaohong Fang

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

    Zequan Yan, Jianjun Wang & Xiaohong Fang

  5. School of Chemistry and Materials Science, University of Science and Technology of China, Hefei, China

    Jianjun Wang

Authors
  1. Hongwei Yang
    View author publications

    Search author on:PubMed Google Scholar

  2. Zequan Yan
    View author publications

    Search author on:PubMed Google Scholar

  3. Xiaolong Liu
    View author publications

    Search author on:PubMed Google Scholar

  4. Weifeng Liu
    View author publications

    Search author on:PubMed Google Scholar

  5. Panru Lin
    View author publications

    Search author on:PubMed Google Scholar

  6. Han Xue
    View author publications

    Search author on:PubMed Google Scholar

  7. Yayun Wu
    View author publications

    Search author on:PubMed Google Scholar

  8. Yifei Jiang
    View author publications

    Search author on:PubMed Google Scholar

  9. Qingrui Fan
    View author publications

    Search author on:PubMed Google Scholar

  10. Jianjun Wang
    View author publications

    Search author on:PubMed Google Scholar

  11. Xiaohong Fang
    View author publications

    Search author on:PubMed Google Scholar

Contributions

X.F. and J.W. designed the research. X.F., J.W., Q.F. and Y.J. supervised the project. H.Y., Z.Y., X.L. and W.L. performed the experiments. Z.Y. and Q.F. synthesized the particles. H.Y., Z.Y., P.L., X.L., Y.W., H.X. and Y.J. analysed the data. H.Y., Y.J., Q.F., J.W. and X.F. wrote the paper. H.Y., Q.F. and J.W. drew Figs. 1a and 5a. All the authors contributed to discussing and editing the paper.

Corresponding authors

Correspondence to Yifei Jiang, Qingrui Fan, Jianjun Wang or Xiaohong Fang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Photonics thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Note, Supplementary Discussion, Figs. 1–27, and Tables 1 and 2.

Source data

Source Data Figs. 1–5

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yang, H., Yan, Z., Liu, X. et al. Single-chain ultrasmall fluorescent polymer dots enable nanometre-resolution cellular imaging and single protein tracking. Nat. Photon. 19, 1336–1344 (2025). https://doi.org/10.1038/s41566-025-01767-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41566-025-01767-1

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing