Skip to main content

Thank you for visiting 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:

A general strategy to develop cell permeable and fluorogenic probes for multicolour nanoscopy


Live-cell fluorescence nanoscopy is a powerful tool to study cellular biology on a molecular scale, yet its use is held back by the paucity of suitable fluorescent probes. Fluorescent probes based on regular fluorophores usually suffer from a low cell permeability and an unspecific background signal. Here we report a general strategy to transform regular fluorophores into fluorogenic probes with an excellent cell permeability and a low unspecific background signal. Conversion of a carboxyl group found in rhodamines and related fluorophores into an electron-deficient amide does not affect the spectroscopic properties of the fluorophore, but allows us to rationally tune the dynamic equilibrium between two different forms: a fluorescent zwitterion and a non-fluorescent, cell-permeable spirolactam. Furthermore, the equilibrium generally shifts towards the fluorescent form when the probe binds to its cellular targets. The resulting increase in fluorescence can be up to 1,000-fold. Using this simple design principle, we created fluorogenic probes in various colours for different cellular targets for wash-free, multicolour, live-cell nanoscopy.

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

Access options

Buy this article

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

Fig. 1: Design strategies to develop cell permeable fluorophores.
Fig. 2: Cell permeable 6-TAMRA derivatives for no-wash live-cell microscopy.
Fig. 3: Cell-permeable probes with wavelengths that range from cyan to the near infrared for no-wash live-cell microscopy.
Fig. 4: No-wash live-cell confocal and STED microscopy.

Similar content being viewed by others

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information. Additional information and files are available from the corresponding author upon reasonable request.


  1. Wang, L., Frei, M. S., Salim, A. & Johnsson, K. Small-molecule fluorescent probes for live-cell super-resolution microscopy. J. Am. Chem. Soc. 141, 2770–2781 (2018).

    Article  Google Scholar 

  2. Liu, Z., Lavis, L. D. & Betzig, E. Imaging live-cell dynamics and structure at the single-molecule level. Mol. Cell 58, 644–659 (2015).

    Article  CAS  Google Scholar 

  3. Xue, L., Karpenko, I. A., Hiblot, J. & Johnsson, K. Imaging and manipulating proteins in live cells through covalent labeling. Nat. Chem. Biol. 11, 917–923 (2015).

    Article  CAS  Google Scholar 

  4. Pellett, P. A. et al. Two-color STED microscopy in living cells. Biomed. Opt. Express 2, 2364–2371 (2011).

    Article  Google Scholar 

  5. van de Linde, S., Heilemann, M. & Sauer, M. Live-cell super-resolution imaging with synthetic fluorophores. Annu. Rev. Phys. Chem. 63, 519–540 (2012).

    Article  Google Scholar 

  6. Jones, S. A., Shim, S. H., He, J. & Zhuang, X. Fast, three-dimensional super-resolution imaging of live cells. Nat. Methods 8, 499–508 (2011).

    Article  CAS  Google Scholar 

  7. McNeil, P. L. & Warder, E. Glass-beads load macromolecules into living cells. J. Cell Sci. 88, 669–678 (1987).

    PubMed  Google Scholar 

  8. Lucy, D. B. et al. Small molecule injection into single-cell C. elegans embryos via carbon-reinforced nanopipettes. PLoS ONE 8, e75712 (2013).

    Article  Google Scholar 

  9. Kollmannsperger, A. et al. Live-cell protein labelling with nanometre precision by cell squeezing. Nat. Commun. 7, 10372–10378 (2016).

    Article  CAS  Google Scholar 

  10. Hori, Y. & Kikuchi, K. Protein labeling with fluorogenic probes for no-wash live-cell imaging of proteins. Curr. Opin. Chem. Biol. 17, 644–650 (2013).

    Article  CAS  Google Scholar 

  11. Lavis, L. D. Teaching old dyes new tricks: biological probes built from fluoresceins and rhodamines. Annu. Rev. Biochem. 86, 825–843 (2017).

    Article  CAS  Google Scholar 

  12. Lukinavicius, G. et al. A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins. Nat. Chem. 5, 132–139 (2013).

    Article  CAS  Google Scholar 

  13. Grimm, J. B. et al. A general method to fine-tune fluorophores for live-cell and in vivo imaging. Nat. Methods 14, 987–994 (2017).

    Article  CAS  Google Scholar 

  14. Lukinavicius, G. et al. Fluorogenic probes for live-cell imaging of the cytoskeleton. Nat. Methods 11, 731–733 (2014).

    Article  CAS  Google Scholar 

  15. Lukinavicius, G. et al. SiR-Hoechst is a far-red DNA stain for live-cell nanoscopy. Nat. Commun. 6, 8497–8503 (2015).

    Article  CAS  Google Scholar 

  16. Butkevich, A. N. et al. Fluorescent rhodamines and fluorogenic carbopyronines for super-resolution STED microscopy in living cells. Angew. Chem. Int. Ed. 55, 3290–3294 (2016).

    Article  CAS  Google Scholar 

  17. Lukinavicius, G. et al. Fluorogenic probes for multicolor imaging in living cells. J. Am. Chem. Soc. 138, 9365–9368 (2016).

    Article  CAS  Google Scholar 

  18. Johnson, L. V., Walsh, M. L. & Chen, L. B. Localization of mitochondria in living cells with rhodamine 123. Proc. Natl Acad. Sci. USA 77, 990–994 (1980).

    Article  CAS  Google Scholar 

  19. Poot, M. et al. Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J. Histochem. Cytochem. 44, 1363–1372 (1996).

    Article  CAS  Google Scholar 

  20. Umezawa, K., Yoshida, M., Kamiya, M., Yamasoba, T. & Urano, Y. Rational design of reversible fluorescent probes for live-cell imaging and quantification of fast glutathione dynamics. Nat. Chem. 9, 279–286 (2017).

    Article  CAS  Google Scholar 

  21. Umezawa, K., Kamiya, M. & Urano, Y. A reversible fluorescent probe for real-time live-cell imaging and quantification of endogenous hydropolysulfides. Angew. Chem. Int. Ed. 57, 9346–9350 (2018).

    Article  CAS  Google Scholar 

  22. Grzybowski, M. et al. A highly photostable near-infrared labeling agent based on a phospha-rhodamine for long-term and deep imaging. Angew. Chem. Int. Ed. 57, 10137–10141 (2018).

    Article  CAS  Google Scholar 

  23. Kim, H. N., Lee, M. H., Kim, H. J., Kim, J. S. & Yoon, J. A new trend in rhodamine-based chemosensors: application of spirolactam ring-opening to sensing ions. Chem. Soc. Rev. 37, 1465–1472 (2008).

    Article  CAS  Google Scholar 

  24. Beija, M., Afonso, C. A. M. & Martinho, J. M. G. Synthesis and applications of rhodamine derivatives as fluorescent probes. Chem. Soc. Rev. 38, 2410–2433 (2009).

    Article  CAS  Google Scholar 

  25. Li, H. et al. An acid catalyzed reversible ring-opening/ring-closure reaction involving a cyano-rhodamine spirolactam. Org. Biomol. Chem. 11, 1805–1809 (2013).

    Article  CAS  Google Scholar 

  26. Ammazzalorso, A., De Filippis, B., Giampietro, L. & Amoroso, R. N-acylsulfonamides: Synthetic routes and biological potential in medicinal chemistry. Chem. Biol. Drug Des. 90, 1094–1105 (2017).

    Article  CAS  Google Scholar 

  27. Verkade, J. M. M. et al. A polar sulfamide spacer significantly enhances the manufacturability, stability, and therapeutic index of antibody–drug conjugates. Antibodies 7, 12–23 (2018).

    Article  Google Scholar 

  28. Keppler, A. et al. A general method for the covalent labeling of fusion proteins with small molecules in vivo. Nat. Biotechnol. 21, 86–89 (2003).

    Article  CAS  Google Scholar 

  29. Los, G. V. et al. HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382 (2008).

    Article  CAS  Google Scholar 

  30. Keppler, A., Pick, H., Arrivoli, C., Vogel, H. & Johnsson, K. Labeling of fusion proteins with synthetic fluorophores in live cells. Proc. Natl Acad. Sci. USA 101, 9955–9959 (2004).

    Article  CAS  Google Scholar 

  31. Milroy, L. G. et al. Selective chemical imaging of static actin in live cells. J. Am. Chem. Soc. 134, 8480–8486 (2012).

    Article  CAS  Google Scholar 

  32. Cai, S. X. et al. Design and synthesis of rhodamine 110 derivative and caspase-3 substrate for enzyme and cell-based fluorescent assay. Bioorg. Med. Chem. Lett. 11, 39–42 (2001).

    Article  CAS  Google Scholar 

  33. Stagge, F., Mitronova, G. Y., Belov, V. N., Wurm, C. A. & Jakobs, S. SNAP-, CLIP- and Halo-Tag labelling of budding yeast cells. PLoS ONE 8, e78745 (2013).

    Article  CAS  Google Scholar 

  34. Liu, Y. et al. The cation–π interaction enables a Halo-Tag fluorogenic probe for fast no-wash live cell imaging and gel-free protein quantification. Biochemistry 56, 1585–1595 (2017).

    Article  CAS  Google Scholar 

  35. Escobedo, J. O., Rusin, O., Lim, S. & Strongin, R. M. NIR dyes for bioimaging applications. Curr. Opin. Chem. Biol. 14, 64–70 (2010).

    Article  CAS  Google Scholar 

  36. Koide, Y. et al. Development of NIR fluorescent dyes based on Si-rhodamine for in vivo imaging. J. Am. Chem. Soc. 134, 5029–5031 (2012).

    Article  CAS  Google Scholar 

  37. Paintrand, M., Moudjou, M., Delacroix, H. & Bornens, M. Centrosome organization and centriole architecture: their sensitivity to divalent cations. J. Struct. Biol. 108, 107–128 (1992).

    Article  CAS  Google Scholar 

  38. Malecki, M. J. et al. Leukemia-associated mutations within the NOTCH1 heterodimerization domain fall into at least two distinct mechanistic classes. Mol. Cell. Biol. 26, 4642–4651 (2006).

    Article  CAS  Google Scholar 

Download references


We acknowledge funding from the Max Planck Society. We thank S. Jakobs for providing the U2OS Vimentin-HaloTag cells. J. Hubrich and C.-M. Gürth supported the cell culture and preparation of neurons. We are grateful to S. Pitsch for the gift of SiR700 and to L. Reymond for the gift of carbopyronine. L.W. and M.T. were supported by fellowships of the Alexander von Humboldt Foundation.

Author information

Authors and Affiliations



All the authors discussed the results and commented on the manuscript. L.W. and K.J. designed the strategy and fluorophore structures. L.W., M.T. and L.X performed the chemical syntheses. L.W. and M.T. characterized the dyes and performed the confocal microscopy with subsequent data analysis. E.D. and J.R. performed the STED microscopy with subsequent data analysis. B.K. developed the cell lines.

Corresponding authors

Correspondence to Lu Wang or Kai Johnsson.

Ethics declarations

Competing interests

K.J. and L.W. are inventors of the patent ‘Cell-permeable fluorogenic fluorophores’ (EP Patent Application 18210676.5, pending), which was filed by the Max Planck Society.

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 information providing details of the experimental methods, Supplementary Figs. 1–30, Tables 1–4 and refs. 1–4.

Reporting Summary

Supplementary Video 1

No-wash real-time multicolour confocal microscopy of U2OS FlpIn Cox8-Halo-SNAP-expressing cells stained with Hoechst 33342 (0.2 μg ml–1)/MaP555-tubulin (1 μM)/MaP618-actin (500 nM)/MaP700-Halo (250 nM).

Supplementary Video 2

No-wash real-time multicolour confocal microscopy of U2OS FlpIn Cox8-Halo-SNAP-expressing cells stained with Hoechst 33342 (0.2 μg ml–1)/MaP510-Halo (250 nM)/MaP555-actin (1 μM)/SiR-Lyso (1 μM).

Supplementary Video 3

No-wash real-time multicolour confocal microscopy of U2OS FlpIn Halo-SNAP-NLS-expressing cells stained with MaP510-Halo (250 nM)/MaP555-tubulin (1 μM)/MaP618-actin (500 nM)/SiR-Lyso (1 μM).

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, L., Tran, M., D’Este, E. et al. A general strategy to develop cell permeable and fluorogenic probes for multicolour nanoscopy. Nat. Chem. 12, 165–172 (2020).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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