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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.

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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.

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.


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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




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.

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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).

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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).

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