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

Nat Chem. 2020 Feb;12(2):165-172. doi: 10.1038/s41557-019-0371-1. Epub 2019 Dec 2.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line, Tumor
  • Cell Membrane / metabolism*
  • Cell Membrane Permeability
  • Fluorescence
  • Fluorescent Dyes / chemical synthesis
  • Fluorescent Dyes / chemistry*
  • Humans
  • Microscopy, Confocal
  • Microscopy, Fluorescence
  • Rhodamines / chemical synthesis
  • Rhodamines / chemistry*


  • Fluorescent Dyes
  • Rhodamines