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Reduced dyes enhance single-molecule localization density for live superresolution imaging.

Lina Carlini1, Alexander Benke, Luc Reymond

  • 1Laboratory of Experimental Biophysics, Institute of Physics of Biological Systems, École Polytechnique Fédérale de Lausanne (EPFL), National Centre of Competence in Research (NCCR) in Chemical Biology, Lausanne (Switzerland).

Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry
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Summary

Cell-permeable rhodamine dyes can be chemically reduced to a non-fluorescent state and then re-oxidized in living cells. This process enhances single-molecule localization microscopy and tracking by increasing localization density and photon counts.

Keywords:
fluorophoreslive-cell imagingrhodaminesingle-molecule studiessuperresolution microscopy

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Area of Science:

  • Biochemistry
  • Cell Biology
  • Microscopy

Background:

  • Cell-permeable rhodamine dyes are essential tools in live-cell imaging.
  • Their fluorescence can be modulated by chemical reduction and oxidation.
  • Optimizing dye properties is crucial for advanced imaging techniques.

Purpose of the Study:

  • To investigate the impact of reductive quenching and subsequent re-oxidation of rhodamine dyes on live-cell super-resolution imaging.
  • To evaluate the benefits for single-molecule localization microscopy (SMLM) and single-molecule tracking (SMT).

Main Methods:

  • Utilized sodium borohydride (NaBH4) for reductive quenching of rhodamine dyes.
  • Observed spontaneous re-oxidation in living cells.
  • Compared imaging performance with reduced and unmodified dyes using point-localization super-resolution microscopy and single-molecule tracking.

Main Results:

  • Reductive quenching converted rhodamine dyes to a non-fluorescent leuco-rhodamine form.
  • Spontaneous oxidation in cells restored fluorescence.
  • Achieved up to ten-fold higher densities of single molecule localizations.
  • Observed increased photons per localization compared to unmodified dyes.
  • Demonstrated significant improvements in the resolution and quality of live-cell super-resolution images.
  • Increased the density of trajectories in single-molecule tracking experiments.

Conclusions:

  • Reversible reductive quenching and re-oxidation of rhodamine dyes offer a powerful strategy to enhance live-cell super-resolution imaging.
  • This method improves key parameters for SMLM and SMT, leading to higher resolution and more robust tracking.