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Area of Science:

  • Microbiology
  • Biophysics
  • Molecular Biology

Background:

  • In vivo studies of biomolecules are essential for understanding biological functions.
  • Fluorescent proteins are commonly used for tracking molecules, but organic fluorophores offer advantages in size and photostability.
  • Expanding single-molecule imaging techniques in live cells is critical for biological discovery.

Purpose of the Study:

  • To develop a versatile and high-throughput method for internalizing organic fluorophore-labeled DNA and proteins into live microbial cells.
  • To enable extended single-molecule observation times and advanced biophysical measurements in vivo.
  • To demonstrate the applicability of the method in both prokaryotic (E. coli) and eukaryotic (S. cerevisiae) systems.

Main Methods:

  • Electroporation was employed to introduce organic fluorophore-labeled DNA fragments and proteins into live Escherichia coli.
  • Single-molecule fluorescence imaging techniques were used to analyze copy numbers, diffusion profiles, and structures of internalized molecules.
  • Single-molecule Förster resonance energy transfer (smFRET) measurements were performed in the cytoplasm of live bacteria.
  • The method was validated by internalizing labeled molecules into yeast Saccharomyces cerevisiae.

Main Results:

  • The new method allows for internalization of organic fluorophore-labeled biomolecules into live E. coli and S. cerevisiae.
  • Single-molecule observation times were extended by two orders of magnitude compared to green fluorescent protein.
  • Continuous monitoring of molecular processes from seconds to minutes became feasible.
  • Successful single-molecule Förster resonance energy transfer measurements were achieved for both DNA and proteins in live bacterial cytoplasm.

Conclusions:

  • The developed electroporation method provides a straightforward, versatile, and high-throughput approach for in vivo single-molecule studies using organic fluorophores.
  • This technique significantly enhances the duration and scope of single-molecule observations in live microbial cells.
  • The method's successful application in both bacteria and yeast broadens the potential for investigating complex biological questions at the single-molecule level in diverse biological systems.