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Related Concept Videos

Super-resolution Fluorescence Microscopy01:37

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Super-resolution fluorescence microscopy (SRFM) provides a better resolution than conventional fluorescence microscopy by reducing the point spread function (PSF). PSF is the light intensity distribution from a point that causes it to appear blurred. Due to PSF, each fluorescing point appears bigger than its actual size, and it is the PSF interference of nearby fluorophores that causes the blurred image. Various approaches to achieving higher resolution through SRFM have recently been...
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High Precision FRET at Single-molecule Level for Biomolecule Structure Determination
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Super-resolved Imaging of Molecular Interactions Using FRET-SOFI.

Hana Valenta1, Marco Dalla Vecchia1, Robin Van den Eynde1

  • 1Department of Chemistry, KU Leuven, Leuven, 3001, Belgium.

Nano Letters
|November 17, 2025
PubMed
Summary
This summary is machine-generated.

Super-resolution optical fluctuation imaging (SOFI) now measures Förster resonance energy transfer (FRET) by combining blinking and non-blinking fluorophores. This advance allows nanoscale imaging of molecular interactions in live cells using sensitive wide-field microscopes.

Keywords:
FRETSOFIfluorescencelive-cellreversibly photoswitchable fluorescent proteinssuper-resolution

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Area of Science:

  • Biophysics
  • Optical Microscopy
  • Molecular Imaging

Background:

  • Super-resolution fluorescence microscopy enables imaging beyond the diffraction limit.
  • Förster resonance energy transfer (FRET) is crucial for studying molecular interactions.
  • Combining FRET with super-resolution imaging presents significant technical challenges.

Purpose of the Study:

  • To develop and validate a novel method for measuring FRET using super-resolution optical fluctuation imaging (SOFI).
  • To investigate the feasibility of FRET-SOFI for imaging molecular interactions in live cells.
  • To assess the advantages and limitations of FRET-SOFI compared to conventional FRET imaging.

Main Methods:

  • Developed a FRET-SOFI approach utilizing a blinking donor and a non-blinking acceptor.
  • Created a theoretical model to understand FRET-SOFI signal characteristics and distortions.
  • Demonstrated FRET-SOFI by imaging intermolecular association of a FRET pair in live cells.

Main Results:

  • FRET-SOFI successfully measured FRET with subdiffraction resolution in live cells.
  • The theoretical model showed FRET-SOFI is less sensitive to donor leakage and direct acceptor excitation.
  • A complex relationship between FRET efficiency and the observed signal was noted.

Conclusions:

  • FRET-SOFI is a viable technique for nanoscale FRET measurements.
  • This method expands the capabilities for imaging molecular interactions at the nanoscale.
  • The technique requires only a sensitive wide-field microscope, increasing accessibility.