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Updated: Jul 31, 2025

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Super-Resolved FRET Imaging by Confocal Fluorescence-Lifetime Single-Molecule Localization Microscopy.

Cecilia Zaza1, Germán Chiarelli2, Ludovit P Zweifel3

  • 1Centro de Investigaciones en Bionanociencias (CIBION), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Godoy Cruz 2390, Ciudad Autónoma de Buenos Aires, C1425FQD, Argentina.

Small Methods
|May 3, 2023
PubMed
Summary
This summary is machine-generated.

This study introduces super-resolved Fluorescence Resonance Energy Transfer (FRET) imaging using single-molecule localization microscopy. This advanced technique overcomes the limitations of conventional methods, offering higher spatial resolution for molecular interaction analysis.

Keywords:
DNA origamiDNA-PAINTFLIMFRETconfocal microscopysingle molecule fluorescencesuper-resolution microscopy

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Area of Science:

  • Biophysics
  • Molecular Imaging
  • Nanotechnology

Background:

  • Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Lifetime Imaging Microscopy (FLIM) visualize molecular interactions but are limited by diffraction.
  • Conventional methods provide ensemble-averaged data, restricting spatial resolution, accuracy, and dynamic range.

Purpose of the Study:

  • To develop a super-resolved FRET imaging approach.
  • To overcome the spatial resolution limitations of conventional FRET and FLIM techniques.

Main Methods:

  • Utilized single-molecule localization microscopy (SMLM) with an early prototype time-resolved confocal microscope.
  • Employed DNA Points Accumulation for Imaging in Nanoscale Topography (DNA-PAINT) with fluorogenic probes for enhanced background reduction and binding kinetics.
  • Used a single laser for donor excitation and a broad detection band for donor and acceptor emission retrieval.

Main Results:

  • Demonstrated super-resolved FRET imaging capabilities.
  • Achieved FRET event detection through lifetime information analysis.
  • Showcased the compatibility of DNA-PAINT with confocal microscopy scanning speeds.

Conclusions:

  • The developed SMLM-based FRET imaging method significantly enhances spatial resolution compared to conventional techniques.
  • This approach provides a powerful tool for detailed analysis of molecular interactions at the nanoscale.
  • The integration of DNA-PAINT with time-resolved confocal microscopy offers a promising avenue for advanced molecular imaging.