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

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.