Super-resolved Imaging of Molecular Interactions Using FRET-SOFI

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.