Measuring Molecular Interactions with Subcellular Resolution: Single-Cell FRET Using the Quantitative Three-Filterset...

Area of Science:

• Cellular Biology
• Biophysics
• Biochemistry

Background:

• Molecular interactions, not just expression levels, are crucial for cellular signaling.
• Förster resonance energy transfer (FRET) is a sensitive technique for measuring molecular proximity (2-10 nm).
• Quantitative FRET, specifically spectral spillover-corrected methods, offers robust measurements of molecular interactions.

Purpose of the Study:

• To provide a comprehensive protocol for implementing quantitative FRET measurements.
• To enable accurate assessment of molecular interactions and expression levels in cellular processes.
• To introduce the RiFRET ImageJ/Fiji plugin for streamlined FRET data analysis.

Main Methods:

• Utilizing spectral spillover-corrected quantitative (three-filterset) FRET.
• Applying the method in flow cytometry and fluorescence microscopy.
• Employing the freely available RiFRET plugin for image processing and FRET map derivation.

Main Results:

• Pixel-by-pixel quantitative FRET efficiency values are obtained.
• FRET-corrected fluorescent intensities reflecting molecular expression levels are derived.
• The RiFRET plugin facilitates user-friendly and automated processing of large image datasets.

Conclusions:

• Quantitative FRET provides valuable insights into molecular interactions beyond simple expression levels.
• The presented protocol and RiFRET plugin offer a versatile and accessible approach for FRET analysis.
• This method enhances the understanding of cellular signaling pathways through precise molecular interaction studies.