Simultaneous quantitative live cell imaging of multiple FRET-based biosensors

Area of Science:

• Biophysics
• Molecular Biology
• Fluorescence Spectroscopy

Background:

• Förster Resonance Energy Transfer (FRET) is crucial for studying molecular interactions.
• Existing FRET analysis methods can face limitations with complex multi-color systems and crosstalk.
• Accurate quantification of FRET efficiency and fluorophore concentration is essential for reliable biological measurements.

Purpose of the Study:

• To develop and validate a novel multi-color spectral FRET analysis method.
• To enable accurate measurement of FRET efficiencies and fluorophore concentrations in systems with multiple sensors.
• To demonstrate the method's capability for simultaneous imaging of various cellular signaling events.

Main Methods:

• Development of a novel routine for computing 3D excitation/emission spectral fingerprints of FRET.
• Unmixing of the 3D FRET spectrum to determine fluorophore concentrations and scaled FRET efficiencies.
• Application of the method to intramolecular FRET sensors and FRET-based calcium sensors with novel fluorescent protein combinations.

Main Results:

• The developed method accurately computes FRET efficiencies without crosstalk.
• Demonstrated accurate measurement of relative fluorophore concentrations and scaled FRET efficiencies.
• Successfully applied to simultaneous imaging of spatially colocalized changes in intracellular calcium, cAMP, and PKA activity.

Conclusions:

• The novel spectral FRET analysis method provides accurate quantification of FRET efficiencies and concentrations.
• This technique overcomes crosstalk limitations in multi-color FRET analysis.
• Enables advanced multi-analyte intracellular imaging for studying complex biological processes.