Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Fluorescence resonance energy transfer (FRET) using fluorescent proteins (FPs) is crucial for studying molecular interactions in live cells.
• Quantitative FRET measurements require robust and reliable FP pairs with well-characterized photophysical properties.

Purpose of the Study:

• To evaluate the E(0)GFP-mCherry FP pair for quantitative FRET analysis.
• To validate its performance in both in vitro and in vivo settings using advanced microscopy techniques.

Main Methods:

• Characterization of E(0)GFP-mCherry photophysics, including spectral overlap and environmental independence.
• Application of acceptor photobleaching (APB) and fluorescence lifetime imaging microscopy (FLIM) for FRET efficiency determination.
• Development of a FRET standard using a tandem construct with a cleavable linker for calibration.

Main Results:

• The E(0)GFP-mCherry pair exhibits excellent spectral overlap (R(0) = 51 Å), minimal crosstalk, and stable emission spectra.
• Both APB and FLIM methods, including the phasor approach to FLIM, accurately determined FRET efficiency.
• The developed FRET standard provided reliable benchmark values, confirming the accuracy of APB and FLIM data.

Conclusions:

• The E(0)GFP-mCherry pair demonstrates ideal characteristics for sensitive and quantitative FRET imaging.
• Its high accuracy in vitro and in vivo makes it suitable for advanced FRET applications, particularly with FLIM.
• This FP pair enables effective quantitative FRET imaging under various experimental conditions.