Dual-Label Single-Molecule Imaging Method for Quantifying Apparent Fluorescence Efficiency of Fluorescent Proteins

Area of Science:

• Biophysics
• Cell Biology
• Molecular Imaging

Background:

• Fluorescent proteins (FPs) are essential for quantitative biological research, including protein stoichiometry, signal transduction, and protein-protein interaction studies.
• Accurate measurement of FP fluorescence efficiency is critical for reliable quantitative analyses in live and fixed cells.

Purpose of the Study:

• To introduce a novel dual-color single-molecule imaging method (DC-FEFP) for precise quantification of FP fluorescence efficiency.
• To evaluate the fluorescence efficiency of commonly used FPs and assess the impact of cell fixation on their photophysical properties.

Main Methods:

• Development of a dual-color single-molecule imaging technique (DC-FEFP) utilizing high signal-to-noise ratio FPs or self-labeling tags.
• Application of DC-FEFP to quantify FP fluorescence efficiency at the single-molecule level in both living and fixed cellular environments.

Main Results:

• mNeonGreen demonstrated the highest fluorescence efficiency among three tested FPs in living cells.
• Cell fixation significantly altered the photophysical properties of FPs, impacting their fluorescence efficiency.
• DC-FEFP provides high precision and versatility for FP fluorescence efficiency measurements.

Conclusions:

• DC-FEFP is a robust tool for accurate single-molecule quantification of FP fluorescence efficiency.
• The findings highlight the importance of considering fixation-induced changes in FP properties for quantitative imaging.
• This method offers reliable data for calibrating protein stoichiometry using single-molecule imaging.