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

Fluorescent proteins (FPs) are indispensable tools for life science research, crucial for quantitative analyses, such as protein stoichiometry determination, signal transduction, and protein-protein interactions. In this work, we introduce a new method of dual-color single-molecule imaging to assess the apparent fluorescence efficiency of FPs (DC-FEFP). By integrating high signal-to-noise ratio (SNR) FPs or self-labeling tags, this approach enables us to precisely quantify the fluorescence efficiency of various FPs at the single-molecule level in both living and fixed cells. With DC-FEFP, we found that mNeonGreen has the highest fluorescence efficiency among three commonly used FPs in living cells, as well as the significant impact of fixation on the photophysical properties of FPs. DC-FEFP is a high-precision and versatile tool for quantifying the fluorescence efficiency of FPs. It is capable of providing accurate information to calibrate protein stoichiometry based on single-molecule imaging.