REAL-TIME QUANTITATIVE FLUORESCENCE RESONANCE ENERGY TRANSFER MEASUREMENTS USING FLUORESCENCE MICROSCOPY
荧光共振能量转移效率的实时定量测量

Fluorescence resonance energy transfer (FRET) is widely used in studies of biomolecular structure and dynamics. By combining fluorescence microscopy with FRET it is possible to obtain quantitative temporal and spatial information about the binding and interaction of protein, lipids, enzymes, DNA, and RNA in vivo. With the recent development of a variety of mutant green fluorescent proteins (GFPs), FRET microscopy provides the potential to measure the dynamic interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are actually part of the molecules themselves. However, present intensity-based FRET quantitative measurements suffer from cross talk of the donor and acceptor emission spectra, which cannot be corrected in a real time. We present a simple method to correct this cross talk in a real time. The data were obtained with only one standard filter set in a fluorescence microscopy. Four coefficients were introduced to eliminate the cross talk, which are constants over various FRET strengths and can be calculated ahead of the experiments based on the emission spectra of the donor and acceptor, and the spectra features of the detection channels. Our quantitative FRET measurements approach has the potential to track dynamic interactions in biological system.