研究目的
To develop a robust method (SPEES-FRET) for quantitative FRET measurement in living cells with strong autofluorescence, enabling accurate determination of FRET efficiency (E) and concentration ratio (RC) between acceptor and donor molecules.
研究成果
The SPEES-FRET method demonstrates strong robustness against cellular autofluorescence, providing stable and accurate measurements of FRET efficiency and concentration ratios in living cells, even for dim cells or those with low FRET efficiency. This makes it a valuable tool for studying protein-protein interactions in cells with strong autofluorescence.
研究不足
The method's accuracy may be affected by the maturity of fluorescent proteins and photobleaching. Additionally, the method requires calibration with reference samples and may not be directly applicable to all cell types without adjustment.
1：Experimental Design and Method Selection:
The study employs spectral unmixing of excitation-emission spectra (ExEm-unmixing) to resolve FRET signals into components of donor, acceptor, donor-acceptor sensitization, and autofluorescence.
2：Sample Selection and Data Sources:
HEK293 and HepG2 cells are used, with HEK293 cells exhibiting strong autofluorescence and HepG2 cells showing low autofluorescence.
3：List of Experimental Equipment and Materials:
A self-assembled spectral wide-field microscope (SM) equipped with specific filters and a CCD camera is used for measurements.
4：Experimental Procedures and Operational Workflow:
The method involves measuring spectral fingerprints of donor and acceptor in cells with low autofluorescence, spontaneous spectral fingerprints in cells with strong autofluorescence, and applying spectral unmixing to FRET samples.
5：Data Analysis Methods:
Linear unmixing of excitation-emission spectra into four components and calculation of E and RC using predetermined correction factors.
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