Objective Acceptor-sensitized 3-cube fluorescence resonance energy transfer (FRET) imaging (also termed E-FRET imaging) is a popular FRET quantification method in living cells that uses fluorescence intensity. We recently developed a measurement of calibration factors (termed as mTA-G method) that eliminates the influence of the emission transmission characteristics of the instrument used on quantitative E-FRET measurement, significantly increasing the success rate and accuracy of quantitative E-FRET measurement in living cells. Because of its inherent ability to resolve the excitation-emission spectra of donor and acceptor, as well as donor-acceptor sensitization, spectral unmixing of simultaneous excitation and emission spectra (mExEm-spFRET) has been used for quantitative FRET measurement without the need for additional reference for correcting the excitation crosstalk. We evaluated the two methods’ robustness by implementing them on a self-assembled quantitative FRET measurement system with cells expressing different constructs.

Methods The research methods of this paper are mainly divided into four sections: Cell culture and plasmids transfection, predetermining spectral crosstalk and spectral fingerprints, measuring calibration factors and system parameters, superior robustness of mExEm-spFRET to E-FRET method. First, MCF-7 cells were cultured in 6-well plates. For transfection, cells were separately transfected with four different FRET plasmids using transfection reagent. Then, living MCF-7 cells separately expressing YFP (Y) and CFP (C) were used to predetermine the spectral crosstalk coefficients (a, b, c and d) and spectral fingerprints (S_D, S_A, and S_S) were shown in Fig.1 and Fig.2. Next, calibration factors (G and γ) were measured using cells expressing C4Y, C10Y, C40Y, and C80Y (Fig.3). The cells expressing C4Y were used to measure system parameters (f_SC and r_K) (Fig.4). Finally, to evaluate the robustness of mExEm-spFRET and E-FRET methods, we performed quantitative mExEm-spFRET and E-FRET measurements respectively for the same cells separately expressing four kinds of plasmids under different signal-to-noise ratios (R_SN) on different days (Fig. 5 and Table 3).

Results and Discussion The E and R_C values of different FRET plasmid in the cells in Fig. 3 measured by mExEm-spFRET and E-FRET method were shown in Table 1, respectively. For cells 1 and 2, the E and R_C values measured by both methods were consistent with the reported E values and the expected R_C values. Still, the E values measured by E-FRET were generally larger than those calculated by the mExEm-spFRET method. These results indicate that both methods are applicable for live-cell FRET measurement. Table 2 shows different constructs’ statistical E and R_C values in living MCF-7 cells under different R_SN. For the cells under R_SN>3, the two methods obtained consistent FRET efficiency (E) values, but E-FRET obtained smaller donor/acceptor concentration ratio(R_C) values than the expected for individual constructs; for the cells under R_SN<3, the two methods obtained consistent R_C values, but the deviation of individual plasmid E values obtained by E-FRET was slightly larger. These results further demonstrate E-FRET has slightly less robustness than the mExEm-spFRET method, especially for the cells under a low R_SN. We repeated the above measurements on our system on March 10 ^th and obtained consistent results with FRET results measured on December 12 by mExEm-spFRET (Table 3). But the R_C values of C80Y obtained by E-FRET were inconsistent with expected values. These results show the superior robustness of mExEm-spFRET to E-FRET method especially for the cells with low E (E<0.14). Because the fluorescence expression of YFP is very unstable and easily disturbed by the background (BG) signal, particularly for the cells with low R_SN, resulting in the inaccurate results measured by the E-FRET method. Because of the excellent robustness of mExEm-spFRET, just as described above, the mExEm-spFRET method still obtained accurate results for the C80Y construct in the cells with a low R_SN.

Conclusions In this report, we evaluated the robustness of both E-FRET and mExEm-spFRET methods by implementing E-FRET and mExEm-spFRET measurements, respectively, with two excitation wavelengths using the same cells expressing different constructs under different R_SN. For the cells under R_SN>3, the two methods obtained consistent FRET efficiency (E) values, but E-FRET obtained smaller R_C values than the expected for individual constructs; for the cells under R_SN<3, the two methods obtained consistent R_C values, but the deviation of individual plasmid E values obtained by E-FRET was slightly larger. E-FRET and mExEm-spFRET methods are very applicable for live-cell FRET measurement and the superior robustness of mExEm-spFRET to E-FRET method, especially for the cells with low R_SN and E (R_SN<3, E<0.14).