活细胞定量FRET成像方法鲁棒性评估

Robustness Evaluation of Quantitative Fluorescence Resonance Energy Transfer Imaging Methods in Live Cells

基于受体敏化的3-cube(通道)荧光共振能量转移(FRET)成像方法(通常称为E-FRET方法)是活细胞定量FRET检测中主流的成像分析技术。基于激发发射光谱线性分离的定量FRET检测方法(mExEm-spFRET)因天然地克服光谱串扰的能力,在活细胞定量FRET检测中具备非常好的鲁棒性。利用表达不同模型质粒的乳腺癌MCF-7活细胞,在不同信噪比(R_(SN))的条件下分别进行了定量E-FRET和mExEm-spFRET测量,以FRET效率(E)和质粒供受体浓度比(R_(C))参量作为指标,评估二种方法的鲁棒性。对于R_(SN)>3的细胞,两种方法得到一致的E值,但是E-FRET方法得到的个别质粒R_(C)值偏小;对于R_(SN)<3的细胞,两种方法都能得到一致的R_(C)值,但是E-FRET方法得到的个别质粒E值误差率大于0.1,与文献值偏差稍大。E-FRET与mExEm-spFRET具有几乎一致的活细胞定量FRET检测能力,但是mExEm-spFRET的鲁棒性优于E-FRET方法。

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 andγ)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(RC)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).