Wallrabe Horst, Sun Yuansheng, Fang Xiaolan, Periasamy Ammasi, Bloom George
Keck Center for Cellular Imaging, University of Virginia, Charlottesville, VA ; Department of Biology, University of Virginia, Charlottesville, VA.
Proc SPIE Int Soc Opt Eng. 2012 Feb 9;8226. doi: 10.1117/12.906432. Epub 2012 Jan 21.
With traditional 2-color Förster Resonance Energy Transfer (FRET) microscopy, valuable quantitative analyses can be conducted. Correlations of donor (D), acceptor (A) and their ratios (D:A) with energy transfer efficiency (E%) or distance (r) allows measurement of changes between control and experimental samples; also, clustered vs. random assembly of cellular components can be differentiated. Essentially, only the above three parameters D, A and D:A vs. E% are the basis for these deductions. 3-color FRET uses the same basic parameters, but exponentially expands the opportunities to quantify interrelationships among 3 cellular components. We investigated a number of questions based on the results of a triple combination (F1-F2-F3) of TFP-NWASP/Venus-IQGAP1/mCherry-Actin - all involved in the nucleation of actin - to apply the extensive analysis assay possible with 3-color FRET. How do changing N-WASP or IQGAP1 fluorescence levels affect actin fluorescence? What is the effect on E% of NWASP-actin by IQGAP1 or E% of IQGAP1-actin by N-WASP? These and other questions are explored in the context of all proteins of interest being in FRET distance vs. any two in the absence of the third. 4 cases are compared based on bleed-through corrected FRET: (1) all 3 interact, (2) only F1-F3 and F2-F3 [not F1-F2], (3) only F1-F2 and F2-F3 interact [not F1-F3], (4) only F1-F2 and F1-F3 interact [not F2-F3]. Other than describing the methodology in detail, several biologically relevant results are presented showing how E% (i.e. distance), fluorescence levels and ratios are affected in each of the cases. These correlations can only be observed in a 3-fluorophore combination. 3-color FRET will greatly expand the investigative range of quantitative analysis for the life-science researcher.
利用传统的双色荧光共振能量转移（FRET）显微镜，可以进行有价值的定量分析。供体（D）、受体（A）及其比例（D:A）与能量转移效率（E%）或距离（r）之间的相关性，能够测量对照样品与实验样品之间的变化；此外，还可以区分细胞成分的聚集组装与随机组装。本质上，只有上述三个参数D、A和D:A与E%是这些推导的基础。三色FRET使用相同的基本参数，但极大地扩展了量化三种细胞成分之间相互关系的机会。我们基于TFP-NWASP/Venus-IQGAP1/mCherry-Actin的三重组合（F1-F2-F3）的结果研究了许多问题——所有这些都参与肌动蛋白的成核——以应用三色FRET可能进行的广泛分析检测。N-WASP或IQGAP1荧光水平的变化如何影响肌动蛋白荧光？IQGAP1对NWASP-肌动蛋白的E%或N-WASP对IQGAP1-肌动蛋白的E%有什么影响？在所有感兴趣的蛋白质都处于FRET距离与任意两者在没有第三者的情况下，探讨了这些及其他问题。基于扣除渗漏的FRET比较了4种情况：（1）所有三者相互作用，（2）只有F1-F3和F2-F3相互作用[不是F1-F2]，（3）只有F1-F2和F2-F3相互作用[不是F1-F3]，（4）只有F1-F2和F1-F3相互作用[不是F2-F3]。除了详细描述方法外，还展示了一些与生物学相关的结果，表明在每种情况下E%（即距离）、荧光水平和比例是如何受到影响的。这些相关性只能在三荧光团组合中观察到。三色FRET将极大地扩展生命科学研究人员定量分析的研究范围。
