Tramier Marc, Coppey-Moisan Maïté
Institut Jacques Monod, UMR 7592 CNRS, University Paris 6/University Paris 7, 2 Place Jussieu, 75251 Paris Cedex 05, France.
Methods Cell Biol. 2008;85:395-414. doi: 10.1016/S0091-679X(08)85017-0.
In this chapter, we present the basic physical principles of the fluorescence anisotropy imaging microscopy (FAIM) and its application to study FP-tagged protein dynamics and interaction in live cells. The Förster mechanism of electronic energy transfer can occur between like chromophores (homo-fluorescence resonance energy transfer, homo-FRET) inducing fluorescence depolarization and can be monitored by fluorescence anisotropy. The energy transfer rate is fast compared to the rotational time of proteins, and therefore its detection as a fast depolarization process in the fluorescence anisotropy can be easily discriminated from rotational motion. Quantitative analysis of fluorescence anisotropy decays provides information on structural parameters: distance between the two interacting chromophores and spatial orientation between the chromophores within dimeric proteins. Fluorescence anisotropy decay is not easy to measure in living cells under the microscope and the instrumentations are necessarily sophisticated. In contrast, any type of microscope can be used to measure the steady-state anisotropy. Interestingly, two-photon excitation steady-state FAIM is a powerful tool for qualitative analysis of macromolecule interactions in living cells and can be used easily for time-lapse homo-FRET.
在本章中，我们介绍了荧光各向异性成像显微镜（FAIM）的基本物理原理及其在研究活细胞中荧光蛋白标签标记的蛋白质动力学和相互作用方面的应用。电子能量转移的Förster机制可在同类发色团之间发生（同型荧光共振能量转移，同型FRET），从而导致荧光去极化，并且可以通过荧光各向异性进行监测。与蛋白质的旋转时间相比，能量转移速率很快，因此将其检测为荧光各向异性中的快速去极化过程可以很容易地与旋转运动区分开来。荧光各向异性衰减的定量分析提供了有关结构参数的信息：两个相互作用的发色团之间的距离以及二聚体蛋白质中发色团之间的空间取向。在显微镜下在活细胞中测量荧光各向异性衰减并不容易，并且仪器设备必然很复杂。相比之下，任何类型的显微镜都可用于测量稳态各向异性。有趣的是，双光子激发稳态FAIM是用于定性分析活细胞中大分子相互作用的强大工具，并且可以很容易地用于延时同型FRET。
