Hachet-Haas Muriel, Converset Noël, Marchal Olivier, Matthes Hans, Gioria Sophie, Galzi Jean-Luc, Lecat Sandra
Département Récepteurs et Protéines Membranaires, Institut Gilbert-Laustriat, UMR 7175 CNRS/Université-Strasbourg I, BP10413, F-67412 ILLKIRCH Cedex, France.
Microsc Res Tech. 2006 Dec;69(12):941-56. doi: 10.1002/jemt.20376.
Fluorescence resonance energy transfer (FRET) between an adequate pair of fluorophores is an indication of closer proximity than colocalization and is used by biologists to study fluorescently modified protein interactions inside cells. We present a method for visualization of FRET images acquired by confocal sensitized emission, involving excitation of the donor fluorophore and detection of the energy transfer as an emission from the acceptor fluorophore into the FRET channel. Authentic FRET signal measurements require the correction from the FRET channel of the undesired bleed-through signals (BT) resulting from both the leak-through of the donor emission and the direct acceptor emission. Our method reduces the interference of the user to a minimum by analyzing the entire image, pixel by pixel. It proposes imaging treatments and the display of control images to validate the BT calculation and the image corrections. It displays FRET images as a function of the colocalization of the two fluorescent partners. Finally, it proposes an alternative to normalization of the FRET intensities to compare FRET signal variations between samples. This method called "FRET and Colocalization Analyzer" has been implemented in a Plug-in of the freely available ImageJ software. It is particularly adapted when transient expression of the fluorescent proteins is used thereby giving very variable expression levels or when the colocalization of the two partners is varying in proportion, in amount, and in size, as a function of time. The method and program are validated using the analysis of the spatio-temporal interactions between a G-protein coupled receptor, the tachykinin NK2 receptor, and the beta-arrestin 2 as an example.
一对合适的荧光团之间的荧光共振能量转移（FRET）表明它们比共定位更接近，生物学家利用它来研究细胞内荧光修饰的蛋白质相互作用。我们提出了一种用于可视化通过共聚焦敏化发射获得的FRET图像的方法，该方法涉及供体荧光团的激发以及将能量转移检测为受体荧光团向FRET通道的发射。真实的FRET信号测量需要从FRET通道校正由供体发射的泄漏和受体直接发射导致的不期望的渗漏信号（BT）。我们的方法通过逐像素分析整个图像将用户的干扰降至最低。它提出了成像处理方法并显示控制图像以验证BT计算和图像校正。它将FRET图像显示为两个荧光伴侣共定位的函数。最后，它提出了一种替代FRET强度归一化的方法，以比较样品之间的FRET信号变化。这种称为“FRET和共定位分析器”的方法已在免费的ImageJ软件的插件中实现。当使用荧光蛋白的瞬时表达从而产生非常可变的表达水平时，或者当两个伴侣的共定位随时间在比例、数量和大小上发生变化时，该方法特别适用。该方法和程序通过分析G蛋白偶联受体、速激肽NK2受体和β-抑制蛋白2之间的时空相互作用进行了验证。
