Chen Ye, Periasamy Ammasi
W.M. Keck Center for Cellular Imaging, University of Virginia, Charlottesville, Virginia 22904, USA.
Microsc Res Tech. 2004 Jan 1;63(1):72-80. doi: 10.1002/jemt.10430.
Two-photon excitation fluorescence resonance energy transfer (2P-FRET) imaging microscopy can provide details of specific protein molecule interactions inside living cells. Fluorophore molecules used for 2P-FRET imaging have characteristic absorption and emission spectra that introduce spectral cross-talk (bleed-through) in the FRET signal that should be removed in the 2P-FRET images, to establish that FRET has actually occurred and to have a basis for distance estimations. These contaminations in the FRET signal can be corrected using a mathematical algorithm to extract the true FRET signal. Another approach is 2P-FRET fluorescence lifetime imaging (FLIM). This methodology allows studying the dynamic behavior of protein-protein interactions in living cells and tissues. 2P-FRET-FLIM was used to study the dimerization of the CAATT/enhancer binding protein alpha (C/EBPalpha). Results show that the reduction in donor lifetime in the presence of acceptor reveals the dimerization of the protein molecules and also determines more precisely the distance between the donor and acceptor. We describe the development and characterization of the 2P-FRET-FLIM imaging system with the Bio-Rad Radiance2100 confocal/multiphoton microscopy system.
双光子激发荧光共振能量转移（2P-FRET）成像显微镜能够提供活细胞内特定蛋白质分子相互作用的细节。用于2P-FRET成像的荧光团分子具有特征性的吸收和发射光谱，这会在FRET信号中引入光谱串扰（渗漏），应在2P-FRET图像中予以去除，以确定FRET是否实际发生，并为距离估计提供依据。FRET信号中的这些干扰可以通过数学算法进行校正，以提取真实的FRET信号。另一种方法是2P-FRET荧光寿命成像（FLIM）。这种方法能够研究活细胞和组织中蛋白质-蛋白质相互作用的动态行为。2P-FRET-FLIM被用于研究CCAAT/增强子结合蛋白α（C/EBPα）的二聚化。结果表明，在存在受体的情况下供体寿命的缩短揭示了蛋白质分子的二聚化，并且还更精确地确定了供体与受体之间的距离。我们描述了使用伯乐Radiance2100共聚焦/多光子显微镜系统的2P-FRET-FLIM成像系统的开发与特性。
