Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization
Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization
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DOI：
10.1002/jemt.10430
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发表时间：
2004-01-01
影响因子：
2.5
通讯作者：
Periasamy, A
中科院分区：
文献类型：
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作者：
Chen, YE;Periasamy, A
双光子激发荧光共振能量转移 (2P-FRET) 成像显微镜可以提供活细胞内特定蛋白质分子相互作用的详细信息。用于 2P-FRET 成像的荧光团分子具有特征吸收和发射光谱，会在 FRET 信号中引入光谱串扰（渗透），应在 2P-FRET 图像中消除该串扰，以确定 FRET 确实发生并为距离估计奠定基础。 FRET 信号中的这些污染可以使用数学算法进行校正，以提取真实的 FRET 信号。另一种方法是 2P-FRET 荧光寿命成像 (FLIM)。这种方法允许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 用于研究 CAATT/增强子结合蛋白 α (C/EBPα) 的二聚化。结果表明，在受体存在的情况下，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和受体之间的距离。我们描述了使用 Bio-Rad Radiance2100 共焦/多光子显微镜系统的 2P-FRET-FLIM 成像系统的开发和表征。
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.