Imaging Core

意义 光学显微镜促进了我们对细胞结构和相关功能的理解。 研究表明，细胞事件，如信号转导和基因转录，需要蛋白质组装成特定的大分子复合物。传统的生物物理或生物化学方法还没有提供直接进入蛋白质伴侣在其自然环境中的相互作用，但光显微镜技术使我们能够在生理条件下研究分子。新的成像技术，加上新的基因编码的发展， 荧光标记和传感器，以及计算机软件的能力不断提高， 图像获取和分析使研究人员能够对蛋白质分子的功能和过程进行更复杂的研究，范围从基因表达到第二信使级联和细胞间信号传导（DelPozo等人，2002; Struck等人，1981; Roessel和Brand，2002; Ting等人， 2001年）。一种高灵敏度和非侵入性的蛋白质分子成像方法是福斯特（荧光）共振能量转移（FRET）显微镜。 FRET是一个距离依赖的物理过程，能量从一个激发的分子荧光团（供体）非辐射地转移到另一个荧光团 （受体）通过分子间长程偶极-偶极耦合。FRET可以 准确测量分子接近度（1-10 nm），通常当供体和受体位于福斯特半径（供体一半激发能转移到受体的距离，约3-6 nm）内时。的 FRET的效率取决于分子间分离的逆六次方（Forster，1965; Lakowicz，1999; Stryer，1978）， 使其成为研究产生分子邻近性变化的各种生物现象的灵敏方法（Cummings等人，2002; Day等人，2003; Miyawaki等人，1999; Wallrabe等人，2003年a）。如果发生FRET，供体荧光被淬灭，受体荧光被敏化（增加）（Periasamy和Day，2005）。供体和受体标记的探针的共定位可以在~0.09 μ m内看到，并且可以验证近距离的分子缔合。

Significance Light microscopy advanced our understanding of cellular structure and associated functions. Research has shown that cellular events, such as signal transduction and gene transcription, require the assembly of proteins into specific macromolecular complexes. Traditional biophysical or biochemical methods have not provided direct access to interactions of protein partners in their natural environments but light microscopic techniques allow us to study molecules under physiological conditions. New imaging technologies, coupled with the development of new genetically encoded fluorescent labels and sensors, and the increasing capability of computer software for image acquisition and analysis, have enabled researchers to conduct more sophisticated studies of the functions and processes of protein molecules, ranging from gene expression to second-messenger cascades and intercellular signaling (DelPozo et al., 2002; Struck et al., 1981; Roessel and Brand, 2002; Ting et al., 2001). One highly sensitive and non-invasive method of protein molecular imaging is Forster (fluorescence) resonance energy transfer (FRET) microscopy. FRET is a distance-dependent physical process, where energy is transferred nonradiatively from an excited molecular fluorophore (donor) to another fluorophore (acceptor) by means of intermolecular longrange dipole-dipole coupling. FRET can accurately measure molecular proximity (1-10 nm), typically when donor and acceptor are positioned within the Forster radius (the distance at which half the excitation energy of the donor is transferred to the acceptor, ~3-6 nm). The efficiency of FRET is dependent on the inverse sixth power of intermolecular separation (Forster, 1965; Lakowicz, 1999; Stryer, 1978), making it a sensitive method for investigating a variety of biological phenomena that produce changes in molecular proximity (Cummings et al., 2002; Day et al 2003; Miyawaki et al., 1999; Wallrabe et al., 2003a). If FRET occurs, donor fluorescence is quenched and acceptor fluorescence is sensitized (increased) (Periasamy and Day, 2005). Co-localization of the donor- and acceptor-labeled probes can be seen within ~0.09 um and molecular associations at close distances can be verified.