Breusegem Sophia Y, Levi Moshe, Barry Nicholas P
Department of Medicine, Division of Renal Diseases and Hypertension, University of Colorado Health Sciences Center, Denver, Colo 80262, USA.
Nephron Exp Nephrol. 2006;103(2):e41-9. doi: 10.1159/000090615. Epub 2006 Mar 10.
With few and commercially available add-ons, both confocal and full-field fluorescence microscopes can be adapted to provide more information on the biological sample of interest. In this review we discuss the possibilities offered by two additional functionalities to fluorescence microscopes, fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging mi croscopy (FLIM). FCS measurements at a single point in a sample allow kinetic and diffusion properties of fluorescently labeled molecules to be determined, as well as their concentration and aggregation state. Data from multiple points of the sample can be acquired using scanning-FCS, image correlation spectroscopy, and raster image correlation spectroscopy. These techniques cover phenomena with characteristic durations from sub-microsecond to second time scales. The power of FLIM lies in the fact that the measured fluorescent lifetime of a fluorophore is sensitive to the molecular environment of that fluorophore. FLIM is a robust means to quantify Forster resonance energy transfer and thus determine protein-protein interactions or protein conformational changes. In addition, FLIM is very valuable for functional imaging of ion concentrations in cells and tissues as it can be applied in heterogeneously labeled samples. In summary, FCS and FLIM allow information to be gathered beyond localization, including diffusional mobility, protein clustering and interactions, and molecular environment.
只需少量市面上可买到的附加组件，共聚焦显微镜和全场荧光显微镜都能够进行改装，以便提供更多有关感兴趣生物样品的信息。在本综述中，我们将讨论荧光显微镜的另外两种功能——荧光相关光谱法（FCS）和荧光寿命成像显微镜技术（FLIM）所带来的可能性。在样品中的单个点进行FCS测量，可以确定荧光标记分子的动力学和扩散特性，以及它们的浓度和聚集状态。使用扫描FCS、图像相关光谱法和光栅图像相关光谱法，可以从样品的多个点获取数据。这些技术涵盖了从亚微秒到秒时间尺度的具有特征持续时间的现象。FLIM的强大之处在于，所测量的荧光团的荧光寿命对该荧光团的分子环境敏感。FLIM是一种用于量化福斯特共振能量转移从而确定蛋白质-蛋白质相互作用或蛋白质构象变化的可靠方法。此外，FLIM对于细胞和组织中离子浓度的功能成像非常有价值，因为它可以应用于异质标记的样品。总之，FCS和FLIM能够收集除定位之外的信息，包括扩散迁移率、蛋白质聚集和相互作用以及分子环境。
