Fluorescence correlation spectroscopy with autofluorescent proteins.
Fluorescence correlation spectroscopy with autofluorescent proteins.
复制标题
具有自发荧光蛋白质的荧光相关光谱。
DOI：
10.1007/b102212
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发表时间：
2005
期刊：
影响因子：
--
通讯作者：
P. Schwille
中科院分区：
文献类型：
--
作者：
T. Kohl;P. Schwille
荧光相关光谱 (FCS) 是一种在单分子水平上运行的多功能技术，成功应对了现代生物学研究的许多挑战。基于对扩散进出衍射极限激光焦点的移动荧光分子的检测，该方法可以解析细胞及其隔室内的粒子动力学。先前的FCS研究描述了蛋白质功能的各种参数，即迁移性、运输和定位现象、生化底物的酶促转换以及分子缔合和解离反应。 FCS 在细胞内系统应用方面的最新进展特别利用了检测自发荧光蛋白及其基因编码的细胞蛋白融合物。本综述讨论了 FCS 分析在荧光蛋白上的最新应用，特别强调了化学和物理特性。详细讨论了所提出方法的固有局限性，并概述了实验系统优化的策略。
Fluorescence correlation spectroscopy (FCS) is a versatile technique operating at the single-molecule level, that successfully meets many challenges of modern biological research. Based on the detection of mobile fluorescent molecules diffusing in and out of a diffraction-limited laser focus, the method allows to resolve particle dynamics within cells and their compartments. Previous FCS studies have described various parameters of protein function, namely mobility, transport and localization phenomena, enzymatic turnovers of biochemical substrates and molecular association and dissociation reactions. Recent progress in the application of FCS to intracellular systems has particularly taken advantage of detecting autofluorescent proteins and their genetically encoded fusions to cellular proteins. This review discusses recent applications of FCS analysis with and on fluorescent proteins, particularly highlighting chemical and physical properties. Inherent limitations of the presented approaches are discussed in detail and strategies for optimisation of experimental systems outlined.