Quantitative fluorescence imaging of protein diffusion and interaction in living cells
Quantitative fluorescence imaging of protein diffusion and interaction in living cells
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DOI：
10.1038/nbt.1928
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发表时间：
2011-09-01
影响因子：
46.9
通讯作者：
Knop, Michael
中科院分区：
文献类型：
--
作者：
Capoulade, Jeremie;Wachsmuth, Malte;Knop, Michael
细胞内的扩散过程和局部动态平衡会导致分子空间分布不均匀，这对于细胞分裂、分化和迁移中的核组织和信号传导等过程至关重要 (1)。为了理解这些机制，需要对蛋白质丰度、迁移率和相互作用进行空间分辨定量测量，但目前的方法研究动态参数的能力有限。在这里，我们描述了一种基于光片照明（2）的显微镜，它允许大规模并行荧光相关光谱（FCS）（3）测量，并用它来可视化哺乳动物细胞和离体果蝇组织中蛋白质的扩散和相互作用。对细胞核中异染色质蛋白 HP1 α（参考文献 4）的迁移率进行成像，我们可以提供高分辨率扩散图，揭示具有类似异染色质 HP1 α-染色质相互作用的常染色质区域。我们期望 FCS 成像将成为精确表征细胞反应扩散过程的有用方法。
Diffusion processes and local dynamic equilibria inside cells lead to nonuniform spatial distributions of molecules, which are essential for processes such as nuclear organization and signaling in cell division, differentiation and migration(1). To understand these mechanisms, spatially resolved quantitative measurements of protein abundance, mobilities and interactions are needed, but current methods have limited capabilities to study dynamic parameters. Here we describe a microscope based on light-sheet illumination(2) that allows massively parallel fluorescence correlation spectroscopy (FCS)(3) measurements and use it to visualize the diffusion and interactions of proteins in mammalian cells and in isolated fly tissue. Imaging the mobility of heterochromatin protein HP1 alpha (ref. 4) in cell nuclei we could provide high-resolution diffusion maps that reveal euchromatin areas with heterochromatin-like HP1 alpha-chromatin interactions. We expect that FCS imaging will become a useful method for the precise characterization of cellular reaction-diffusion processes.