Time-resolved three-dimensional molecular tracking in live cells.
Time-resolved three-dimensional molecular tracking in live cells.
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DOI：
10.1021/nl103247v
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发表时间：
2010-11-10
期刊：
影响因子：
10.8
通讯作者：
Werner JH
中科院分区：
文献类型：
--
作者：
Wells NP;Lessard GA;Goodwin PM;Phipps ME;Cutler PJ;Lidke DS;Wilson BS;Werner JH
我们报道了一种用于追踪活细胞内单个量子点（QD）标记蛋白质的方法，该方法采用四个重叠的共聚焦体积单元，并每5毫秒进行一次主动反馈，以追踪三维分子运动。相较于基于电荷耦合器件（CCD）相机的三维分子追踪方法，此方法具有显著优势，包括更大的Z轴追踪范围（本文展示为10微米）、显著更低的激发功率（本文使用15微瓦），以及对被追踪分子进行时间分辨光谱分析（如荧光寿命测量或荧光相关光谱分析）的能力。特别地，我们首次展示了活细胞内单个量子点标记蛋白质的荧光光子反聚束现象，并证明了能够以生物学相关的转运速率追踪单个染料标记的核苷酸（Cy5 - dUTP）。为了展示这些方法在探索活细胞时空动态方面的强大功能，我们对大鼠肥大细胞表面及内部的单个量子点标记的免疫球蛋白E（IgE）受体进行了追踪。质膜上受体的轨迹揭示了细胞表面拓扑结构的三维纳米级特征。在信号转导级联反应的后期阶段，量子点标记的IgE - FcεRI簇在配体介导的内吞作用过程中被捕获，并在其以约950纳米/秒的速度通过细胞的囊泡转运过程中得到追踪。
We report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses four overlapping confocal volume elements and active feedback once every 5 milliseconds to follow three dimensional molecular motion. This method has substantial advantages over 3D molecular tracking methods based upon CCD cameras, including increased Z tracking range (10 μm demonstrated here), substantially lower excitation powers (15 μW used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the molecules being tracked. In particular, we show for the first time fluorescence photon anti-bunching of individual QD labeled proteins in live cells and demonstrate the ability to track individual dye labeled nucleotides (Cy5-dUTP) at biologically relevant transport rates. To demonstrate the power of these methods for exploring the spatio-temporal dynamics of live cells, we follow individual QD-labeled IgE receptors both on and inside rat mast cells. Trajectories of receptors on the plasma membrane reveal three dimensional, nano-scale features of the cell surface topology. During later stages of the signal transduction cascade, clusters of QD labeled IgE-FcεRI were captured in the act of ligand-mediated endocytosis and tracked during rapid (~950 nm/s) vesicular transit through the cell.
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影响因子：
56.9
作者：
Elf, Johan;Li, Gene-Wei;Xie, X. Sunney
通讯作者：
Xie, X. Sunney
DOI：
10.1073/pnas.0912894107
发表时间：
2010-04-13
影响因子：
11.1
作者：
Sahl, Steffen J.;Leutenegger, Marcel;Eggeling, Christian
通讯作者：
Eggeling, Christian
影响因子：
32.4
作者：
Andrews, Nicholas L.;Pfeiffer, Janet R.;Lidke, Diane S.
通讯作者：
Lidke, Diane S.
影响因子：
7.8
作者：
Fujiwara, Takahiro;Ritchie, Ken;Murakoshi, Hideji;Jacobson, Ken;Kusumi, Akihiro
通讯作者：
Kusumi, Akihiro
影响因子：
64.8
作者：
Eggeling, Christian;Ringemann, Christian;Hell, Stefan W.
通讯作者：
Hell, Stefan W.