Tracking Image Correlation: Combining Single-Particle Tracking and Image Correlation
Tracking Image Correlation: Combining Single-Particle Tracking and Image Correlation
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DOI：
10.1016/j.bpj.2013.04.005
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发表时间：
2013-06-04
影响因子：
3.4
通讯作者：
Lamb, D. C.
中科院分区：
文献类型：
--
作者：
Dupont, A.;Stirnnagel, K.;Lamb, D. C.
生物分子之间的相互作用和协调对大多数细胞功能是至关重要的。对活细胞中蛋白质相互作用的观察可能会更好地理解潜在的机制。经过相互作用伙伴的荧光标记和活细胞显微镜，通常用定量的全局方法分析共定位。最近的研究已经解决了移动生物分子的个体共定位问题，通常是通过使用单粒子跟踪(SPT)和比较两个颜色通道中的荧光强度来实现的。在这里，我们介绍了一种新的方法，结合SPT和相关方法来获得沿双色粒子单一轨迹的动态三维共局域化分析。在3D跟踪后，通过两个检测通道的局部3D图像互相关来计算每个粒子所在位置的共局化。对于每个被分析的粒子，输出由3D轨迹、时间分辨率3D共焦信息和两个通道中的荧光强度组成。此外，互相关分析表明，两个荧光标记的三维相对运动精度为30 nm。我们将该方法应用于跟踪活细胞中的病毒融合事件，并展示了它在密集和噪声环境中获得单个粒子的时间分辨共定位状态的能力。
The interactions and coordination of biomolecules are crucial for most cellular functions. The observation of protein interactions in live cells may provide a better understanding of the underlying mechanisms. After fluorescent labeling of the interacting partners and live-cell microscopy, the colocalization is generally analyzed by quantitative global methods. Recent studies have addressed questions regarding the individual colocalization of moving biomOlecules, usually by using single-particle tracking (SPT) and comparing the fluorescent intensities in both color channels. Here, we introduce a new method that combines SPT and correlation methods to obtain a dynamical 3D colocalization analysis along single trajectories of dual-colored particles. After 3D tracking, the colocalization is computed at each particle's position via the local 3D image cross correlation of the two detection channels. For every particle analyzed, the output consists of the 3D trajectory, the time-resolved 3D colocalization information, and the fluorescence intensity in both channels. In addition, the cross-correlation analysis shows the 3D relative movement of the two fluorescent labels with an accuracy of 30 nm. We apply this method to the tracking of viral fusion events in live cells and demonstrate its capacity to obtain the time-resolved colocalization status of single particles in dense and noisy environments.