Department of Chemistry, Center for NanoScience and Center for Integrated Protein Science Munich, Ludwig Maximilians Universität, Munich, Germany.
Biophys J. 2013 Jun 4;104(11):2373-82. doi: 10.1016/j.bpj.2013.04.005.
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.
生物分子的相互作用和协调对于大多数细胞功能至关重要。在活细胞中观察蛋白质相互作用可以更好地理解潜在的机制。在对相互作用的伙伴进行荧光标记并进行活细胞显微镜观察后，通常通过定量全局方法分析共定位。最近的研究已经解决了关于移动生物分子的个体共定位的问题，通常是通过使用单粒子跟踪（SPT）并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，该方法结合 SPT 和相关方法，可沿双荧光粒子的单个轨迹获得动态的 3D 共定位分析。在 3D 跟踪之后，通过两个检测通道的局部 3D 图像互相关计算每个粒子位置处的共定位。对于分析的每个粒子，输出结果包括 3D 轨迹、时间分辨的 3D 共定位信息以及两个通道中的荧光强度。此外，相关分析以 30nm 的精度显示了两个荧光标记的 3D 相对运动。我们将该方法应用于活细胞中病毒融合事件的跟踪，并证明了它在密集和嘈杂的环境中获取单个粒子的时间分辨共定位状态的能力。
