Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, B-9000 Gent, Belgium.
Int J Mol Sci. 2013 Aug 8;14(8):16485-514. doi: 10.3390/ijms140816485.
Interactions between objects inside living cells are often investigated by looking for colocalization between fluorescence microscopy images that are recorded in separate colours corresponding to the fluorescent label of each object. The fundamental limitation of this approach in the case of dynamic objects is that coincidental colocalization cannot be distinguished from true interaction. Instead, correlation between motion trajectories obtained by dual colour single particle tracking provides a much stronger indication of interaction. However, frequently occurring phenomena in living cells, such as immobile phases or transient interactions, can limit the correlation to small parts of the trajectories. The method presented here, developed for the detection of interaction, is based on the correlation inside a window that is scanned along the trajectories, covering different subsets of the positions. This scanning window method was validated by simulations and, as an experimental proof of concept, it was applied to the investigation of the intracellular trafficking of polymeric gene complexes by endosomes in living retinal pigment epithelium cells, which is of interest to ocular gene therapy.
活细胞内物体之间的相互作用通常通过寻找荧光显微镜图像之间的共定位来进行研究，这些图像是根据每个物体的荧光标记分别记录在不同颜色的图像中。在动态物体的情况下，这种方法的基本限制是不能区分偶然的共定位与真实的相互作用。相反，通过双色单粒子追踪获得的运动轨迹之间的相关性提供了相互作用的更强指示。然而，活细胞中经常出现的现象，如无定形相或瞬时相互作用，可能会将相关性限制在轨迹的小部分上。这里提出的用于检测相互作用的方法是基于在沿着轨迹扫描的窗口内的相关性，该窗口覆盖了位置的不同子集。该扫描窗口方法通过模拟进行了验证，并且作为实验概念验证，它被应用于研究活视网膜色素上皮细胞中的内体中聚合基因复合物的细胞内运输，这对于眼部基因治疗很有意义。
