UNSW Australia, Biomedical Imaging Facility, Mark Wainwright Analytical Center, Sydney, Australia; Department of Physics, McGill University, Montreal, Canada.
Department of Physics, McGill University, Montreal, Canada; Department of Physiology, McGill University, Montreal, Canada.
Methods. 2018 May 1;140-141:126-139. doi: 10.1016/j.ymeth.2018.02.011. Epub 2018 Feb 16.
Molecular, vesicular and organellar flows are of fundamental importance for the delivery of nutrients and essential components used in cellular functions such as motility and division. With recent advances in fluorescence/super-resolution microscopy modalities we can resolve the movements of these objects at higher spatio-temporal resolutions and with better sensitivity. Previously, spatio-temporal image correlation spectroscopy has been applied to map molecular flows by correlation analysis of fluorescence fluctuations in image series. However, an underlying assumption of this approach is that the sampled time windows contain one dominant flowing component. Although this was true for most of the cases analyzed earlier, in some situations two or more different flowing populations can be present in the same spatio-temporal window. We introduce an approach, termed velocity landscape correlation (VLC), which detects and extracts multiple flow components present in a sampled image region via an extension of the correlation analysis of fluorescence intensity fluctuations. First we demonstrate theoretically how this approach works, test the performance of the method with a range of computer simulated image series with varying flow dynamics. Finally we apply VLC to study variable fluxing of STIM1 proteins on microtubules connected to the plasma membrane of Cystic Fibrosis Bronchial Epithelial (CFBE) cells.
分子、囊泡和细胞器流对于细胞功能（如运动和分裂）中使用的营养物质和基本成分的输送至关重要。随着荧光/超分辨率显微镜技术的最新进展，我们可以以更高的时空分辨率和更好的灵敏度来解析这些物体的运动。以前，通过对图像序列中的荧光波动进行相关分析，时空图像相关光谱学已被应用于绘制分子流图。然而，这种方法的一个基本假设是，采样的时间窗口包含一个主要的流动成分。尽管对于之前分析的大多数情况来说这是正确的，但在某些情况下，同一时空窗口中可能存在两个或更多不同的流动群体。我们引入了一种方法，称为速度景观相关（VLC），它通过扩展荧光强度波动的相关分析来检测和提取采样图像区域中存在的多个流动成分。首先，我们从理论上演示了这种方法的工作原理，并用一系列具有不同流动动力学的计算机模拟图像系列测试了该方法的性能。最后，我们应用 VLC 来研究连接到囊性纤维化支气管上皮 (CFBE) 细胞质膜的微管上的 STIM1 蛋白的可变通量。
