Multi-Image Colocalization and Its Statistical Significance
Multi-Image Colocalization and Its Statistical Significance
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DOI：
10.1016/j.bpj.2010.07.006
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发表时间：
2010-09-22
影响因子：
3.4
通讯作者：
Moore, Edwin D. W.
中科院分区：
文献类型：
--
作者：
Fletcher, Patrick A.;Scriven, David R. L.;Moore, Edwin D. W.
准确定位细胞内分子是现代生物学的主要任务之一，共定位分析是其主要和最常用的工具之一。尽管这种方法很流行，但解释往往是不确定的，因为很少分析两幅或多幅图像之间的共定位，以确定观测值是否可能偶然发生。为了解决这个问题，我们开发了一种基于蒙特卡罗随机化的稳健方法来测量共定位的统计显著性。该方法适用于基于体素、基于强度、基于对象和最近邻指标。我们将所有这些扩展到测量具有三种颜色的图像的共定位。我们还引入了三个新指标；Blob共定位，其中Blob由被三维体素群包围的局部最大值组成；簇直径，用于测量三个或更多图像中荧光团的簇；以及簇间距离来衡量这些簇之间的距离。通过在大范围内改变图像阈值来测试这些指标的稳健性，这在共定位的统计意义上没有变化。blob共定位与体素和曼德斯共定位指标的比较表明，不同的测量方法产生一致的结果，显著性和非显著性值相似。使用我们的方法，我们不仅能够确定标记的分子是否以大于偶然的概率共定位，而且还能够确定它们是否被隔离在不同的隔室中。该程序是用c++编写的，源代码和Linux版本都是免费的。
Accurately localizing molecules within the cell is one of main tasks of modern biology, and colocalization analysis is one of its principal and most often used tools. Despite this popularity, interpretation is often uncertain because colocalization between two or more images is rarely analyzed to determine whether the observed values could have occurred by chance. To address this, we have developed a robust methodology, based on Monte Carlo randomization, to measure the statistical significance of a colocalization. The method works with voxel-based, intensity-based, object-based, and nearest-neighbor metrics. We extend all of these to measure colocalization in images with three colors. We also introduce three new metrics; blob colocalization, where the blob consists of a local maximum surrounded by a three-dimensional group of voxels; cluster diameter, to measure the clustering of fluorophores in three or more images; and the intercluster distance to measure the distance between these clusters. The robustness of these metrics was tested by varying the image thresholds over a broad range, which produced no change in the statistical significance of the colocalizations. A comparison of blob colocalization with voxel and Manders colocalization metrics shows that the different measures produce consistent results with similar values for significance and nonsignificance. Using our methodology, we are able to determine not only whether the labeled molecules colocalize with a probability greater than chance, but also whether they are sequestrated into different compartments. The program, written in C++, is freely available as source, as well as in a Linux version.