Quantitative Determination of Spatial Protein-Protein Correlations in Fluorescence Confocal Microscopy
Quantitative Determination of Spatial Protein-Protein Correlations in Fluorescence Confocal Microscopy
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DOI：
10.1016/j.bpj.2009.10.037
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发表时间：
2010-02-03
影响因子：
3.4
通讯作者：
Stefani, Enrico
中科院分区：
文献类型：
--
作者：
Wu, Yong;Eghbali, Mansoureh;Stefani, Enrico
为了量化空间蛋白质-蛋白质接近（共定位）在成对的显微图像的两组不同的荧光团标记的蛋白质，我们表明，图像强度的互相关和自相关函数包括快速和缓慢衰减的组件。快速成分由特异性标记的蛋白质簇引起，而慢速成分由图像异质性和广泛分布的背景引起。为了更好地评估两个特异性标记的蛋白质之间的空间邻近性，我们通过将相关函数中的尖峰细化为高斯函数来提取快速衰减的分量，然后将其用于获得蛋白质-蛋白质邻近性指数和Pearson相关系数。我们还采用了中值滤波方法作为背景减少的通用方法，以最大限度地减少非特异性荧光。我们通过分析计算机模拟图像和生物图像来说明我们的方法。
To quantify spatial protein-protein proximity (colocalization) in paired microscopic images of two sets of proteins labeled by distinct fluorophores, we showed that the cross-correlation and the autocorrelation functions of image intensity consisted of fast and slowly decaying components. The fast component resulted from clusters of proteins specifically labeled, and the slow component resulted from image heterogeneity and a broadly-distributed background. To better evaluate spatial proximity between the two specifically labeled proteins, we extracted the fast-decaying component by fining the sharp peak in correlation functions to a Gaussian function, which was then used to obtain protein-protein proximity index and the Pearson's correlation coefficient. We also employed the median-filter method as a universal approach for background reduction to minimize nonspecific fluorescence. We illustrated our method by analyzing computer-simulated images and biological images.