Philimonenko A A, Janácek J, Hozák P
Department of Cell Ultrastructure and Molecular Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
J Struct Biol. 2000 Dec;132(3):201-10. doi: 10.1006/jsbi.2000.4326.
The ultrastructural localization of various antigens in a cell using antibodies conjugated to gold particles is a powerful instrument in biological research. However, statistical or stereological tools for testing the observed patterns for significant clustering or colocalization are missing. The paper presents a method for the quantitative analysis of single or multiple immunogold labeling patterns using interpoint distances and tests the method using experimental data. The clustering or colocalization of gold particles was detected using various characteristics of the distribution of distances between them. Pair correlation and cross-correlation functions were used for exploratory analysis; second order reduced K (or cross-K) functions were used for testing the statistical significance of observed events. Confidence intervals of function values were estimated by Monte Carlo simulations of the Poisson process for independent particles, and results were visualized in histograms. Furthermore, a suitability of K functions modified by censoring or weighting was tested. The reliability of the method was assessed by evaluating the labeling patterns of nascent DNA and several nuclear proteins with known functions in replication foci of HeLa cells. The results demonstrate that the method is a powerful tool in biological investigations for testing the statistical significance of observed clustering or colocalization patterns in immunogold labeling experiments.
利用与金颗粒偶联的抗体对细胞中各种抗原进行超微结构定位，是生物学研究中的一种强有力手段。然而，目前缺少用于检验所观察到的显著聚类或共定位模式的统计或体视学工具。本文提出了一种利用点间距离对单重或多重免疫金标记模式进行定量分析的方法，并使用实验数据对该方法进行了检验。利用金颗粒之间距离分布的各种特征来检测金颗粒的聚类或共定位情况。配对相关函数和交叉相关函数用于探索性分析；二阶约化K（或交叉K）函数用于检验所观察事件的统计显著性。通过对独立颗粒的泊松过程进行蒙特卡罗模拟来估计函数值的置信区间，并以直方图形式呈现结果。此外，还测试了经删失或加权修改的K函数的适用性。通过评估新生DNA和几种在HeLa细胞复制位点具有已知功能的核蛋白的标记模式，对该方法的可靠性进行了评估。结果表明，该方法是生物学研究中检验免疫金标记实验中所观察到的聚类或共定位模式统计显著性的有力工具。
