Journal of Structural Biology, Vol. 132, No. 3, December 2000, pp. 201-210 (ISSN: 1047-8477; doi:10.1006/jsbi.2000.4326)

Statistical Evaluation of Colocalization Patterns in Immunogold Labelling Experiments

Anatoly A. Philimonenko^{1, 3}, Jiri Janácek², and Pavel Hozák¹

¹Department of Cell Ultrastructure & Molecular Biology, Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic;
²Department of Biomathematics, Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic;
³Department of Evolutionary Animal Genetics, Institute of Cytology and Genetics; Academy of Sciences of Russia, Siberian Division, Novosibirsk, Russia

Correspondence should be addressed to Dr. P. Hozák at Institute of Experimental Medicine, Department of Cell Ultrastructure and Molecular Biology, Vídenská 1083, 142 20 Prague 4-Krc, Czech Republic. Phone/Fax: +420-241-062-219. E-mail: hozak@img.cas.cz

Abstract

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. Copyright 2000 Academic Press.

Key Words:  cluster; colocalization; DNA replication; immunogold labeling; pattern analysis; stereology; ultrastructure