The ultrastructural localization of various antigens in a cell using antibo
dies conjugated to gold particles is a powerful instrument in biological re
search. However, statistical or stereological tools for testing the observe
d patterns for significant clustering or colocalization are missing. The pa
per presents a method for the quantitative analysis of single or multiple i
mmunogold labeling patterns using interpoint distances and tests the method
using experimental data, The clustering or colocalization of gold particle
s was detected using various characteristics of the distribution of distanc
es between them. Pair correlation and cross-correlation functions were used
for exploratory analysis; second order reduced K (or cross-K) functions we
re used for testing the statistical significance of observed events. Confid
ence intervals of function values were estimated by Monte Carlo simulations
of the Poisson process for independent particles, and results were visuali
zed in histograms. Furthermore, a suitability of K functions modified by ce
nsoring or weighting was tested. The reliability of the method was assessed
by evaluating the labeling patterns of nascent DNA and several nuclear pro
teins with known functions in replication foci of HeLa cells. The results d
emonstrate that the method is a powerful tool in biological investigations
for testing the statistical significance of observed clustering or colocali
zation patterns in immunogold labeling experiments. (C) 2000 Academic Press
.