Abstract
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.