Analysis of conditional colocalization relationships and hierarchies from three-color microscopy images
Colocalization is a cornerstone approach in cell biology for the analysis of multicolor microscopy images. It provides information on the localization of molecules within various subcellular compartments and allows the interrogation of molecular interactions in their spatiotemporal cellular context. However, the overwhelming majority of colocalization analyses are designed for two-color microscopy images, which limits their applicability and the type of information that they may reveal, leading to underutilization of multicolor microscopy images. Here we describe an approach for analyzing the colocalization relationships between three molecular entities, termed 'conditional colocalization analysis,' based on spatial point pattern analysis of detected objects in microscopy images. Going beyond the question of whether colocalization is present or not, it addresses the question of whether the colocalization between two molecular entities is influenced, positively or negatively, by their respective colocalization with a third entity. We showcase two applications of conditional colocalization analysis, one addressing the question of the compartmentalization of molecular interactions, and one investigating the hierarchy of molecular interactions in a multimolecular complex. The software for conditional colocalization analysis is freely accessible online at https://github.com/kjaqaman/conditionalColoc.