Analysis of conditional colocalization relationships and hierarchies in three-color microscopy images

Colocalization analysis of multicolor microscopy images is a cornerstone approach in cell biology. It provides information on the localization of molecules within various subcellular compartments and allows the interrogation of molecular interactions in their cellular context. However, almost all colocalization analyses are designed for two-color images. This limits their applicability and the type of information that they reveal, leading to underutilization of multicolor microscopy images. Here we describe an approach, termed “conditional colocalization analysis,” for analyzing the colocalization relationships between three molecular entities in three-color microscopy images. Going beyond the question of whether colocalization is present or not, it addresses the question of whether the colocalization between two entities is influenced, positively or negatively, by their colocalization with a third entity. We showcase two applications of conditional colocalization analysis, one addressing the question of compartmentalization of molecular interactions, and one investigating the hierarchy of molecular interactions in a multimolecular complex. The software for conditional colocalization analysis is available at . ### Competing Interest Statement The authors have declared no competing interest.