Abstract
Super-resolution microscopy coupled with multiplexing techniques can resolve specific spatial arrangements of different components within molecular complexes. However, reliable quantification and analysis of such specific organization is extremely problematic because it is frequently obstructed by random co-localization incidents between crowded molecular species and the intrinsic heterogeneity of molecular complexes. To address this, we present a Triple-Pair-Correlation (TPC) analysis approach for unbiased interpretation of the spatial organization of molecular assemblies in crowded three-color super-resolution (SR) images. We validate this approach using simulated data, as well as SR images of DNA replication foci in human cells. This demonstrates the applicability of TPC in deciphering the specific spatial organization of molecular complexes hidden in dense multi-color super-resolution images.