Chromosomes from bacteria to humans are organized at the sub-megabase scale into genomic domains. We will present examples of how super-resolution microscopies can be combined with new DNA labeling technologies and genome-wide chromosome capture methods to shed light into the mechanisms of formation and regulation of genome architecture. In particular, we will present our recent advances in multifocus plane microscopy for 3D single-molecule localization microcopy, two-color STORM to study genome structure, and high-thoughput 3D-SIM-based image analysis to investigate chromosome segregation.