The spatial organization of chromatin has become recognized as a major factor in the regulation of gene expression. While chromosome conformation capture techniques, such as high-throughput chromosome conformation capture (Hi-C), have advanced our understanding of the role of 3D organization of chromatin on chromosomal function, Hi-C data are typically ensemble in nature and relies on intense computational methods to derive genomic structural information. The field is beginning to shift in recognizing the potential value of imaging methods to study spatial organization of chromatin. Imaging allows acquisition and analysis at the individual cell level while providing direct visualization and analysis of 3D chromatin organization. This feature will highlight how single-molecule localization microscopy (SMLM) using the Bruker Vutara SMLM system in conjunction with sequential labeling via microfluidics is revolutionizing our understanding of nuclear spatial organization and its role in regulation of gene function.