The simultaneous observation of 3D chromatin arrangement and transcription in single cells is critical to understand how DNA is organized inside cells and how this organization influences or affects other processes, such as transcriptional regulation. We have recently introduced an innovative technology known as HiM , which enables the sequential tagging, three-dimensional (3D) visualization, and precise localization of multiple genomic DNA regions alongside RNA expression within individual cells. In this document, we present a comprehensive guide outlining the creation of probes, as well as sample preparation and labeling. Finally, we provide a step-by-step guide to conduct a complete HiM acquisition using our open-source software package, Qudi-HiM, which controls the robotic microscope handling the entire acquisition procedure.