Microscopy imaging of chromatin offers valuable insights into its spatial organization in the nucleus, a novel epigenetic dimension influencing the genome’s functions. Particularly, visualization at the nanoscale in single cells is uniquely complementary to molecular profiling methods averaging chromatin configuration and composition over thousands of cells. How are chromatin and chromosomal domains distributed in relation to gene expression? How variable are these configurations? How do chromatin domains evolve in structure, composition, and distribution during cellular differentiation or cellular responses to environmental stimuli? Super-resolution microscopy techniques, like stimulated emission depletion (STED), are key in answering such questions. However, such imaging techniques are not often used in the field of plant cell biology compared to mammalian counterparts, which has greatly advanced our understanding of the 3D principles in genome organization. In an effort to bridge this gap, we provide a clear guide for isolating, embedding, immunostaining, and STED imaging intact leaf nuclei from Arabidopsis thaliana in 3D.