A high-resolution visualization of mitochondrial genome spatial organization in S. cerevisiae. Using widefield OMX microscopy, we captured 41 Z-planes to resolve the relationship between the mitochondrial network (mt-dsRed, red) and individual mtDNA nucleoids. To track the genome, LacO repeats were integrated upstream of the COX2 gene and labeled via a mitochondrially targeted LacI-3xGFP fusion. Following precise segmentation in Imaris, the volumetric data was rendered in Blender to reveal the 3D architecture and distribution of the mitochondrial DNA within the organelle.