Mitochondria are energy-producing organelles that are essential for eukaryotic cell function. Central to their role is mitochondrial DNA (mtDNA), a small, circular genome present in multiple copies per cell that encodes proteins and RNAs necessary for oxidative phosphorylation. In multicellular organisms, mtDNA is continuously synthesized in both proliferating and post-mitotic cells and varies in copy number between tissue types. MtDNA distribution and synthesis are linked to mitochondrial fission and fusion dynamics in budding yeast and in cancer cell models, however, whether these processes are linked in post-mitotic somatic cells remains unclear. Here, we present molecular imaging tools for the study of mtDNA maintenance within live Caenorhabditis elegans. We have coupled Mitotracker and SYBR Gold stains with Airyscan superresolution microscopy to establish "ground truth" tissue-specific patterns of mtDNA distribution. Then, using transgenic strains in which the mtDNA binding protein
hmg-5/TFAM has been fluorescently tagged with wrmScarlet at the endogenous locus we identify tissue-specific regimes of transcription factor distribution within mitochondria and relate them to fusion-fission dynamics. We find that some transgenic strategies, such as the overexpression of mtDNA binding factors via extrachromosomal array, perturb normal mitochondrial DNA levels; we present a set of best practices in strain construction. These validated imaging tools lay the foundation to test whether differences in mitochondrial structure between tissues are predictive of mtDNA abundance and/or distribution. This work has the potential to inform tissue-specific pathologies of mtDNA disease in humans.