Cross-comparison of strains used for mitochondrial imaging in Caenorhabditis elegans during aging

Measurements of mitochondrial morphology are a powerful proxy for assessing mitochondrial health, particularly during aging when organelle dynamics are disrupted. Caenorhabditis elegans provides an ideal system for in vivo mitochondrial imaging, but widely used high-copy transgenic strains can induce artifacts that confound interpretation because of their impact on cellular and organismal health and physiology. Here, we present and validate a suite of C. elegans strains expressing single-copy, matrix-localized GFP in the muscle, intestine, and hypodermis using the MosSCI technology. These strains enable robust, tissue-specific visualization of mitochondrial morphology without the caveats associated with multi-copy reporters. We benchmark their performance against existing models and demonstrate that our mitochondrial reporters are similarly capable of assessing age-associated mitochondrial morphology, while avoiding defects in cellular and physiological health associated with the multi-copy reporters. Furthermore, we assess how aging methods, bacterial diets, and inhibition of fusion and fission machinery impact mitochondrial morphology during aging. Our findings provide a standardized and physiologically relevant platform for studying mitochondrial dynamics during aging in C. elegans.