Clonal Imaging of Mitochondria in the Dissected Fly Wing

Mitochondria are essential for long-term neuronal function and survival. They are maintained in neurons, including long axonal stretches, through dynamic processes such as fission, fusion, biogenesis, and mitophagy. Here, we describe a protocol for the in-depth morphological analysis of individual mitochondria in axons in vivo. Most mitochondrial analysis of axons is currently performed in vitro with neurons in a developmental state. Therefore, an understanding of the axonal mitochondrial network during aging in fully differentiated neurons and the long-term consequence of gene knockout is often not developed. By using a clonal system paired with fluorescent genetically encoded markers in the Drosophila wing, we can visualize individual neurons (out of the whole bundle), including their long axons and the mitochondria that they contain, using confocal imaging. The clonal system also allows visualization of neurons with genetic perturbations that would otherwise be lethal if present in the whole organism, allowing investigators to bypass lethality. This protocol can further be adapted to measure the physiological and biochemical state of the mitochondria. Mitochondrial morphology and health in axons are tightly linked to aging, axon injury, and neurodegeneration; therefore, this method can be used to investigate mitochondrial dysfunction associated with novel genes or those linked to neurodegenerative disease and axonopathy.