The long cellular architecture of neurons requires regulation in part through transport and anchoring events to distribute intracellular organelles. During development, cellular and sub-cellular events such as organelle additions and their recruitment at specific sites on the growing axons occur over different time scales and often show inter-animal variability thus making it difficult to identify specific phenomena in population averages. To measure the variability in sub-cellular events such as organelle positions, we developed a microfluidic device to feed and immobilize for high-resolution imaging over several days. The microfluidic device enabled long-term imaging of individual animals and allowed us to investigate organelle density using mitochondria as a testbed in a growing neuronal process Sub-cellular imaging of an individual neuron in multiple animals, over 36 hours in our microfluidic device, shows the addition of new mitochondria along the neuronal process and an increase in the accumulation of synaptic vesicles at synapses. Long-term imaging of individual touch receptor neurons shows that the addition of new mitochondria takes place along the entire neuronal process length at a rate of ∼0.6 mitochondria per hour. The threshold for the addition of a new mitochondrion occurs when the average separation between the two pre-existing mitochondria exceeds 24 µm. Our assay provides a new opportunity to move beyond simple observations obtained from assays to allow the discovery of genes that regulate positioning of mitochondria in neurons.Axonal transport of mitochondria is required for the normal function and health of a developing animal with continuously growing axonal processes. Existing technologies are unable to monitor the addition of a new mitochondrion in a growing axon , as it requires continuous or intermittent tracking of the same individual neuron over several hours to days. We have developed a microfluidic device that enables long-term high-resolution imaging of individual in an anesthetic-free setting. Using this device, we observe that the addition of a new mitochondrion can occur anywhere along the entire neuronal process, likely mediated by actively transported mitochondria, and at docking sites that occur with high probability when the separation between adjacent mitochondria crosses 24 µm threshold.