In order to maintain cell homeostasis, lysosomes must be trafficked throughout the cell and associated with different compartments for the delivery and breakdown of macromolecules. This process is especially important in polar cells, such as neurons, that require the trafficking of lysosomes to distal locations to perform their functions. Here, we employed LysoTracker DND-99, a live-cell fluorometric dye that stains acidic organelles, to label lysosomes prior to time-lapse imaging on a Nikon A1 confocal microscope and subsequent assessment of total distance travelled using the TrackMate ImageJ plugin.