A Drosophila S2 cell with lysosomes labeled with LysoTracker Deep Red dye. Four maximum intensity projection images at different time points are shown. The triangles highlight two noticeable events. Images were acquired at 15 volumes per second (volume size 35 µm × 35 µm × 7 µm, 34 slices per volume and camera running at 500 frames per second). At this imaging speed, we could reliably perform 3D tracking of lysosome movement dynamics. See also Supplementary Movies 8–10. Three experiments were repeated independently, with similar results.