Figure 1. Schematic workflow of the method.

Cells were plated 48-h before the experiment onto dishes suitable for confocal microscopy. 24 h before the experiment, cells were transiently transfected with specific proteins to label the cytoplasmic organelle of interest (e.g. early and late endosomes). In the case of lysosomes, 20 min before the experiment, cell were incubated with Lysotrackercontaining medium. A typical confocal microscopy experiment consists in the acquisition of a stack of images (time lapse) of a cytoplasmic portion of a labelled cell, with experimental parameters (temporal resolution, pixel size, etc.) set to the optimal values (see Technical Validation section) for further analysis (e.g. localization and tracking, iMSD, image analysis).