Supplementary Figure 13: Effect of temporal undersampling and spatial blurring on automated tracking of EGFP–Rab11 particles.

a) First frame of image series corresponding to Fig. 3. b) Effect of sampling at 100 Hz (i.e. native recording rate, top), digitally downsampling to 20 Hz (middle) and digitally downsampling to 10 Hz (bottom). Temporal evolution of each track is indicated with color bar at right, red and orange arrows highlight examples of automatically detected tracks (top) that are artificially shortened (middle) and eventually lost (bottom) due to undersampling. Particle tracking was performed on a different cell imaged in instant TIRF-SIM (c, first frame in 300 frame series) and subsequently pinholes and emission microlenses were removed so that the same sample was imaged (for another 300 frames) in diffraction-limited TIRFM (d). 1477 trajectories were detected in instant TIRF-SIM whereas only 1031 were detected in TIRFM. For clarity, only those tracks that were automatically detected and that cover a displacement > 0.3 μm, are shown in the figure (121 in c, 76 in d). We observed a similar increase in detected particle trajectories in an additional cell (1317 total trajectories in instant TIRF-SIM vs. 1107 in TIRF). Scale bars: 5 μm in a, c and d, 1 μm in b. See also Supplementary Videos 12–13. Experiments were repeated at least 3 times with similar results; representative data are shown.