Supplementary Figure 14: Diffractive or motion blur obscures vesicle motion at the plasma membrane.
From: Single-shot super-resolution total internal reflection fluorescence microscopy
a) Still image from 100-frame instant TIRF-SIM series, acquired at 37 °C. Plasma membrane and vesicles are stained with CellTracker CM-DiI in a live U2OS cell. The 'hot' colormap in ImageJ was used for image display due to the large dynamic intensity range in the sample. b) Higher magnification view of white rectangular region in a) comparing instant TIRF-SIM (top), 4 successive frames binned in time (middle) to simulate motion blur, and the same region blurred to simulate diffraction-limited acquisition (bottom). Blue arrows highlight vesicle pair resolved in instant TIRF-SIM but not in diffraction-limited image. Green arrow highlights vesicle pair resolved in instant TIRF-SIM but blurred due to motion or diffractive blur. White arrow highlights artifact - an apparent vesicle - due to motion blur. Scale bars: 5 μm in a), 1 μm in b). See also Supplementary Video 15, 16. Experiments were repeated 3 times with similar results; representative data are shown.
