Fig. 3: Investigation of endocytosis and exocytosis with etSTED.
This shows an etSTED experiment in HeLa cells expressing Dynamin1-EGFP or CD63-pHluorin and using the dynamin_rise or rapid_signal_spikes analysis pipeline. a, Schematic diagram of the dynamin-mediated endocytosis process of interest, with the increase in intensity due to accumulation of dynamin1 at the endocytic site. b, Schematic diagram of the experiment timeline of one widefield frame. Ith, intensity threshold; tth, time threshold. c, Example of a triggering widefield frame in an etSTED experiment with HeLa cells expressing Dynamin1-EGFP. d, Two representative events shown in widefield zooms with three frames leading up to the event (right frame). e, Triggered 5.9 Hz 3D STED timelapses of plasma membrane dynamics where cholesterol (cholesterol-Abberior STAR RED) is labeled. n = 53 events, n = 22 cells. f, Schematic diagram of an exocytosis event of interest, with the increase in fluorescence intensity due to unquenching of pHluorin upon pH neutralization of late endosomes (LEs) and multivesicular bodies (MVBs). g, Schematic diagram of the experiment timeline of one widefield frame. h, Example of a triggering widefield image in etSTED experiment with HeLa cells expressing CD63-pHluorin. i, Two representative events shown in widefield zooms (cyan, left) and analysis ratiometric image zooms (gray, right) with the last two frames before the event. j, Triggered 11 Hz 3D STED timelapses of plasma membrane dynamics and accumulation where cholesterol (cholesterol-Abberior STAR RED) is labeled. n = 232 events, N = 29 cells. Asterisks (*) indicate deconvolved frames, and apply to all following frames in the same timelapse. Scale bars, 10 µm (c,h), 2 µm (d,i), 250 nm (e,j).
