LSV swelling, and pore diameter quantification and normalization. (A) 3D segmentation of an LSV before and after fusion showing vesicle swelling. Representative images (confocal intensity-projection) and 3D segmentation of an LSV before (left) and after (right) fusion. The vesicle expands by 27% in 2D area and 47% in calculated 3D volume, implying that the observed increase in 2D is a result of LSV swelling and not LSV movement in or out of the imaging plane. Glue-GFP (green) and LifeAct-Ruby (magenta; UAS-based expression driven by c135-GAL4). Time mm:ss; relative to fusion. Scale bar is 1 µm. (B) The step-by-step alignment and normalization plots used to produce the pore dynamics plot in Fig. 1 D. Top left: Pore diameter measurements (µm) are plotted over time (seconds; from fusion). Top right: The curves are aligned over time (seconds; relative to maximal pore diameter). Bottom left: The curves are normalized (% of max pore diameter). Bottom right: The mean relative curve is presented with SEM (gray). n (pores) = 12.