Validation of VAMP2-pHluorin as a new probe to visualize GSVs. 3T3-L1 adipocytes were electroporated with VAMP2 and/or GLUT4 and imaged by SDCM and TIRFM. (A) VAMP2-GFP and GLUT4-DsRed colocalize extensively (arrows). (B–D) Insulin-stimulated VAMP2-pHluorin translocation. 3T3-L1 adipocytes electroporated with VAMP2-pHluorin were imaged by 3D time-lapse SDCM. (B) Maximum z-projection images were taken before (0 min) and after (5–30 min) 100 nM insulin addition. (C) Fluorescence intensity line profiles (yellow line in B) were plotted for images before (green) and 30 min after (red) insulin. (D) The ratio of edge-to-middle intensities from line profiles of 10 cells was plotted (error bars indicate mean ± SEM; ***, p < 0.001). (E and F) VAMP2-pHluorin colocalizes with GLUT4-DsRed and allows detection of full fusion (E) and kiss-and-run (F) fusion events using dual-color TIRFM. Bars: (A and B) 10 µm; (E and F) 1 µm.