Confirmation that detected exocytic events are bona fide. Inverted contrast images of E15.5 cortical neuron cultured for 48 h in vitro expressing VAMP2-pHluorin were acquired every 100 ms by TIRF microscopy with 110 nm penetration depth to visualize VAMP2-mediated exocytosis, which is marked by a rapid increase, diffusion, and decrease in fluorescence intensity. Left: Treatment with NH4Cl alkalinizes intracellular compartments, reversing the quenching of VAMP2-pHluorin and making VAMP2-pHluorin–containing vesicles fluorescent before fusion pore opening. Right: TeNT treatment, which cleaves the cytoplasmic face of VAMP2, blocks SNARE complex formation and VAMP2-mediated exocytosis.
