Approaches to imaging exocytosis with FM dyes. (A) FM dye release kinetics depend on dye hydrophobicity. FM dyes with lower hydrophobicity (FM1-84 > FM1-43 > FM2-10) departition faster from the membrane on exocytosis. (B) Partial FM dye release in kiss-and-run. Exocytosis through a transient kiss-and-run fusion pore releases only a fraction of the dye, leaving partially filled vesicles to release the remaining dye in successive rounds of fusion. (C) Probing partial FM dye release by quenching. If partial dye release occurs as a consequence of kiss-and-run, vesicles retain some dye after exocytosis (left). If the hydrophilic quencher bromophenol blue (BPB) is included in the bathing solution (right), it quenches any dye remaining in the vesicles (including any membrane-bound dye molecules), leading to a difference in fluorescence (when compared with the situation without BPB). For full collapse with complete loss of dye, application of BPB would not lead to a difference in fluorescence. (D) Complete dye retention. As shown by experiments combining electrophysiology and imaging, a fraction of vesicles might release a neurotransmitter, without the ability to lose or to take up FM dye. These results were interpreted in favor of exocytosis through a restrictive fusion pore. (E) Direct imaging of exocytosing vesicles by total internal reflection microscopy revealed little FM dye release into the fluid, but rather a lateral diffusion in the plasma membrane (with the dye later departitioning from the membrane at a distance from the point of exocytosis), indicative of full collapse fusion.