(A) Chemical structure of FM1-43. The molecule is composed of a positively charged head group, which prevents the dye from permeating membranes, and a lipophilic tail, which ensures dye partitioning into membranes. These components are connected by a double bond bridge, the number of double bonds determining the spectral characteristics. (B) FM dye staining and destaining. (1) The dye is added to the solution bathing the preparation. It does not have access to the intracellular space. (2) The preparation is stimulated. Vesicles exocytose and come in contact with the dye. (3) Vesicles endocytose and take up dye. (4) The dye is washed from the extracellular solution; it departitions from the plasma membrane but remains trapped in internalized vesicles (staining). (5) The stained preparation is stimulated in the absence of the dye. Vesicles fuse with the membrane and release dye (destaining). (6) Destained vesicles are internalized; a new experiment of staining and destaining can be performed. (C) Frog motor nerve terminal shown during staining. Images were acquired and processed identically, and are, therefore, directly comparable. (Top) The preparation had been bathed in FM dye-containing solution for 1 min. The surface membrane of the nerve terminal is faintly stained. (Middle) After a 1-min tetanus (30 Hz), followed by 15 min of rest in dye-containing solution, the terminal is brightly stained, but background fluorescence is also high. (Bottom) After dye washout, the background staining is greatly reduced, and clusters of vesicles can be observed as distinct fluorescent spots. (D) FM-labeled nerve terminals in different preparations: (Top) Drosophila larval NMJ. Scale bar, 3 µm. (Middle) Lizard (Anolis) NMJ. Scale bar, 2 µm. (Bottom) Cultured hippocampal neurons (fluorescent spots represent single presynaptic boutons). Scale bar, 5 µm. (Photograph courtesy of Dr. V. Murthy.) (E) Nerve terminal destaining. A stained preparation was stimulated at 2 Hz for ∼40 min in the presence of 10-µM curare to prevent muscle movement. The images were taken approximately every 5 min. The nerve terminal loses most of its initial fluorescence. Scale bar, 5 µm. (E, Reprinted, with permission, from Betz and Henkel 1994, © The Rockefeller University Press.)