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In mouse chromaffin cells expressing green synaptopHluorin in the granules, cell membranes were stained from the outside with red lipophilic dyes that rapidly redistribute by flip-flop between both leaflets. Fusion of secretory granules was monitored by evanescent wave microscopy. Exocytosis was triggered by superfusion with high Kþ solution, and double images were taken at 491 and 560 nm excitation, respectively. Fluorescence signals of the membrane probes recorded in the red channel were spatially and temporally aligned with respect to fusion events in the green channel to yield average movies with high signal-to-noise ratio. We found the membrane fluorescent signals to be slightly increased in diffraction-limited spots at locations of docked granules for up to a second prior to fusion. The fluorescent signals, however, rapidly decreased to background levels upon fusion of the granules at the respective sites, with the fluorescence dissipating from the center to the periphery. Our results are best explained by mixing of lipids prior to fusion in a hemifused state.

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