Figure 1. Principle of combined dual-beam optical trapping and fluorescence microscopy to detect membrane fusion.

(a) Schematic (not to scale) of two polystyrene beads (grey) coated with a lipid bilayer (blue) trapped in focused laser beams (orange). Protein fragments comprising Doc2b (green) were added to the aqueous compartment and could bind phospholipids in the presence of Ca²⁺ (yellow). Upon bead separation (with constant velocity v), the force (F) on the left bead was measured. Unless otherwise indicated, experiments were performed at 0.74 μM Doc2b with membranes composed of 80% PC and 20% PS and in 250 μM Ca²⁺. (b) High-force events were accompanied by the formation of a micrometre-long membrane stalk, revealed by simultaneous fluorescence imaging in the presence of the fluorescent phospholipid NBD-PE. (c) Using unlabelled membranes, a fluorescently tagged Doc2b fragment, 0.39 μM C2AB-EGFP, bound efficiently to the entire bilayer including the membrane stalk. Scale bars, 1 μm.