Schematic illustration of the sequential steps of micromanipulation and microinjection required for IV formation. (a) Placement of the micropipette tip into the bulk sample solution containing surface-attached GUV-MLVs. (b) Positioning of the micropipette tip near the GUV surface. (c) A positive pressure (+ p) is applied to the micropipette while putting the pipette into direct contact with the GUV membrane, which results in a flow of calcium ion solution and inflation of the first IV. (d) Applying a continuous injection of calcium ion solution results in synthesis of a multitude of IVs, which are filling the interior of the GUV. The illustrations were drawn in Adobe Illustrator CS6.