Fluorescence microscopy images illustrating calcium-ion assisted formation of IVs. (a) Formation of the IVs upon a 25 s injection of calcium ion solution, supplemented with 10 µM Alexa-488 for visualization when placing the micropipette tip in contact with the GUV surface. (b) IVs co-loaded with streptavidin − 488 (10 µM). Injection time is 12 s. (c) IVs co-loaded with a glutamate solution (10 µM with Alexa 488 NHS ester co-solute). Injection time is 3 s. The images were brightness/contrast-enhanced for improved visualization. The white lines outline the position of the micropipette tip in the experiments. The images were prepared using the NIH ImageJ software, VirtualDub 1.10.4, and Adobe Illustrator CS6. The scale bars represent 5 µm.