Fluorescence microscopy images demonstrating the formation of IVs and their connectivity. (a) By pulling the micropipette (0.3 µm in diameter) away from the GUV, the IVs attached to the micropipette tip were pulled out of the GUV interior and displayed the vesicle connectivity of “beads on a string”. (b) A 5 s injection of calcium ion solution using a micropipette with an inner tip diameter of 2 µm resulted in the formation of polydisperse IVs. The images were prepared using the NIH ImageJ software, VirtualDub 1.10.4, and Adobe Illustrator CS6. The scale bars represent 5 µm.