Microinjection into giant vesicles and light microscopy investigation of enzyme-mediated vesicle transformations.

'Giant vesicles' have diameters of several micrometers and can be observed by light microscopy. Their size may allow manipulation of individual vesicles and direct observation of the progress of a chemical reaction in real time. We set out to test this possibility using enzymatic hydrolysis of vesicle components as a model system. We describe a novel micromanipulation technique that allows us to microinject femtoliter amounts of a reagent solution adjacent to or into giant vesicles with diameters ranging from 10 to 60 microm. The vesicle transformations can be monitored directly in real time by light microscopy and recorded by video analysis. Snake venom phospholipase A2 was added to vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine, and the enzymatic hydrolysis of components of the lipid bilayer was observed over time. A specific effect on the targeted giant vesicle was seen and video recorded, while the neighbouring vesicles remained unaffected. Addition of the enzyme to the outside of a vesicle caused it to burst, whereas injection of the enzyme inside a vesicle resulted in a slow and constant decrease in its size, until it eventually disappeared from the resolution power of the light microscope. These results show that it is possible to micromanipulate an individual vesicle, and to follow visually the progress of an enzymatic reaction occurring on the vesicle bilayer over time.