Giant Unilamellar Vesicle Electroformation: What to Use, What to Avoid, and How to Quantify the Results

Abstract

Since its inception more than thirty years ago, electroformation has become the most commonly used method for growing giant unilamellar vesicles (GUVs). Although the method seems quite straightforward at first, researchers must consider the interplay of a large number of parameters, different lipid compositions, and internal solutions in order to avoid artifactual results or reproducibility problems. These issues motivated us to write a short review of the most recent methodological developments and possible pitfalls. Additionally, since traditional manual analysis can lead to biased results, we have included a discussion on methods for automatic analysis of GUVs. Finally, we discuss possible improvements in the preparation of GUVs containing high cholesterol contents in order to avoid the formation of artifactual cholesterol crystals. We intend this review to be a reference for those trying to decide what parameters to use as well as an overview providing insight into problems not yet addressed or solved.

Keywords: GUVs; cholesterol; electrical parameters; electroformation; electroformation duration; internal solution; lipid composition; lipid deposition; quantitative analysis; temperature.

Figure 1

(a) Deposition of lipid droplets onto the electrode surface. (b) Evaporation of organic solvent under vacuum. (c) Construction of the electroformation chamber. (d) Electroformation chamber filled with an internal solution and connected to an alternating current function generator. (e) An image of fluorescently labeled giant unilamellar vesicles (GUVs) obtained using fluorescence microscopy. The scale bar denotes 50 µm.

Figure 2

(a) Asymmetrical electrodes layout. The top indium tin oxide (ITO) electrode has a smaller surface than the bottom one, so it has to be surrounded by glass coverslips in order to close off the chamber. (b) Electroformation chamber with a coplanar interdigitated ITO electrode.

Figure 3

(a) Droplet deposition. (b) Droplet deposition with smearing afterward to better spread the lipid film. (c) Deposition of lipids by pressing a patterned silicon stamp on the electrode surface. (d) Spin-coating of lipid solution by fast rotation of the electrode immediately after the deposition.

Figure 4

Deposition of lipids utilizing the coffee ring effect. After drying, most of the material is carried away toward the periphery and a ring-like stain is formed. This is known as the coffee-ring effect. By depositing progressively larger droplets, the ring from the previous droplets gets smeared and flattened, thus leaving behind an area of uniform lipid film thickness.