Analysis of Giant Unilamellar Vesicles: from droplet to vesicle

Abstract

In bottom-up reconstitution, a minimal cell is crafted from only essential components. Lipid vesicles are often used in this approach to mimic a cell’s membrane due to their structural resemblance. Most prominently giant unilamellar vesicles (GUVs), which are lipid vesicles with a single lipid bilayer and a size in the range of 1-100 µm, are used in this field. Although there are several methods for producing these GUVs, the cross droplet interface crossing encapsulation (cDICE) is the most robust method, even though it being invented only several years ago. To further improve the reliability of this method and allow for easier troubleshooting, two tools were created. The first tool, an imaging setup that should image emulsion droplets of the cDICE setup at high speed, was able to use both epi- and trans-illumination for the imaging of static emulsion droplets in a model system. The used exposure times are already on the µs-timescale, which is probably sufficient for application on the cDICE setup. Diffculties in alignment of the imaging setup on the cDICE-setup, due to the lack of fine translation, lead to the fact that the setup was not yet successfully employed on the cDICE-setup. The second tool, which performs an analysis on the positions and sizes of GUVs from confocal microscopy data, has proven to track the vesicles generally well. In higher density samples some problems arise, such as the false detection of aggregates, but these can easily be solved by manual inspection from the user. In the end the data analysis tool is ready to use and allows for easier analysis of the parameter space of the cDICE, but the second tool still needs some adaptions, for example an implementation of trans-illumination in the cDICE and a manner for finer adjustment of the alignment.