We have developed a strategy to determine lengths and orientations of tie lines in the coexistence region of liquid-ordered and liquid-disordered phases of cholesterol containing ternary lipid mixtures. The method combines confocal-fluorescence-microscopy image stacks of giant unilamellar vesicles (GUVs), a dedicated 3D-image analysis, and a quantitative analysis based in equilibrium thermodynamic considerations. This approach was tested in GUVs composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-palmitoyl-sn-glycero-3- phosphocholine/cholesterol. In general, our results show a reasonable agreement with previously reported data obtained by other methods. For example, our computed tie lines were found to be nonhorizontal, indicating a difference in cholesterol content in the coexisting phases. This new, to our knowledge, analytical strategy offers a way to further exploit fluorescence-microscopy experiments in GUVs, particularly retrieving quantitative data for the construction of three lipid-component-phase diagrams containing cholesterol. © 2012 Biophysical Society.
Husen, P., Arriaga, L. R., Monroy, F., Ipsen, J. H., & Bagatolli, L. A. (2012). Morphometric image analysis of giant vesicles: A new tool for quantitative thermodynamics studies of phase separation in lipid membranes. Biophysical Journal, 103(11), 2304–2310. https://doi.org/10.1016/j.bpj.2012.10.031