Fidorra M, Garcia A, Ipsen J, Härtel S & L Bagatolli.
Biochimica et Biophysica Acta (BBA) – Biomembranes, 1788(10):2142-9. [Epub 2009 Aug 21]
We report a novel analytical procedure to measure the surface areas of coexisting lipid domains in giant unilamellar vesicles (GUVs) based on image processing of 3D fluorescence microscopy data. The procedure involves the segmentation of lipid domains from fluorescent image stacks and reconstruction of 3D domain morphology using active surface models. This method permits the reconstruction of the spherical surface of GUVs and determination of the area fractions of coexisting lipid domains at the level of single vesicles. Obtaining area fractions enables the scrutiny of the lever rule along lipid phase diagram’s tie lines and to test whether or not the coexistence of lipid domains in GUVs correspond to equilibrium thermodynamic phases. The analysis was applied to DLPC/DPPC GUVs displaying coexistence of lipid domains. Our results confirm the lever rule, demonstrating that the observed membrane domains correspond to equilibrium thermodynamic phases (i.e., solid ordered and liquid disordered phases). In addition, the fact that the lever rule is validated from 11 to 14 randomly selected GUVs per molar fraction indicates homogeneity in the lipid composition among the explored GUV populations. In conclusion, our study shows that GUVs are reliable model systems to perform equilibrium thermodynamic studies of membranes.
Acknowledgments: The authors thank N. Contreras from Area Kreativa for support with the figures and Dr. J. Cowan for his help revising English style and grammar. Research in the laboratory of L.A.B. is funded by grants from Forskningsrådet for Natur og Univers (FNU, Denmark), Forskningsrådet for Sundhed og Sygdom (FSS), and the Danish National Research Foundation (which supports MEMPHYS-Center for Biomembrane Physics). Research in SCIAN-Lab (S.H.) is funded by FONDECYT (1060890 and 1090246) and FONDEF (D07I1019) both CONICYT (Chile), and the Millennium Scientific Initiative (ICMP04-068-F). The interdisciplinary collaboration between L.A.B.’s and S.H.’s laboratories was supported by FONDECYT (7060265 and 7080031), CONICYT Chile and MEMPHYS, Center for Biomembrane Physics, University of Southern Denmark, Odense, Denmark.