The Ternary Lipid Phase Diagram by AFM

Abstract

Understanding the physical properties and lateral structure of lipid bilayers is the subject of continuing interest, not least due to its emerging role in modifying the function of membrane proteins. A wide variety of experimental techniques have been used to determine phase diagrams of relevant ternary lipid mixtures that exhibit properties of the cell membrane. However, there are still many open questions and ambiguities. We use a variety of modes of atomic force microscopy (AFM) to study domain formation in ternary lipid mixtures. AFM has the unique advantages of having high lateral resolution, of being label free, and of being able to simultaneously measure membrane mechanical properties. A commonly stated disadvantage is that it is necessarily limited to supported bilayers, and that the proximity of the substrate will alter the phase behaviour of membrane. Various strategies have been employed over the years to obviate this effect, such as forming membrane stacks, tethering, and polymer cushioning. In this work we show that a model cell membrane on a mica substrate exhibits the same phase transitions as that observed in GUV's. We use temperature control and Fast-scan AFM across the whole phase diagram to study phase transitions, from binary systems to ternary mixtures exhibiting 2-phase (Ld-Lo, Ld-gel and Lo-gel) and 3-phase regions, and hence construct a complete ternary phase diagram. Dynamics previously undetected on support bilayers have been observed, such as Ostwald ripening and domain coalescence, domain boundary fluctuations and critical fluctuations. Nucleation (including tree-ring growth) and spinodal decomposition mechanisms are characterised. Our work demonstrates that AFM is not only a valid approach to studying phase separation in bilayers but that it is able to complement existing techniques and theories and provide new insights by directly visualizing lateral lipid bilayer structure.