Sub-ten-nanometer heterogeneity of solid supported lipid membranes determined by solution atomic force microscopy

• Nanoscopic structures less than 20 nm in lipid membranes are difficult to observe.
• Sub-ten-nanometer heterogeneity in planar bilayers is obtained using solution AFM.
• Nanoscopic-to-macroscopic size transition is obtained by altering lipid composition.
• Heterogeneous bilayers in nanoscopic regime contains fluctuation-like structures.

Visually detecting nanoscopic structures in lipid membranes is important for elucidating lipid–lipid interactions, which are suggested to play a role in mediating membrane rafts. We use solution atomic force microscopy (AFM) to study lateral and normal organization in multicomponent lipid membranes supported by mica substrate. Nanoscopic heterogeneity is observed in a three-component system composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/brain-sphingomyelin (bSM)/cholesterol (Chol). We find sub-ten-nanometer correlation lengths that are used to describe membrane lateral organization. In addition, we find that the correlation length is independent on cholesterol concentration, while the height fluctuation (variation) is not. To explore the mechanism that controls the size of membrane heterogeneity, we extend our study to a four-component system composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/POPC/bSM/Chol. By systematically adjusting the relative amount of DOPC and POPC, we obtain macroscopic-to-nanoscopic size transition of membrane heterogeneity. In contrast to the results from vesicle based fluorescence microscopy, we find that the structural transition is continuous both in the lateral and normal directions. We compare our nanoscopic structures to two theoretical models, and find that both the critical fluctuations and the nanodomain models are not sufficient to account for our solution AFM data. Finally, we propose a nanoheterogeneity model that could serve as the organization principle of the observed nanoscopic structures in multicomponent lipid membranes.

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