Combined AFM and Two‐Focus SFCS Study of Raft‐Exhibiting Model Membranes

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Abstract
Dioleoylphosphatidylcholine/sphingomyelin/cholesterol (DOPC/SM/cholesterol) model membranes exhibit liquid–liquid phase separation and therefore provide a physical model for the putative liquid-ordered domains present in cells. Here we present a combination of atomic force microscopy (AFM) imaging, force measurements, confocal fluorescence imaging and two-focus scanning fluorescence correlation spectroscopy (two-focus SFCS) to obtain structural and dynamical information about this model membrane system. Partition coefficients and diffusion coefficients in the different phases were measured with two-focus SFCS for numerous fluorescent lipid analogues and proteins, while being directly related to the lateral organization of the membrane and its mechanical properties probed by AFM. Moreover we show how the combination of these different approaches is effective in reducing artifacts resulting from the use of a single technique.