Domain Structure in Model Membrane Bilayers Investigated by Simultaneous Atomic Force Microscopy and Fluorescence Imaging

Abstract

Simultaneous atomic force microscope (AFM) and submicron confocal fluorescence imaging of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid domain structures in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) is presented. Lipids labeled by fluorescent probes either at the headgroups or tailgroups enable domain contrast in fluorescence imaging on the basis of partitioning between the gel (DPPC) and disordered liquid (DOPC) phases. However, correlation with AFM topographic information reveals that they do not always faithfully report exact gel domain size or shape. Furthermore, we find that the fluorescence contrast decreases significantly with domain size, such that small domains observed with AFM are not observed in fluorescence images despite adequate optical resolution. We attribute these effects in part to broadened partitioning of the probe lipids across the domain boundaries. Binding of fluorescent Alexa 488-conjugated cholera toxin B subunits to GM1 gangliosides in DPPC domains correlates we...