Abstract: The structure and long-time dynamics of suspended lipid bilayers made of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE) in the gel phase was interrogated using electrical measurements co-recorded with second harmonic generation (SHG) micrographs. SHG microscopy was shown to be an efficient method for distinguishing between the regions of bilayer and annulus as well as probing structural and dynamical variations within a single bilayer. Micromachined silicon chips and two types of plastic partitions, delrin and polyethylene, were investigated as substrate materials. Silicon chips yielded the most stable bilayers, lasting for 1 or 2 days. The membrane characteristics, including the amount of incorporated solvent within the bilayer, the dynamics of bilayer formation, and the stability of the bilayer, were found to be strongly substrate-dependent. In a second set of experiments, SHG-active membrane dyes, di-8-ANEPPS and di-4-ANEPPS, were used to interrogate the domain structure of the same suspended bilayers using scanning two-photon fluorescence (2PF) and SHG microscopy, again correlated with capacitance measurements. Using these dyes, an enhancement in the SHG signal due to membrane potential was recorded, and the rates of molecular diffusion for di-4-ANEPPS through the membrane were investigated. Polarization-resolved SHG imaging was used to determine the orientation of the dye molecules within the membrane.
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