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Comparative height measurements of dip-pen nanolithography-produced lipid membrane stacks with atomic force, fluorescence, and surface-enhanced ellipsometric contrast microscopy.

Dip-pen nanolithography (DPN) with phospholipids has been shown to be a powerful tool for the generation of biologically active surface patterns, but screening of the obtained lithographic structures is still a bottleneck in the quality control of the prepared samples. Here we performed a comparative study with atomic force microscopy (AFM), fluorescence microscopy (FM), and surface-enhanced ellipsometric contrast (SEEC) microscopy of phospholipid membrane stacks consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with high admixing of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (DNP Cap PE) produced by DPN. We present a structural model of membrane stacking based on the combined information gained from the three microscopic techniques. Domains of phase-separated DNP Cap PE can be detected at high DNP Cap PE admixing that are not present at medium or low admixings. While the optical methods allow for a high-throughput screening of lithographic structures (compared to AFM), it was found that, when relying on FM alone, artifacts due to phase-separation phenomena can be introduced in the case of thin membrane stacks.