Label-Free Interference Microscopy and Single-Molecule Displacement Mapping Elucidate the Microscopic Structure and Dynamics of Stacked Lipid Bilayers

Stacked (phospho)lipid bilayers are ubiquitous and of both fundamental and applicational importance. We demonstrate fast, facile label-free microscopic visualization and quantification of glass-supported stacked lipid bilayers (SSLBs) in aqueous environments through interference reflection microscopy (IRM). Quantitative analysis of three-wavelength IRM shows good agreement with theory, achieving ∼10% signal contrast for each ∼5 nm-thick stacked bilayer and definite designation of local bilayer numbers. Harnessing this capability, we show that SSLBs, with ordered domains tens of micrometers in size, are readily formed under diverse conditions of spin-coating, evaporation from organic solutions, and deposition from liposome dispersions. Moreover, we find that SSLBs are stable under physiological ionic strengths but are destabilized in low-salt conditions and prone to delamination in pure water, under which condition local peeling and wrinkling occur for the stacked bilayers but not for the substrate-adhered basal bilayer. Consequently, washing away the stacked bilayers with water leaves behind a uniform single supported lipid bilayer (SLB). Single-molecule displacement/diffusivity mapping shows that this single bilayer exhibits diffusivity indistinguishable from conventional SLBs. In contrast, stacked bilayers exhibit ∼60% higher diffusivity, indicative of a more native environment than SLBs due to decoupling from substrate interactions. Together, our results reveal the ubiquity, microscopic structure, and properties of SSLBs, providing fundamental insights and pointing to potential applications.