Dithionite quenching of NBD-labeled lipids reveals artificial lipid droplet purity and neutral lipid surface accessibility

Artificial lipid droplets (aLDs) provide a controllable platform for studying lipid biochemistry, but their use is limited by contamination with other membrane structures and the lack of quantitative methods to assess sample purity. Here, we establish dithionite quenching of NBD-labeled lipids as a simple approach to evaluate aLD purity. The approach relies on dithionite’s ability to selectively quench NBD fluorophores exposed in the phospholipid monolayer of aLDs and in the outer leaflet of liposome bilayers, but not those protected within the inner leaflet of liposome bilayers. Consistent with liposome contamination, bulk aLD preparations exhibit incomplete quenching, which can be separated by sucrose gradient centrifugation into liposome-like and droplet-enriched populations based on quenching behavior. Guided by this assay, sonication conditions were optimized to increase aLD purity and reduce liposome contamination. A biotin–streptavidin immobilization strategy further enabled stable imaging of individual aLDs. Finally, we applied dithionite quenching to probe the accessibility of neutral lipids within aLDs. This revealed hydrophobicity-dependent quenching kinetics of neutral lipids, with less hydrophobic diacylglycerols showing greater surface exposure within aLDs than more hydrophobic triacylglycerols and cholesterol esters. Taken together, these establish dithionite quenching of NBD-labeled lipids as a simple quantitative method for assessing aLD purity and demonstrate its utility for studying lipid accessibility.