Resistive-Pulse Sensing Coupled with Fluorescence Lifetime Imaging Microscopy for Differentiation of Individual Liposomes

Characterization of individual biological nanoparticles can be significantly improved by coupling complementary analytical methods. Here, we combine resistive-pulse sensing (RPS) with fluorescence lifetime imaging microscopy (FLIM) to differentiate liposomes at the single-particle level. RPS measures the particle volume, shape, and surface-charge density, and FLIM determines the fluorescence lifetime of the fluorophore associated with the lipid membrane. The RPS devices are fabricated in-plane on a glass substrate to facilitate coupling of RPS with FLIM measurements. For proof-of-concept, we studied liposomes containing various cholesterol concentrations with membrane-intercalated Di-8-ANEPPS, whose fluorescence lifetime is known to be sensitive to cholesterol concentrations in the membrane. RPS-FLIM revealed that increasing cholesterol concentrations in the liposome from 0% to 50% increased the fluorescence lifetimes from 2.1 ± 0.2 to 3.4 ± 0.5 ns, respectively. Moreover, RPS-FLIM discerned liposome populations with the same cholesterol concentration but labeled with dyes that have different fluorescence lifetimes (Di-8-ANEPPS and COE-S6), parsing two particle populations with statistically identical volumes, cholesterol concentration, and lipid composition. Interrogation with RPS-FLIM occurred with individual particles making a single pass through the detection region and overcomes issues with fluorescence spectral overlap that limits traditional methods. We envision RPS-FLIM as a versatile and scalable technique with the potential to differentiate biological particles at the single-particle level to simultaneously inform on particle size, surface-charge density, membrane composition, and identity.