Fast Hyperspectral and Super-Resolved Mapping of Lipid Membrane Polarity with Single-Molecule Sensitivity

Cell membranes display nanoscale heterogeneity in lipid composition and organization that regulates vital biological processes yet remain challenging to resolve with conventional imaging. We introduce spectral phasor single-molecule localization microscopy (SP-SMLM), a hyperspectral and super-resolution method that combines wavefront-like optical filtering with single-molecule imaging for simultaneous spatial and spectral analysis. A lab-built three-channel imager with sine/cosine filters encodes emission spectra of single molecules into the phasor space, enabling high-throughput, high-SNR mapping of membrane polarity at sub-50 nm spatial and 15 s temporal resolutions. Through simulation, we validate that the phasor angle correlates with the spectral mean for single dye molecules. When applied to Nile red-stained COS-7 cells, SP-SMLM revealed organelle-specific polarity differences and dynamic remodeling of the lipid composition within live cells. The method’s hyperspectral capability, rapid acquisition, and compatibility with 2D/3D imaging platforms position SP-SMLM as a powerful tool for studying membrane heterogeneity and dynamics in live cells.