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 remains 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-sec 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 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.