Document Type
Article
Publication Date
4-27-2026
Keywords
high throughput, single-molecule spectroscopy, spectral phasor, hyperspectral super-resolution imaging, lipid membrane, heterogeneous polarity mapping
Abstract
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.
Citation
Elric Dion Pott, Meek Yang, James Ethan Batey, Joie Embree, Bin Dong; Fast Hyperspectral and Super-Resolved Mapping of Lipid Membrane Polarity with Single-Molecule Sensitivity. Chemical & Biomedical Imaging 27 April 2026; 4 (4): 584–589. https://doi.org/10.1021/cbmi.5c00130
Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial-No Derivative Works 4.0 International License.
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