Single-Molecule 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes

Jin Lu, Hesam Mazidi, Tianben Ding, Oumeng Zhang, Matthew D. Lew

Research output: Contribution to journalArticlepeer-review

20 Scopus citations


In soft matter, thermal energy causes molecules to continuously translate and rotate, even in crowded environments, thereby impacting the spatial organization and function of most molecular assemblies, such as lipid membranes. Directly measuring the orientation and spatial organization of large collections (>3000 molecules μm−2) of single molecules with nanoscale resolution remains elusive. In this paper, we utilize SMOLM, single-molecule orientation localization microscopy, to directly measure the orientation spectra (3D orientation plus “wobble”) of lipophilic probes transiently bound to lipid membranes, revealing that Nile red's (NR) orientation spectra are extremely sensitive to membrane chemical composition. SMOLM images resolve nanodomains and enzyme-induced compositional heterogeneity within membranes, where NR within liquid-ordered vs. liquid-disordered domains shows a ≈4° difference in polar angle and a ≈0.3π sr difference in wobble angle. As a new type of imaging spectroscopy, SMOLM exposes the organizational and functional dynamics of lipid-lipid, lipid-protein, and lipid-dye interactions with single-molecule, nanoscale resolution.

Original languageEnglish
Pages (from-to)17572-17579
Number of pages8
JournalAngewandte Chemie - International Edition
Issue number40
StatePublished - Sep 28 2020


  • localization microscopy
  • nanodomains
  • rotational diffusion
  • sphingomyelinase
  • supported lipid bilayer


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