Cellular membranes are organized into compositional and functional domains on the scale of 10-300 nm. The structure of these domains can be interrogated by imaging the brightness or fluorescence spectra of lipophilic probes bound to the lipid membrane. However, the 3D rotational motions of molecules within these membranes provide “orientation spectra” that also reveal interactions of the probe with surrounding lipid molecules and local chemical and physical conditions – the so-called “nanoenvironment” in the lipid membrane.
In this work, we report the development of single-molecule orientation localization microscopy (SMOLM) to image the compositional heterogeneity of artificial lipid membranes. SMOLM combines an orientation-sensitive PSF with single-molecule localization microscopy (SMLM), enabling simultaneous measurements of the 3D orientation, rotational motion, and localization of lipophilic dyes in lipid membranes. We discover that the 3D orientation and rotational motions of Nile red are sensitive to lipid composition, lipid packing, and cholesterol concentrations, consistent with an umbrella model of lipid membranes. We further utilize Nile red in SMOLM to distinguish and map the liquid-ordered (Lo) and liquid-disordered (Ld) domains in DOPC/DPPC/cholesterol membranes with resolution beyond the diffraction limit. Moreover, we extend SMOLM to monitor in-situ enzyme activity on lipid molecules within the membrane. SMOLM directly observes the conversion of cholesterol-rich Lo domains to ceramide-rich Lo domains by sphingomyelinase, revealing compositional heterogeneity that cannot be observed by conventional SMLM. Future extensions to other chemical or biological systems will enable SMOLM and molecular orientation spectroscopy to elucidate nanoscale molecule-environment interactions to reveal underlying macromolecule function and interaction dynamics.
Paper reference: Lu, J., Mazidi, H., Ding, T., Zhang, O. and Lew, M.D. (2020), Single‐Molecule 3D Orientation Imaging Reveals Nanoscale Compositional Heterogeneity in Lipid Membranes . Angew. Chem. Int. Ed.. doi:10.1002/anie.202006207