Sub-Diffraction Limited Imaging of Lipid Domains using Ratiometric STED

Abstract

Cellular membranes are comprised of heterogeneously distributed lipid domains that are important for cellular functions. While recent literature has greatly augmented the importance of the spatial separation and the structural features of membrane rafts, the biophysical techniques available for studying membrane nanodomains still remain sparse. Studying lipid clustering and domain formation at a sub-diffraction level continues to be a challenge despite great advances in the field of super-resolution microscopy. In this study, we report the use of a commercially available solvatochromic dye (Nile Red) as a lipid domain sensor in conjunction with stimulated emission depletion (STED) microscopy. By maintaining a fixed concentration of constantly exchanging fluorophores in bulk, we all but eliminate photobleaching from the STED depletion laser, an issue that is commonly associated with STED microscopy. We further leverage the solvatochromic nature of the dye, by monitoring its spectral shift to report on the lipid order changes as a result of changes in lipid composition. As a proof of concept, we performed ratio metric STED imaging on surface-immobilized large unilamellar vesicles (LUVs) prepared with varying lipid compositions to test the validity of our approach. Our results demonstrate that our simple, yet novel approach provides spatial resolution at a <100nm level, while simultaneously reporting on the lipid order of the immobilized LUVs. We anticipate that this technique will be applicable to a wide range of biological systems, and will yield valuable insights into the spatial and chemical organization of lipid rafts in cell membranes. This work was supported by the NIH R01 AI150453 grant to GBM.