Molecular crowding is an inherent feature of the cell interior. Synthetic cells as provided by giant unilamellar vesicles (GUVs) encapsulating macromolecules (polyethylene-glycol and dextran) represent an excellent mimetic system to study membrane transformations associated with molecular crowding and protein condensation. Similarly to cells, such GUVs loaded with macromolecules exhibit highly curved structures such as internal nanotubes. In addition, upon liquid-liquid phase separation as inside living cells, the membrane of GUVs encapsulating an aqueous two-phase system deforms to form apparent kinks at the contact line of the interface between the two aqueous phases. These structures, nanotubes and kinks, have dimensions below optical resolution and if resolved, can provide information about material properties such as membrane spontaneous curvature and intrinsic contact angle describing the wettability contrast of the encapsulated phases to the membrane. Previous experimental studies were based on conventional optical microscopy which cannot resolve these membrane and wetting proper-ties. Here, we studied these structures with super-resolution microscopy, namely stimulated emission depletion (STED) microscopy, together with microfluidic manipulation. We demonstrate the cylindrical nature of the nanotubes with unprecedented detail based on the superior resolution of STED and automated data analysis. The spontaneous curvature deduced from the nanotube diameters is in excellent agreement with theoretical predictions. Furthermore, we were able to resolve the membrane 'kink' structure as a smoothly curved membrane demonstrating the existence of the intrinsic contact angle. We find very good agreement between the directly measured values and the theoretically predicted ones based on the apparent contact angles on the micrometer scale. During different stages of cellular events, biomembranes undergo a variety of shape transformations such as the formation of buds and nanotubes regulated by membrane necks. We demonstrate that these highly curved membrane structures are amenable to STED imaging and show that such studies provide important insights in the membrane properties and interactions underlying cellular activities.
Super‐resolution imaging of highly curved membrane structures in giant vesicles encapsulating molecular condensates