Optimization of 2D and 3D cell culture to study membrane organization with STED microscopy.

Epithelia cells assemble into sheets that compartmentalize organs and generate tissue barriers. This is achieved by forming polarized membrane domains, which are connected by junctional complexes. While much is known about the organization of the basal membrane due to its easy accessibility by high and super-resolution microscopy, the apical and lateral membrane domains remain poorly characterized. Here we describe our methods to study the molecular organization of apical and lateral membrane domains by combining 2D and 3D epithelial cell culture with super-resolution STED microscopy. We show that inverted cell monolayers enable live cell imaging of the apical membrane with a resolution sufficient to resolve the densely packed micro-villi of human enterocytes. Furthermore, 3D cell culture enables us to resolve adhesion complexes in the lateral domain of kidney derived cells. We envision that these methods will help to reveal the supra-molecular structure of lateral and apical membrane domains in epithelial cells