Super-Resolution Optical Sectioning Microscopy Visualizes Nanopores in the Plasma Membrane of Endothelial Cells in situ

The ultrastructure of endothelial cells (ECs) in situ is of great interest due to their involvement in many physiological processes. In some organs, these cells form transcellular pores or fenestrae, allowing for the rapid exchange of molecules between blood and interstitium. Despite their importance, no optical images of these dynamic morphological structures have yet been acquired in situ. Major obstacles to their in situ imaging are the lack of specifical labels for fenestrae and their size well below the optical diffraction limit. Here, we report how we have overcome these challenges and managed to visualize the EC ultrastructure in situ in 25 m thick liver sections. To enable this, a lipophilic, fluorescent membrane dye was infused into the portal vein of murine livers to stain the sinusoidal ECs before the organ was harvested. Tissue sections were subsequently imaged using a novel, super-resolution optical sectioning structured illumination microscope (OS-SIM), providing approx. 170 nm spatial resolution with significantly faster image acquisition compared to confocal microscopy.