title: Multimodal super-resolution optical microscopy visualizes the close connection between membrane and the cytoskeleton in liver sinusoidal endothelial cell fenestrations.
abstract: Liver sinusoidal endothelial cells (LSECs) act as a filter between blood and the hepatocytes. LSECs are highly fenestrated cells; they contain transcellular pores with diameters between 50 to 200 nm. The small sizes of the fenestrae have so far prohibited any functional analysis with standard and advanced light microscopy techniques. Only the advent of super-resolution optical fluorescence microscopy now permits the recording of such small cellular structures. Here, we demonstrate the complementary use of two different super-resolution optical microscopy modalities, 3D structured illumination microscopy (3D-SIM) and single molecule localization microscopy in a common optical platform to obtain new insights into the association between the cytoskeleton and the plasma membrane that supports the formation of fenestrations. We applied 3D-SIM to multi-color stained LSECs to acquire highly resolved overviews of large sample areas. We then further increased the spatial resolution for imaging fenestrations by single molecule localization microscopy applied to select small locations of interest in the same sample on the same microscope setup. We optimized the use of fluorescent membrane stains for these imaging conditions. The combination of these techniques offers a unique opportunity to significantly improve studies of subcellular ultrastructures such as LSEC fenestrations.
authors.affiliation: Biomolecular Photonics, Department of Physics, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors.forename: Viola
authors.initials: V
authors.lastname: Mönkemöller
authors.affiliation: Faculty of Health Sciences, Department of Medical Biology, Vascular Biology Research Group, The Arctic University of Norway, 9037 Tromsø, Norway.
authors.forename: Cristina
authors.initials: C
authors.lastname: Øie
authors.affiliation: Biomolecular Photonics, Department of Physics, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Hübner
authors.affiliation: Biomolecular Photonics, Department of Physics, Bielefeld University, Universitätsstr. 25, 33615 Bielefeld, Germany.|Department of Internal Medicine, and NSF Center for Biophotonics, University of California, Davis, 2700 Stockton Blvd., Ste. 1400, Sacramento, CA 95817, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Huser
authors.affiliation: Faculty of Health Sciences, Department of Medical Biology, Vascular Biology Research Group, The Arctic University of Norway, 9037 Tromsø, Norway.
authors.forename: Peter
authors.initials: P
authors.lastname: McCourt
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000648:ultrastructure*; D003599:Cytoskeleton / Q000648:ultrastructure*; D042783:Endothelial Cells / Q000648:ultrastructure*; D022781:Hepatocytes / Q000648:ultrastructure; D008099:Liver / Q000648:ultrastructure*; D008856:Microscopy, Fluorescence; D064847:Multimodal Imaging; D051381:Rats