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.

Morphology and force probing of primary murine liver sinusoidal endothelial cells

“…However, because of the impossibility in performing proper fenestration measurements in the live cells, in the current work, we were forced to use glutaraldehyde‐fixed LSECs. The LSEC fenestrations are surrounded by the cytoskeleton ring made of actin in the form of a calmodulin‐actomyosin complex that is responsible for their diameter . Therefore, according to Targosz‐Korecka et al, information about their structure should be preserved after fixation.…”

“…In most of the publications, the structure of fenestrations in LSECs was described in fixed cells using electron microscopy, atomic force microscopy (AFM) or recently super‐resolution optical microscopy . However, in fixed (especially fixed‐dried) cells, the structure of fenestrations might be altered .…”

Super-Resolution Microscopy: Shedding Light on the Cellular Plasma Membrane

“…Liver sinusoidal endothelial cells exhibit a pore-like structure which allows the filtration of objects between the blot and hepatocytes based on size selection. The spatial distribution of the cytoskeleton and plasma membrane was observed in liver sinusoidal endothelial cells using combination 3D-SIM and STORM microscopy, revealing a close association between the membrane and cytoskeleton 147 . A periodic structure of actin, spectrin, ankyrin and sodium channels was shown in neuronal axons using STORM microscopy, revealing a strikingly regular assembly of these cytoskeletal elements 148 .…”

STED microscopy: A simplified method for liver sinusoidal endothelial fenestrae analysis

“…The resolution limit of these technics allows to detect fenestrae regardless their small size and their high representation in LSECs membranes. Using three-dimensional structured illumination microscopy (3D-SIM) and direct stochastic optical reconstruction microscopy (dSTORM), Monkemoller et al (2014Monkemoller et al ( , 2015 [Cogger et al, 2010;Zapotoczny et al, 2017] recently observed fenestrae in optical ways and tried to analyse their dynamics and co-localised actin filaments that surround fenestrae.…”