From fixed-dried to wet-fixed to live - comparative super-resolution microscopy of liver sinusoidal endothelial cell fenestrations

• Fenestrations in liver sinusoidal endothelial cells (LSEC) are transcellular nanopores of 50–350 nm diameter that facilitate bidirectional transport of solutes and macromolecules between the bloodstream and the parenchyma of the liver. Liver diseases, ageing, and various substances such as nicotine or ethanol can negatively influence LSECs fenestrations and lead to defenestration. Over the years, the diameter of fenestrations remained the main challenge for imaging of LSEC in vitro. Several microscopy, or rather nanoscopy, approaches have been used to quantify fenestrations in LSEC to assess the effect of drugs and, and toxins in different biological models. All techniques have their limitations, and measurements of the “true” size of fenestrations are hampered because of this. In this study, we approach the comparison of different types of microscopy in a correlative manner. We combine scanning electron microscopy (SEM) with optical nanoscopy methods such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy. In addition, we combined atomic force microscopy (AFM) with SEM and STED, all to better understand the previously reported differences between the reports of fenestration dimensions. We conclude that sample dehydration alters fenestration diameters. Finally, we propose the combination of AFM with conventional microscopy that allows for easy super-resolution observation of the cell dynamics with additional chemical information that can be traced back for the whole experiment. Overall, by pairing the various types of imaging techniques that provide topological 2D/3D/label-free/chemical information we get a deeper insight into both limitations and strengths of each type microscopy when applied to fenestration analysis.

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