Quantitative analysis methods for studying fenestrations in liver sinusoidal endothelial cells. A comparative study.

Liver Sinusoidal Endothelial Cells (LSEC) line the hepatic vasculature providing blood filtration via transmembrane nanopores called fenestrations. These structures are 50-300 nm in diameter, which is below the resolution limit of a conventional light microscopy. To date, there is no standardized method of fenestration image analysis. With this study, we provide and compare three different approaches: manual measurements, a semi-automatic (threshold-based) method, and an automatic method based on user-friendly open source machine learning software. Images were obtained using three super resolution techniques - atomic force microscopy (AFM), scanning electron microscopy (SEM), and structured illumination microscopy (SIM). Parameters describing fenestrations such as diameter, area, roundness, frequency, and porosity were measured. Finally, we studied the user bias by comparison of the data obtained by five different users applying provided analysis methods.

Atomic Force Microscopy Reveals the Dynamic Morphology of Fenestrations in Live Liver Sinusoidal Endothelial Cells.

Here, we report an atomic force microscopy (AFM)-based imaging method for resolving the fine nanostructures (e.g., fenestrations) in the membranes of live primary murine liver sinusoidal endothelial cells (LSECs). From data on topographical and nanomechanical properties of the selected cell areas collected within 1 min, we traced the dynamic rearrangement of the cell actin cytoskeleton connected with the formation or closing of cell fenestrations, both in non-stimulated LSECs as well as in response to cytochalasin B and antimycin A. In conclusion, AFM-based imaging permitted the near real-time measurements of dynamic changes in fenestrations in live LSECs.

Morphology and force probing of primary murine liver sinusoidal endothelial cells.

Liver sinusoidal endothelial cells (LSECs) represent unique type of endothelial cells featured by their characteristic morphology, ie, lack of a basement membrane and presence of fenestrations-transmembrane pores acting as a dynamic filter between the vascular space and the liver parenchyma. Delicate structure of LSECs membrane combined with a submicron size of fenestrations hinders their visualization in live cells. In this work, we apply atomic force microscopy contact mode to characterize fenestrations in LSECs. We reveal the structure of fenestrations in live LSECs. Moreover, we show that the high-resolution imaging of fenestrations is possible for the glutaraldehyde-fixed LSECs. Finally, thorough information about the morphology of LSECs including great contrast in visualization of sieve plates and fenestrations is provided using Force Modulation mode. We show also the ability to precisely localize the cell nuclei in fixed LSECs. It can be helpful for more precise description of nanomechanical properties of cell nuclei using atomic force microscopy. Presented methodology combining high-quality imaging of fixed cells with an additional nanomechanical information of both live and fixed LSECs provides a unique approach to study LSECs morphology and nanomechanics that could foster understanding of the role of LSECs in maintaining liver homeostasis.

Label-free spectroscopic characterization of live liver sinusoidal endothelial cells (LSECs) isolated from the murine liver.

Liver sinusoidal endothelial cells (LSECs) represent a highly specialized and unique type of endothelial cell in terms of their morphology and function. The biochemical and functional characterization of LSECs in vitro is restrained by the rapid change of LSECs' phenotype upon culturing under classical experimental conditions. In this work, we present a novel approach to characterize the biochemical content of murine LSECs, freshly isolated from the liver, with the use of microspectroscopic analysis. For comparison, hepatocytes and Hepatic Stellate Cells (HSCs) were analyzed. Our approach, based on label-free confocal Raman imaging of live cells combined with chemometric analysis, provided insight into the biochemical content of freshly isolated LSECs on a subcellular level. LSECs were featured by a distinct biochemical signature in comparison with other major cell types of the liver. Based on our work we claim that the non-invasive and non-destructive confocal Raman imaging may assist in obtaining chemical information spatially distributed within the cells that characterize the phenotype of primary LSECs as well as other types of liver cells. Furthermore, our approach provides a unique insight into LSECs' morphology and chemical composition that may help to understand their functions.