You are here : Home > The lab > Study of hepatic ultrastructures by FIB-SEM 3D electron microscopy

Yousr Rekik

Study of hepatic ultrastructures by FIB-SEM 3D electron microscopy


Page Web française.
Published on 28 June 2023
Thesis presented June 28, 2023

The development of volume electron microscopy techniques has revolutionized tissue and cell imaging. In particular, FIB-SEM (focused ion beam scanning electron microscopy) imaging allows to elucidate the three-dimensional ultrastructure of biological structures at a nanometric scale. In the framework of my PhD project, two independent studies were performed using the FIB-SEM approach.
In a first study, I worked on silver nanoparticles (AgNP) which are used, for their biocidal function, in everyday products (food packaging, anti-odor clothes...) as well as in medical devices (wound dressings, catheters...). These NP release Ag(I) ions that are responsible for the biocidal effect but are also toxic for mammalian cells. In order to mimic the fate of AgNP in the organism with a focus on their transformations and the excretion of silver species from the liver, we used a 3D model of hepatocyte spheroid which mimics the liver and allows to study biliary excretion. These spheroids were exposed to a silver salt and to two types of AgNP to mimic, respectively, oral and parenteral exposure which are the two main routes of human exposure to these NP. The combination of FIB-SEM technique with other elemental imaging, chemical analysis and speciation techniques allowed us to study the transformations of the different silver species, to visualize their distributions at a subcellular scale, to highlight the storage of silver excess into vacuoles and the biliary excretion of Ag(I) ions and to analyze the impact of silver species on the ultrastructure of hepatocytes, in particular on the mitochondrial network. These results highlight the relevance of our hepatic model to study the transformations of nanomaterials in the liver beyond AgNP.
In a second study, I worked on liver sinusoidal endothelial cells (LSEC) that line the wall of hepatic capillaries or sinusoids. LSEC are highly specialized endothelial cells that have fenestrations or fenestrae allowing for bidirectional exchange between sinusoidal blood and hepatocytes. Studies describing the structure of LSEC are mostly 2D studies that do not allow to fully elucidate the in vivo architecture of these cells and the spatial organization of their fenestrations. The FIB-SEM technique was used to image mouse liver samples focusing on the porosity of sinusoids. A preparation protocol of liver samples that properly preserves the ultrastructure of sinusoids was established. Then, I developed a segmentation method that allowed 3D reconstruction of LSEC at high resolution. The study of the 3D architecture of LSEC and the determination of their fenestrae diameters open new perspectives to further understand the correlation between the function and the structure of these cells under physiological and pathological conditions. Our approach could also help to model the transport of different substances or drugs between blood and hepatocytes and could be used to study the sinusoidal porosity of patients for personalized medicine applications.

Volume electron microscopy, FIB-SEM, liver, 3D cell culture, silver nanoparticles, LSEC, fenestrations

On-line thesis. ​