Morphological alterations in primary hepatocytes upon nanomaterial incubation assessed by digital holographic microscopy and holotomography

Significant improvements in quantitative phase imaging (QPI) technologies established digital holographic microscopy (DHM) and holotomography (HT) as tools for label-free imaging of cell morphology as well as to assess the 3D distribution of intracellular structures. Here, we applied DHM and HT consecutively on the same samples to observe and quantify the impact of nanomaterials on the morphology of primary hepatocytes. The liver has a high vascularization and metabolic activity, and hepatocytes were selected because they may contact and internalize nanoparticles circulating in the blood. Effects of nanoparticles on cells can range from a reduction of viability to alterations in morphology and intracellular structures. Thus, first an automated modular DHM setup was applied for large-area QPI screening of the entire hepatocyte populations while a commercial HT system (Tomocube HT-2H, Tomocube, Korea) was utilized to observe selected tiny 3-dimensional intracellular changes of interest via refractive index tomograms. For the investigations, hepatocytes were isolated from collagenase-perfused rat livers and seeded into HT-compatible cell culture dishes. After cultivation and incubation with different types of nanoparticles (CeO2, Ag, Au) for 24 hours the cells were fixed with a mixture of glutaraldehyde and paraformaldehyde to preserve cell morphology and structure. The results of our study demonstrate that QPI with DHM is capable for efficient large-area 2D screening and to reveal of nanomaterial-related alterations in the entire hepatocyte populations while HT provides high performance complementary 3D insights and the localization of tiny intracellular damages.