Free fatty acids (FFAs) are major energy fuel in the animal body. They are usually stored as triglycerides (TGs) in specialized organelles called lipid droplets (LDs). LDs are normally found in adipocytes; however, they can accumulate ectopically in many other tissues and may contribute to cell death and dysfunction of the tissue. This process is referred as lipotoxicity. Recent histological and ultrastructural studies demonstrate that aberrant accumulation of LDs in hepatic cells influences the structure and morphology of various organelles. The first goal of this study was to describe the changes occurring in hepatic cells when ectopic accumulation of lipid occurs. To that end, a transgenic mouse model bearing a deletion of the enzyme PTEN in hepatocytes (LPTENKO mice), which induces an aberrant accumulation of LDs in hepatocytes (steatosis), was analyzed. As well, further analyses were carried out with hepatic cells exposed to fatty acids as in vitro surrogate models of hepatic steatosis. Finally, we aimed also at verifying a recent hypothesis suggesting through biochemical analyses that well-defined channel-like structures connect different lipid droplets between them. Analyses were performed using a new microscope, the Helios NanoLab 660 SEM, able to generate 3D ultrastructural data of cellular organelles. Using this tool, we focus the attention mostly on ultrastructural modification of the endoplasmic reticulum, of the mitochondrial network and of LDs. The alteration of these organelles may have significant impact on hepatocyte functions and viability which, in turn, can lead to damage to the entire organ, affecting the whole organism.