AFM investigation of endothelial fenestrae

Abstract

Endothelial cells of rat liver sinusoids (LEC) contain fenestrae, which are clustered in sieve plates. These open fenestrae, with an average diameter in the order of 0.1 μm, control the exchange of fluids, solutes and particles between the blood and the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM). SEM can also be used to study dynamic changes in fenestrae by comparing specimens subjected to different experimental conditions. However, the SEM uses fixed, dried and coated specimens. Recently, the atomic force microscope (AFM) was introduced, enabling the analysis of cell surfaces under fluid. We used the AFM for the investigation of fenestrae in cultured LEC, whereas SEM served as a reference.LEC were isolated by collagenase perfusion and purified by a Percoll gradient and selective adherence. After spreading on collagen, LEC were cultured for 8 h. Cells were fixed in 2% glutaraldehyde (12 h), followed by 1% tannic acid (1 h) and postfixed in 1% osmium. Samples were dehydrated in alcohol, critical point dried in ethanol / CO2, and sputter coated with gold, We compared imaging of dried-coated (SEM and AFM), dried-uncoated (AFM) and wet-fixed (AFM) specimens in a series of experiments, with the purpose to prepare for depicting living cells in vitro.