Comparative atomic force and scanning electron microscopy: an investigation on fenestrated endothelial cells in vitro

Abstract

SummaryRat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference.AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wetfixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (6%) and decreased (ÿ15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wetfixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).

An in vitro assay reveals a role for the diaphragm protein PV-1 in endothelial fenestra morphogenesis

“…The potent effect of microfilament disassembly on fenestra induction in bEND5 cells and the spatial and temporal correlation between stress fiber disassembly and sieve plate formation highlighted the importance of actin microfilaments. Whereas previous studies had addressed the role of actin disassembly in sustaining or modulating the number of pores in fenestrated liver endothelial cells (31)(32)(33)35) and isolated kidney glomeruli (36), we demonstrated a role for actin disassembly in de novo fenestra formation. Our observations of a rearrangement of large actin filaments during fenestra induction by the physiological stimulus VEGF and the prevention of such rearrangements and fenestra induction by the F-actin-stabilizing drug phalloidin further supported a role for actin remodeling in fenestra formation.…”

Microfilament–Disrupting Agent Latrunculin A Induces and Increased Number of Fenestrae in Rat Liver Sinusoidal Endothelial Cells: Comparison With Cytochalasin B

“…However, latrunculin A increases the number of fenestrae more rapidly than cytochareports confirmed the increase in the number of fenestrae when LECs were treated with cytochalasin B. 8,[17][18][19][20][21][22][23] lasin B (Fig. 2).…”

“…After 1 hour of treatment, an equal maximum ous experimental conditions. 8,[19][20][21][22][23] The conclusion is allowed number of fenestrae was observed for both agents. The that a change in the number of fenestrae depends on the effect of latrunculin A was obtained at concentrations state of polymerization of the actin cytoskeleton.…”

Visualization of cytoskeletal elements by the atomic force microscope

“…At present, there are three main techniques to study the cellular cytoskeleton: TEM [1,2], immunofluorescence microscopy [3,4], and developed recently, transmission X-ray microscopy [5]. It was found that the cytoskeleton consists of three types of filaments: microtubules, microfilaments, and intermediate filaments.…”