Development of Atomic Force Microscope System Combined with Confocal Laser Scanning Microscope to Simultaneously Measure Mechanical Stiffness and Observe Microstructure of Cultured Cells

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Under flow condition endothelial cells are elongated and oriented to flow direction depending upon the level of shear stress and the duration of exposure. It is well known that cytoskeletal components, especially F-actin filaments, are playing important roles in the process of adaptation of cell shape to mechanical environment. In this project We have mainly focued two topics and obtained the results shown below.1. Dynamic behavior of actin filament in living cellsWe observed dynamic behavior of actin filaments in living cultured endothelial cells during exposure to shear stress. To do this, a vector of a green fluorescent protein (GFP)/actin fusion was introduced into cells using a lipofectoamine. Dynamic behavior of actin filament in an endothelial cell after introducing cytochalasin D was observed through CCD camera under an inverted fluorescent microscope. In different experiments, the endothelial cells were exposed to fluid shear stress of 2 Pa in a parallel plate flow chamber and … More the cytoskeletal structure was observed. Actin filaments located first at peripheral regions seemed to change the alignment after flow exposure and aligned to a long axis of the cell, which was not oriented to the flow direction yet, but already elongated.2. Stress analysis in a cell exposed to shear stressA fluid flow-structural analysis, i.e. a coupled field analysis, was performed to simulate three dimensional stress distribution in endothelial cells exposed to shear stress. The three-dimensional finite element model was generated on the basis of the cell surface geometry measured by an atomic force microscopy. The model consisted of a fluid element and a solid element representing the flow field and the endothelial cells, respectively. Analytical results on stress distribution in the cell showed that high compressive stress appeared both in the upstream side and the downstream side. These results may indicate that the stress distributions in the cells have close correlation with the F-actin distributions. Less