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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The atomic force microscope (AFM) system was originally developed in combination with an inverted confocal laser scanning microscope to simultaneously measure mechanical stiffness and to observe microstructure of cultured cell. To detect an indentation depth of the cantilever, a cantilever moving system was introduced. A special specimen holder was made to hold a commercially available culture dish. The movements of the cantilever and the specimen holder were controlled by a personal computer. This system was applied to statically cultured and shear stress exposed endothelial cells, and the following results were obtained.1. Observation of actin filaments and measurement of three dimensional configuration were performed for a fixed endothelial cell.2. Input and output (I/O) ports of culture medium were newly designed to use a culture dish for flow exposure experiments. Morphology and mechanical properties of cultured endothelial cells were measured using the AFM system. Endothelial cells cultured at static condition had a polygonal shape and more soft mechanical properties around a nucleus than those at peripheral regions. The stiffness of the endothelial cells exposed to shear stress of 2 Pa became higher with the duration time of exposure. Cell shape became elongated to the flow direction and the location of a nucleus moved to downstream side by shear flow.3. A fluid flow-structural analysis was performed. The three-dimensional finite element model was generated on the basis of the cell surface geometry measured by the AFM. 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 mainly in the upstream side. This result may indicate that the stress distributions in the cells have close correlation with the F-actin distributions.