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

原子力显微镜（AFM）系统最初是与倒置共聚焦激光扫描显微镜结合开发的，以同时测量机械刚度和观察培养细胞的微观结构。为了检测悬臂梁的压痕深度，引入了悬臂梁移动系统。制作一个特殊的标本保持器来保持市售的培养皿。悬臂梁和试样保持器的运动由个人计算机控制。将该系统应用于静态培养和切应力暴露的内皮细胞，得到以下结果.对固定的内皮细胞进行肌动蛋白丝的观察和三维结构的测量.新设计了培养基的输入和输出（I/O）端口，以使用培养皿进行流动暴露实验。利用AFM系统测量培养的内皮细胞的形态和力学性能。静态培养的内皮细胞呈多边形，细胞核周围的力学性质较周围软。内皮细胞在2 Pa剪切应力作用下的刚度随作用时间的延长而增加。剪切流使细胞形态向流动方向拉长，细胞核位置向下游移动.进行了流体流动-结构分析。基于AFM测量的细胞表面几何形状生成三维有限元模型。该模型包括一个流体单元和一个固体单元，分别代表流场和内皮细胞。分析结果表明，高压应力主要出现在上游侧。这一结果可能表明细胞内的应力分布与F-actin的分布密切相关。

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.