Stress Estimation of the Cytoskeleton Using a Confocal Microscope

众所周知，细胞或组织的机械特性的变化出现在细胞或组织形式的变化之前。如果可以定量测量细胞的机械特性，就可以早期发现疾病并及时进行治疗。首先，我们研究了一种测量系统，该系统可以使用共聚焦显微镜获得三维物体的横截面图像，并应用外力对细胞骨架进行应力测量。在该系统的基础实验中，通过微针直接加载获得了HEK细胞中肌动蛋白丝的荧光图像。在与未加载图像对比的基础上，测量了细胞的位移和针的弯曲，并计算了细胞在加载点的应力。其次，我们用驱动电机自动化测量系统。通过该系统，我们可以自动获取细胞三维重建的完整数据，并实现对细胞分子分布的实时测量。我们还改进了双激光束光镊系统。采用双激光束，可以同时施加外力和夹持力，并且可以任意方向加载。最后，我们研究了细胞骨架内部结构的建模方法。考虑了受力机理，采用有限元法对结构进行了推导。

It is known that changes in the mechanical characteristics of a cell or tissue appear before changes in the form of a cell or tissue. If the mechanical characteristics of a cell could be measured quantitatively, it would permit early detection of disease and prompt medical treatment. Firstly, we investigate a measurement system that makes it possible to obtain cross-sectional images of 3-demensional object using a confocal microscope with the application of an external force to perform stress measurement of the cytoskeleton of the cell. In basic experiments for the proposed system, fluorescent images of the actin filaments in HEK cells were obtained with direct loading by micro needles. Based on comparison with images obtained without loading, the displacement of the cell and bending of the needle were measured and the stress on the cell at the loading point was calculated. Secondary, we automated the measurement system with driven motors. By the system, we can acquire a whole data for 3-dimensional reconstruction of a cell automatically and also realize a temporal measurement of molecular distribution of a cell. We also improved the optical tweezers system using doubled laser beams. With doubled laser beams, we can realize an external force and grip force at the same time and loading in any directions. Finally, we investigated the modeling method of internal structures of the cytoskeleton. We considered the mechanisms of the stress and inferred the structure using finite element method.