Stress Estimation of the Cytoskeleton Using a Confocal Microscope

已知细胞或组织的机械特性的变化出现在细胞或组织的形式的变化之前。如果细胞的机械特性可以定量测量，就可以及早发现疾病并及时进行治疗。首先，我们研究了一种测量系统，该测量系统使得可以使用共聚焦显微镜获得三维物体的横截面图像，并施加外力以进行细胞骨架的应力测量。在所提出的系统的基础实验中，HEK细胞中的肌动蛋白丝的荧光图像通过微针直接加载获得。基于与未加载时获得的图像的比较，测量细胞的位移和针的弯曲，并计算加载点处细胞上的应力。其次，我们使用驱动电机实现了测量系统的自动化。该系统可以自动获取细胞三维重建所需的完整数据，实现细胞分子分布的实时测量。我们还改进了双光束光镊系统。通过双重激光束，我们可以同时实现外力和握力，并在任何方向上加载。最后，研究了细胞骨架内部结构的建模方法。我们考虑的应力机制和推断的结构，用有限元方法。

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.