エバネッセント光を利用した分子間力3次元測定可能な光学顕微鏡の試作

我们项目的主要目标是建立一种方法，使我们能够在光学显微镜下同时成像和操作单个分子。我们尝试结合两种技术，即光学捕获和倏逝激发显微镜。为此，我们彻底修改了一种在玻璃-水界面处创建渐逝场的旧方法：我们利用盖玻片作为波导，以便 YAG 或氩离子激光束多次进行全内反射，并到达盖玻片上样品所在的所需位置。预计该方法将以比传统方法更少干扰的方式与光学捕获技术相结合，以产生倏逝场。光捕获和新的倏逝激发方法令人满意：我们可以测量肌动球蛋白运动力、肌动蛋白和α-肌动蛋白之间的内聚力，或者我们可以在玻璃-水界面观察单个荧光分子。当两种方法结合起来时，出现了一个以前没有预料到的问题：物镜对用于光阱的红外激光束的像差远远大于我们的预期，并且在我们使用的设置下几乎不可能使用光阱。因此我们寻找其他方法，但发现应该通过设计一种新的物镜来解决这个问题，并针对红外波长区域进行像差校正。目前，利用红外波长光作为荧光显微镜的激发光源，与显微镜制造商的密切合作将是我们最迫切需要实现的目标。

The major goal of our project was to establish a methodology which enables us to simultaneously image and manipulate single molecules under an optical microscope. We attempted to combine two techniques, namely, an optical trapping and evanescent excitation microscopy. To do this, we drastically modified an old methodology to create the evanescent field at glass-water interface : we utilized a coverslip as a waveguide so that a YAG or an argon ion laser beam excuse total internal reflection multiple times and reach the desired position of the coverslip where sample is located. This methodology was anticipated to be coupled to the optical trapping technique in less interfering manner than the conventional method to create the evanescent field. The optical trapping and the new evanescent excitation method satisfactorily well : we could measure the actomyosin motor force, cohesive force between actin and alpha-actinin, or we could observe single fluorescent molecules at the glass-water interface. When the two methods combined, a problem which was not anticipated before appeared : aberration of the objective lens against the infra-red laser beam used for the optical trap was far stronger than our anticipation and it was almost impossible to use optical trap under the setup we utilized. We hence searched other ways, but found the problem should be resolved by designing a new objective lens with aberration is corrected for infra-red wavelength region. At present, the usage of the infra-red wavelength light as a excitation light source for fluorescence microscopy, close cooperation with microscope manufacturer will be the most urgent to approach and achieve our goal.