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。我们试图结合两种技术，即光学捕获和瞬变激发显微镜。为了做到这一点，我们彻底修改了一种旧的方法，以在玻璃-水界面上创建倏逝场：我们使用盖盖作为波导，这样YAG或氩离子激光束就可以多次消除全内反射，并到达样品所在的盖盖的理想位置。该方法有望以比传统方法干扰更少的方式与光捕获技术耦合以产生倏逝场。光学捕获和新的瞬时激发方法令人满意：我们可以测量肌动蛋白的运动力，肌动蛋白和α -肌动蛋白之间的凝聚力，或者我们可以观察玻璃-水界面上的单个荧光分子。当这两种方法结合在一起时，出现了一个之前没有预料到的问题：用于光阱的红外激光束对物镜的像差远远大于我们的预期，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，但发现需要设计一种新的物镜来解决这个问题，并在红外波长区域进行像差校正。目前，利用红外波长的光作为荧光显微镜的激发光源，与显微镜制造商密切合作将是我们最迫切要接近和实现的目标。

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.