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纵单个分子。我们试图将两种技术，即光学捕获和倏逝波激发显微镜，联合收割机。为了做到这一点，我们彻底修改了旧的方法，以在玻璃-水界面处产生倏逝场：我们利用盖玻片作为波导，使得YAG或氩离子激光束多次免除全内反射，并到达样品所在的盖玻片的期望位置。这种方法被预期耦合到光学捕获技术，比传统的方法产生的倏逝场的干扰少的方式。光学捕获和新的倏逝波激发方法令人满意的好：我们可以测量肌动球蛋白的马达力，肌动蛋白和α-辅肌动蛋白之间的凝聚力，或者我们可以观察到单个荧光分子在玻璃-水界面。当这两种方法结合起来时，出现了一个以前没有预料到的问题：物镜透镜对用于光阱的红外激光束的像差远远强于我们的预期，在我们使用的设置下几乎不可能使用光阱。因此，我们寻找其他的方法，但发现这个问题应该通过设计一个新的物镜透镜的红外波长区的像差是正确的解决。目前，使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接近和实现我们目标的最紧迫的事情。

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.