Manufacture of Scanning Force Microscope for Biological Research

(一)我们制造了一种原子力显微镜，它比普通的原子力显微镜扫描速度更快，更不容易漂移。将该原子力显微镜固定在发光荧光显微镜上。为了展示我们的AFM设备的高性能，我们试图获得水溶液中肌球蛋白头部(亚片段1)的高分辨率图像。这些图像显示出精细的结构，与由S1的X射线结晶学得出的S1原子模型中的结构类似。在AFM图像中，我们能够看到ATP结合部位以及S1顶端的大裂隙。这些结果证明我们的AFM设备处于世界上最大的水平。(B)我们进行了一项研究，为开发一台高速AFM做准备，其中一幅图像将在30毫秒内获得。我们设计并制造了一台高速扫描仪，实现了在150毫秒内获得一幅图像的高速成像。作为实验，我们研制了一台原子力显微镜，该原子力显微镜采用了适合高速成像的位移检测系统。我们还研究了适合于高速AFM的悬臂的制备方法。通过这些准备研究，我们能够为视频速率AFM的开发提供支架。(C)为了在真正的单分子水平上测量双分子相互作用力，我们开发了一种在悬臂尖端捕获单个蛋白质分子的方法。使用这种方法，我们成功地量化了单个重肌球蛋白(HMM)分子和肌动蛋白之间的力场。利用这项新技术，我们还发现，ATP与HMM头部的结合诱导了HMM头部的振动结构变化。

(a) We manufactured an atomic force microscope that was able to be scanned faster and less susceptible to drift than ordinary AFM apparatuses. This AFM was attached to an epiluminescence fluorescence microscope. To show the high performance of our AFM apparatus we tried to obtain high resolution images of myosin heads (subfragment-1) in aqueous solution. The images revealed fine structures that was comparable to those in an atomic model of S1 derived from x-ray crystallography of S1. In the AFM images we were able to see the ATP binding site as well as the large cleft at the tip of S1. These results proved that our AFM apparatus was on the level of the greatest in the world.(b) We performed a study preparatory to the development of a high-speed AFM in which one image would be acquired within 30msec. We designed and manufactured a high-speed scanner and attained high-speed imaging where one image was obtained within 150msec. By way of experiment we produced an AFM that employed a displacemant detection system suitable for high-speed imaging. We also examinds methods for preparing cantilevers that were suitable for a high-speed AFM.From these preparative studies we were able to secure a scaffolding for the development of the video-rate AFM.(c) To measure bimolecular interaction force at a truly single molecular level we developed a method to capture a single molecule of protein at the apex of a cantilever tip. Using this method we succeeded in quantifying the force field exerted between a single molecule of heavy meromyosin (HMM) and actin. Using this new technology we also discovered that ATP binding to HMM heads induced vibratile structural changes in HMM heads.