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Manufacture of Scanning Force Microscope for Biological Research

生物研究用扫描力显微镜的制造

基本信息

批准号：
06558099
负责人：
ANDO Toshio
金额：
$ 7.49万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

(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.

(a)我们制造了一个原子力显微镜，能够扫描速度更快，更不容易受到漂移比普通AFM设备。将该AFM附接到外延发光荧光显微镜。为了显示我们的AFM装置的高性能，我们试图获得高分辨率的图像肌球蛋白头（亚片段-1）在水溶液中。图像显示的精细结构与来自S1的X射线晶体学的S1原子模型中的精细结构相当。在AFM图像中，我们能够看到ATP结合位点以及S1尖端的大裂缝。这些结果表明，我们的AFM装置是在世界上最大的水平。(b)我们进行了一项研究准备发展的高速原子力显微镜，其中一个图像将在30毫秒内获得。我们设计并制造了一台高速扫描仪，并实现了150毫秒内获得一幅图像的高速成像。通过实验，我们制作了一台适合于高速成像的原子力显微镜。我们也研究了适合于高速AFM的电子探针的制备方法。从这些制备研究中，我们能够为视频速率AFM的发展提供一个支架。(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的顶点捕获单个蛋白质分子的方法。使用这种方法，我们成功地量化了一个单一分子的重肌球蛋白（HMM）和肌动蛋白之间施加的力场。使用这种新技术，我们还发现，ATP结合到HMM头诱导振动结构的变化，在HMM头。