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Development of an Atomic Force Microscope Suitable for Observation of Biological Samples.

开发适合观察生物样品的原子力显微镜。

基本信息

批准号：
03455011
负责人：
ANDO Toshio
金额：
$ 2.5万
依托单位：
Kanazawa University
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1993
项目状态：
已结题

项目摘要

Atomic force microscopy(AFM)holds great promise for biological science, since it allows us to observe with nanometer resolution the native surface structure of protein in an aqueous environment, and further it can quantify force exerted between the surfaces of protein and the tip of an AFM probe(or a second protein attached to the probe tip). Commercially manufactured atomic force microscopes are, however, inadequate for the biological application, since blind scanning has to be made to locate objects of interest randomly distributed on the sample substratum. To overcome this problem, we have developed an atomic force microscope integrated with an inverted fluorescence microscope. The Z-actuator is a short hollow piezo tube, on the top of which a sample is placed. The objective lens is inserted from the bottom into the hollow space. Since this piezo tube is wide (inner diameter 3.5cm), its top cannot deflect much in the XY directions. As XY actuator two piezo plates pointing at right angles to each other are placed horizontally with their tips touching a hollow plate(glued to the bottom of the Z-piezo tube)on the sides. The location of the probe tip as well as individual protein molecules that are stained with fluorescent dye can be visualized under the fluorescence microscope. By the use of an XY-stage to move a sample and another XY-stage to move the AFM head(including the probe tip), the probe tip can pinpoint a specific object of interest. This ensures an obtained AFM image to be of the object, neither of contaminants nor of an undulation of the substratum. Further, it facilitates the capture of single protein molecule at the tip of the probe. Using the developed AFM,we obtained clear images of a single actin filament and two-headed structure of myosin. We succeeded in capturing a single myosin molecule at the tip of the probe, utilizing the strong affinity between avidin and biotin.

原子力显微镜（AFM）在生物科学中具有很大的应用前景，因为它允许我们在水环境中以纳米分辨率观察蛋白质的天然表面结构，并且还可以量化蛋白质表面和AFM探针尖端（或附着在探针尖端上的第二种蛋白质）之间施加的力。然而，商业制造的原子力显微镜是不够的生物应用，因为盲目扫描必须定位随机分布在样品基质上的感兴趣的对象。为了克服这个问题，我们开发了一种与倒置荧光显微镜集成的原子力显微镜。Z致动器是一个短的中空压电管，样品放置在其顶部。物镜透镜从底部插入中空空间。由于这种压电管很宽（内径3.5cm），其顶部不能在XY方向上偏转太多。作为XY致动器，两个彼此成直角指向的压电板水平放置，其尖端接触侧面的中空板（粘合到Z压电管的底部）。探针尖端的位置以及用荧光染料染色的单个蛋白质分子可以在荧光显微镜下可视化。通过使用XY台移动样品和另一个XY台移动AFM头（包括探针尖端），探针尖端可以精确定位感兴趣的特定对象。这确保了所获得的AFM图像是物体的，既不是污染物的也不是基底的起伏的。此外，它有助于在探针尖端捕获单个蛋白质分子。使用开发的原子力显微镜，我们得到了清晰的图像，一个单一的肌动蛋白丝和双头结构的肌球蛋白。利用亲和素和生物素之间的强亲和力，我们成功地在探针尖端捕获了单个肌球蛋白分子。