Development of scanning fluorescence microscope with sub-micrometer resolution for the topographic micro-analysis

为了获得生物物质表面化学物种的分子和成分信息，发展一种新的高分辨率光学显微观察系统是十分重要的，本研究项目作者根据一种全新的原理，研制了一种亚微米分辨率的高分辨率光学扫描荧光显微镜。其中利用了样品表面近场微激发辐照的二维扫描和高灵敏度的荧光测量。为此，我们设计了一种新型的激发光学头和三维扫描装置。光学头由一根削尖的光纤组成，在真空中用蒸发的铝覆盖，在其顶部有一个微小的针孔（~ 100 nm）。光学头端安装在头戴式光学显微镜的Z台上，其上配备有压电Z定位器。样品固定在交联压电X-Y扫描头中央的样品保持器上，该仪器可提供各种薄膜样品结构表面的分子尺度高分辨率三维荧光图像。给出了光盘、荧光点阵、面包等样品表面的光学扫描显微图像，并以面包为例进行了分析。首次展示了具有亚微米分辨率的活体微生物的光学立体图像。

In order to obtain localized-molecular and compositional information on chemical species at the surface of biological substances it is of importance to develop a new high-resolution optical microscopic observation system.In this research project, the author has been developed a high-resolution optical scanning fluorescence microscope with sub-micrometer resolution, on the basis of a quite new principle. in which two-dimensional scanning of micro-excitation irradiation in near-field of the surface of samples and high sensitive fluorimetric measurement have been utilized. For this purpose, a new excitation optical tip and a three dimensional scanning unit have been designed. The optical tip consists of a sharpened optical fiber covered with evaporated aluminum in vacuo, on which the top has a micro-pinhole (- 100nm). The optical tip is mounted on the Z-stage of a head-stand optical microscope, on which a piezoelectric Z-positioner is equipped. The sample is mounted on the sample holder equipped in the center of a crosslinked piezoelectric X-Y scanning head.This instrument provides a three dimensional fluorescent image of the structural surface of various kinds of thin membrane samples with high resolution on the molecular scale. Several optical-scanning microscopic images on the surface of samples, such as compact disk, fluorescence lattics and, especially bread east as an example of biological samples, are presented. The optical stereoscopic images of living microorganisms with sub-micrometer resolution are demonstrated for the first time.