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局部分子和组成信息，研制一种新的高分辨率光学显微观察系统具有重要意义。在本研究项目中，作者基于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射的二维扫描和高灵敏度的荧光测量。为此，设计了一种新的激发光尖和三维扫描单元。光学针尖由真空中镀有蒸发铝的锐化光纤组成，其顶部有一个微针孔(-100 nm)。所述光学尖端安装在头立式光学显微镜的Z工作台上，在所述Z工作台上安装有压电Z定位器。该仪器将样品安装在安装在交联式压电X-Y扫描头中央的样品固定器上，在分子尺度上提供了各种薄膜样品结构表面的高分辨率三维荧光图像。给出了样品表面的几种光学扫描显微图像，如光盘、荧光晶格，特别是以生物样品为例的面包EAST。首次展示了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.