Development of analytical color fluorescence electron microscope (ACFEM) and its application to the medical and biological fields

为了建立一种新的电子显微细胞化学分析方法，我们研制了一种分析型彩色荧光电子显微镜（ACFEM），并将其应用于生物医学研究。ACFEM以高分辨率扫描电子显微镜为基础，配备阴极发光（CL）收集镜和光谱多通道分析系统，使我们能够获得彩色CL图像、任意波长的单色CL图像、常规SEN图像以及电子束扫描样品表面时的CL光谱。我们在没有荧光染色的情况下观察了来自各种器官和组织的CL，以分析发射CL的物质。发现生物样品中脂滴（LD）发射的CL分别在320 nu和430-450 nu处具有双峰模式光谱。320 nm-CL是胆固醇酯的衍生物，因为320 nm-CL通常在胆固醇酯中检测到， ...更多信息 大鼠肾上腺皮质、黄体、内膜和颗粒细胞的LD在用降胆固醇剂治疗时消失，并在垂体切除后显示强度增加。同样，通过对大鼠视网膜和睾丸的研究，证明430-450 na-CL来源于至少包括维生素A酯的脂质混合物。由于缺乏高特异性的抗脂质抗体，如类固醇和维甲酸，目前尚不能用细胞化学方法分析单个脂滴中的成分。本研究在方法学上解决了这个问题。此外，我们还观察到FITC-IgG在ACF 24下发射CL。这一事实表明，将荧光抗体法直接引入电镜的可能性。近年来，在金刚石评价和新型光敏材料开发方面的研究表明，ACFEM在工业研究领域也有很大的应用前景。少

In order to establish a new method for an electron microscopic cytochemical analysis, we developed an analytical color fluorescence electron microscope(ACFEM)and applied this method to the biomedical research. On the basis of a high resolution scanning electron microscope, the ACFEM, equipped with a cathodoluminescence(CL)-collecting mirror and a spectral multichannel analytical system, enables us to obtain color CL images, monochromatic CL images at any given wavelength, and conventional SEN images, as well as CL spectrum when an electron beam is scanning over the surface of specimens.Using the ACFEM, we observed CL from a varieties of organs and tissues without fluorescent staining to analyze the substances which emit CL. CL was found to be emitted from lipid droplets(LD)in biological specimens with a two-peak pattern spectrum at 320 nu and 430-450 na respectively. It was proved that the 320 nm-CL was derived from cholesterol esters, because 320 nu-CL, which was usually detected in t … More he LD of adrenal cortex, corpus luteum, theca interna and granulosa cells of the rats, disappeared when the animals were treated with hypocholesterolemic agents, and showed an increase of intensity after hypophysectomy. Similarly, the 430-450 na-CL was demonstrated to be derived from a mixture of lipids at least including vitamin A ester by the research dealt with retina and testes of the rats. So far, it was impossible to analyze the components in a single lipid droplet by means of cytochemistry for the reason of lacking antibodies with high specialities against lipids, such as steroids and retinoids. This study methodologically dissolved this problem.Moreover, we also observed that FITC-IgG emits CL under the ACF24. This fact indicates the possibility to directly introduce fluorescent antibody method to electron microscopy. Recently, the study on evaluating diamonds and developing new photo sensitive materials have revealed that the ACFEM could be very useful in the industrial research fields, also. Less