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Development of A High-Resolution Non-Confocal Slicing Microscope

高分辨率非共焦切片显微镜的开发

基本信息

批准号：
05558099
负责人：
TERAKAWA Susumu
金额：
$ 5.5万
依托单位：
Hamamatsu University School of Medicine, Photon Medical Research Center
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

A numerical aperture of 1.4 has been the largest for objective lenses, to date. This set a limit to the spatial resolution at about 0.2mum. It is considered to be theoretically impossible to make an objective lens of any higher numerical aperture, because of limited refractive indices of the materials for optics such as, glass and oil. The spherical aberrtaion is also a practical problem when making a lens of high numerical aperture. In this research, construction of an objective lens of a numerical aperture higher than the conventional value was attempted, with a final purpose to observe exocytosis in the synaptic terminal of neuronal cells using the video microscopy. The idea is to make use of a special glass and oil of high refractive indices, and to ignore the marginal aberration, as the video microscopy requires only the axial image.Two lenses of a numerical aperture of 1.65 were developed successfully. Both can fit to the threaded revolver of the conventional microscope, attaining a magnification of 100 times with a working distance of 100mum. It improved the spatial resolution by about 20%. Several biological specimens were examined with one of these lenses at the differential interference contrast mode. Mitochondria, Golgi-like structures, nuclear membrane, secretory graules were clearly visible. Some cytoskeletal patterns were visible underneath the erythrocyte membrane. Microvilli of the intestinal epithelium and spines of the platelet were better resolved than before.Shortcomings were that the immersion oil was unstable against a strong light, and that the best resolution is realized in a very short range near the coverslip. With this optics, synaptic exocytosis is planned to be examined, soon. The lens will become commercially available upon order(Olympus, HR Lens).

1.4的数值孔径是迄今为止物镜的最大值。这将空间分辨率限制在约0.2 μ m。由于用于光学器件的材料（例如玻璃和油）的有限折射率，理论上不可能制造任何更高数值孔径的物镜透镜。球面像差也是制造高数值孔径透镜时的一个实际问题。在本研究中，尝试构建数值孔径高于常规值的物镜透镜，最终目的是使用视频显微镜观察神经元细胞突触末端的胞吐作用。这种方法的思想是利用一种特殊的玻璃和高折射率的油，并忽略边缘像差，因为视频显微镜只需要轴向图像。两者都可以适合传统显微镜的螺纹旋转器，在100 μ m的工作距离下实现100倍的放大率。空间分辨率提高了20%左右。用其中一个透镜在微分干涉对比模式下检查了几个生物标本。线粒体、高尔基体、核膜、分泌颗粒清晰可见。红细胞膜下可见一些细胞骨架图案。肠上皮细胞的微绒毛和血小板的棘比以前更好地解决了，缺点是浸油对强光不稳定，最佳分辨率是在盖玻片附近的很短的范围内实现的。有了这种光学器件，突触胞吐作用计划很快得到检验。该透镜将在订购后上市（Olympus，HR透镜）。