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).

迄今为止，1.4的数值孔径是目标镜头的最大。这将空间分辨率限制为约0.2MUM。从理论上讲，由于玻璃和油的光学材料的折射率有限，因此不可能使任何更高数值孔径的客观镜头成为任何更高的数值孔的客观镜头。当制造高数值孔的镜头时，球形尺寸也是一个实际问题。在这项研究中，尝试使用视频显微镜在神经元细胞的突触末端观察胞吐作用的最终目的是尝试高于常规值的数值镜头的构造。这个想法是利用高折射率高的特殊玻璃和油，而忽略边际像差，因为视频显微镜仅需要轴向图像。成功开发了数值1.65的两个镜头。两者都可以适合常规显微镜的螺纹左轮手枪，从而获得100倍的放大倍率，其工作距离为100MUM。它提高了空间分辨率约20％。在差异干扰对比模式下，使用其中一种镜片检查了几个生物标本。线粒体，类似高尔基体的结构，核膜，分泌的graules清晰可见。在红细胞膜下方可以看到一些细胞骨架模式。肠上皮和血小板的棘突的微绒毛比以前更好地分辨出来。Shortcoces是，浸入油在强光方面是不稳定的，并且最佳分辨率在盖玻片附近非常短的范围内实现。使用此光学元件，计划很快就会检查突触胞吐作用。镜头将按顺序（Olympus，HR镜头）上市获得。