Development of A High-Resolution Non-Confocal Slicing Microscope

1.4的数值光圈是迄今为止物镜的最大光圈。这将空间分辨率限制在约0.2um。由于玻璃和油等光学材料的折射率有限，理论上不可能制造出任何更大数值孔径的物镜。球差也是制作大数值孔径透镜时的实际问题。在这项研究中，试图构建一个数值孔径高于常规值的物镜，最终目的是用视频显微镜观察神经元细胞突触末端的胞吐作用。利用一种特殊的高折射率玻璃和油，忽略边缘像差，因为视频显微镜只需要轴向图像。成功地研制了两个数值孔径为1.65的透镜。两者都可以装在常规显微镜的螺纹式左轮手枪上，工作距离100微米，放大100倍。空间分辨率提高了约20%。用其中一种透镜在差分干涉对比模式下检查了几个生物标本。线粒体、高尔基体样结构、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。肠上皮的微绒毛和血小板的刺比以前有了更好的分解。缺点是浸泡的油对强光不稳定，并且在盖片附近的很短的距离内实现了最佳的分解。有了这种光学装置，突触的胞吐作用很快就会被检查出来。该镜头将在商业上订购(奥林巴斯，HR镜头)。

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).