Development of A High-Resolution Non-Confocal Slicing Microscope

到目前为止，1.4的数值孔径是物镜的最大孔径。这将空间分辨率限制在0.2 μ m左右。由于光学材料（如玻璃和油）的折射率有限，理论上不可能制造出数值孔径更大的物镜。在制造大数值孔径透镜时，球差也是一个实际问题。本研究尝试构建一个数值孔径高于常规数值孔径的物镜，最终目的是利用视频显微镜观察神经元突触末梢的胞外分泌。这个想法是利用一种特殊的玻璃和高折射率的油，并且忽略边缘像差，因为视频显微镜只需要轴向图像。成功研制了两个数值孔径为1.65的透镜。两者都可以安装在传统显微镜的螺纹转轮上，达到100倍的放大倍率，工作距离为100微米。它将空间分辨率提高了约20%。几个生物标本被检查与其中一个这些透镜在差干涉对比模式。线粒体、高尔基样结构、核膜、分泌颗粒清晰可见。红细胞膜下可见一些细胞骨架。小肠上皮微绒毛和血小板棘的溶解较前明显改善。缺点是浸泡油在强光下不稳定，并且在盖盖附近的很短范围内实现最佳分辨率。有了这种光学，突触胞吐将很快被研究。该镜头将在订购（奥林巴斯，HR lens）后上市。

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).