Development of A High-Resolution Non-Confocal Slicing Microscope

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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).