New light technology for fluorescence excitation and direct measurement of non-stained organic molecules such as DNA

我们研究了用荧光显微镜测量DNA等有机分子发出的微弱荧光。我们开发了一种使用近红外脉冲激光的双光子荧光显微镜，以及带有自共轭镜的4倍共聚焦显微镜和双通共聚焦显微镜。我们从理论上分析了三种发达的显微镜，并成功地捕获了图像。1)双光子荧光显微镜研制了一种近红外脉冲激光（钛：蓝宝石激光：750nm）双光子荧光显微镜。利用研制的系统，对DAPI染色的小鼠胚胎眼进行了观察。我们成功地通过扫描焦平面获得光学切片图像，而无需对样品进行切片。该系统在样品中提供250 μ m深度的图像。通过对染色细胞核的测量，我们证实该显微镜具有高分辨率。2)带相位共轭镜的4倍共聚焦显微镜研制了一种带相位共轭镜的4倍共聚焦显微镜。我们采用掺Ce的BaTiO_3晶体作为相位共轭镜。利用显微镜测量了系统的轴向响应。结果表明，中心峰的宽度为-0.25 μ m，与理论值（λ /2）一致。然而，反射镜的低反射率（-60%）导致样品中干扰的可见度较低。利用研制的带相位共轭镜的双通共聚焦显微镜对硅藻进行了观察。即使经过两次样品，图像的分辨率也很好。由于相位共轭特性，所研制的显微镜成像无像差。

We investigated fluorescence microscopy for measuring the faint fluorescent light from organic molecules such as DNA.We developed a two-photon fluorescence microscope using a near-infrared pulsed laser, and 4pi confocal and double-pass confocal microscopes with a self-conjugated mirror. We theoretically analyzed three developed microscopes and succeeded in capturing the images.1) Two-photon fluorescence microscopeWe developed a two-photon fluorescence microscope with a near-infrared pulse laser (Ti : Sapphire laser : 750nm). By use of developed system, we observed an eye of mouse embryo stained with DAPI.We succeeded in obtaining optical sectioning images by scanning the focal plane without slicing the sample. The system provides the images at 250 mum depth in the sample. We confirmed that the microscope had high resolution through measurement of a stained nucleus.2) 4pi confocal microscope with a phase-conjugate mirrorWe developed a 4pi confocal microscope with a phase-conjugate mirror. As the phase-conjugate mirror, we used a Ce doped BaTiO_3 crystal. By using of the microscope, we measured an axial response of the system. The result shows that a center peak has -0.25 mum width, which agrees with theoretical value (lambda/2). However, low reflectivity (-60%) of the mirror causes low visibility of interference in the sample.3) Double-pass confocal microscope with a phase-conjugate mirrorWe observed a diatom by using the developed double-pass confocal microscope with a phase-conjugate mirror. The resolution of the images was good even after passing through the sample twice. Due to the phase-conjugate property, the image given with the developed microscope was aberration free.