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

我们研究了用于测量来自DNA等有机分子的微弱荧光的荧光显微镜。我们利用近红外脉冲激光和带有自共轭反射镜的4PI共焦和双程共焦显微镜研制了一台双光子荧光显微镜。1)双光子荧光显微镜我们研制了一种近红外脉冲激光(钛宝石激光：750 nm)双光子荧光显微镜。利用研制的系统，我们观察到一只染有DAPI的小鼠胚胎眼，我们成功地在不对样品切片的情况下，通过扫描焦平面获得了光学切片图像。该系统在样品中提供250微米深度的图像。通过对染色细胞核的测量，证实该显微镜具有较高的分辨率。2)位相共轭4PI共焦显微镜。采用掺Ce的BaTiO_3晶体作为位相共轭反射镜。利用显微镜测量了系统的轴向响应。结果表明，中心峰宽度为-0.25um，与理论值(lambda/2)相符。然而，反射镜的低反射率(-60%)导致样品中干涉的能见度较低。3)位相共轭双程共焦显微镜我们用研制的双程共焦显微镜和位相共轭镜观察了硅藻。即使在通过样品两次后，图像的分辨率也很好。由于具有位相共轭特性，用研制的显微镜得到的图像是无像差的。

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.