Saito Kenta, Arai Yoshiyuki, Zhang Jize, Kobayashi Kentaro, Tani Tomomi, Nagai Takeharu
Research Institute for Electronic Science, Hokkaido University, Japan.
Cell Struct Funct. 2011;36(2):237-46. doi: 10.1247/csf.11026. Epub 2011 Oct 29.
Laser-scanning confocal microscopy has been employed for exploring structures at subcellular, cellular and tissue level in three dimensions. To acquire the confocal image, a coherent light source, such as laser, is generally required in conventional single-point scanning microscopy. The illuminating beam must be focused onto a small spot with diffraction-limited size, and this determines the spatial resolution of the microscopy system. In contrast, multipoint scanning confocal microscopy using a Nipkow disk enables the use of an incoherent light source. We previously demonstrated successful application of a 100 W mercury arc lamp as a light source for the Yokogawa confocal scanner unit in which a microlens array was coupled with a Nipkow disk to focus the collimated incident light onto a pinhole (Saito et al., Cell Struct. Funct., 33: 133-141, 2008). However, transmission efficiency of incident light through the pinhole array was low because off-axis light, the major component of the incident light, was blocked by the non-aperture area of the disk. To improve transmission efficiency, we propose an optical system in which off-axis light is able to be transmitted through pinholes surrounding the pinhole located on the optical axis of the collimator lens. This optical system facilitates the use of not only the on-axis but also the off-axis light such that the available incident light is considerably improved. As a result, we apply the proposed system to high-speed confocal and multicolor imaging both with a satisfactory signal-to-noise ratio.
激光扫描共聚焦显微镜已被用于在亚细胞、细胞和组织水平上三维探索结构。为了获取共聚焦图像，在传统的单点扫描显微镜中通常需要一个相干光源，如激光。照明光束必须聚焦到一个具有衍射极限尺寸的小点上，这决定了显微镜系统的空间分辨率。相比之下，使用尼普科夫圆盘的多点扫描共聚焦显微镜能够使用非相干光源。我们之前证明了成功应用100瓦汞弧灯作为横河共聚焦扫描仪单元的光源，在该单元中，微透镜阵列与尼普科夫圆盘耦合，将准直的入射光聚焦到一个针孔上（斋藤等人，《细胞结构与功能》，33: 133 - 141, 2008）。然而，入射光通过针孔阵列的传输效率较低，因为作为入射光主要成分的离轴光被圆盘的非孔径区域阻挡。为了提高传输效率，我们提出一种光学系统，在该系统中离轴光能够透过围绕位于准直透镜光轴上的针孔的针孔进行传输。这种光学系统不仅便于使用轴上光，也便于使用离轴光，从而使可用的入射光得到显著改善。结果，我们将所提出的系统应用于高速共聚焦和多色成像，两者都具有令人满意的信噪比。
