Otomo Kohei, Hibi Terumasa, Fang Yi-Cheng, Hung Jui-Hung, Tsutsumi Motosuke, Kawakami Ryosuke, Yokoyama Hiroyuki, Nemoto Tomomi
Research Institute for Electronic Science, Hokkaido University, Kita 20 Nishi 10, Kita-ku, Sapporo 001-0020, Japan.
Graduate School of Information Science and Technology, Hokkaido University, Kita 14 Nishi 9, Kita-ku, Sapporo 060-0814, Japan.
Biomed Opt Express. 2018 May 15;9(6):2671-2680. doi: 10.1364/BOE.9.002671. eCollection 2018 Jun 1.
We developed a compact stimulated emission depletion (STED) two-photon excitation microscopy that utilized electrically controllable components. Transmissive liquid crystal devices inserted directly in front of the objective lens converted the STED light into an optical vortex while leaving the excitation light unaffected. Light pulses of two different colors, 1.06 and 0.64 μm, were generated by laser diode-based light sources, and the delay between the two pulses was flexibly controlled so as to maximize the fluorescence suppression ratio. In our experiments, the spatial resolution of this system was up to three times higher than that obtained without STED light irradiation, and we successfully visualize the fine microtubule network structures in fixed mammalian cells without causing significant photo-damage.
我们开发了一种紧凑的受激发射损耗（STED）双光子激发显微镜，它使用了电控组件。直接插入物镜前方的透射式液晶器件将STED光转换为光学涡旋，同时不影响激发光。基于激光二极管的光源产生了两种不同颜色（1.06和0.64μm）的光脉冲，并且灵活控制两个脉冲之间的延迟，以最大化荧光抑制率。在我们的实验中，该系统的空间分辨率比无STED光照射时提高了高达三倍，并且我们成功地在固定的哺乳动物细胞中可视化了精细的微管网络结构，而不会造成明显的光损伤。
