Kawakami Ryosuke, Sawada Kazuaki, Kusama Yuta, Fang Yi-Cheng, Kanazawa Shinya, Kozawa Yuichi, Sato Shunichi, 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 20 Nishi 10, Kita-ku, Sapporo, 001-0020, Japan ; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan ; These authors contributed equally to this work.
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 20 Nishi 10, Kita-ku, Sapporo, 001-0020, Japan ; These authors contributed equally to this work.
Biomed Opt Express. 2015 Feb 20;6(3):891-901. doi: 10.1364/BOE.6.000891. eCollection 2015 Mar 1.
In vivo two-photon microscopy is an advantageous technique for observing the mouse brain at high resolution. In this study, we developed a two-photon microscopy method that uses a 1064-nm gain-switched laser diode-based light source with average power above 4 W, pulse width of 7.5-picosecond, repetition rate of 10-MHz, and a high-sensitivity photomultiplier tube. Using this newly developed two-photon microscope for in vivo imaging, we were able to successfully image hippocampal neurons in the dentate gyrus and obtain panoramic views of CA1 pyramidal neurons and cerebral cortex, regardless of age of the mouse. Fine dendrites in hippocampal CA1 could be imaged with a high peak-signal-to-background ratio that could not be achieved by titanium sapphire laser excitation. Finally, our system achieved multicolor imaging with neurons and blood vessels in the hippocampal region in vivo. These results indicate that our two-photon microscopy system is suitable for investigations of various neural functions, including the morphological changes undergone by neurons during physiological phenomena.
体内双光子显微镜是一种以高分辨率观察小鼠大脑的优势技术。在本研究中，我们开发了一种双光子显微镜方法，该方法使用基于增益开关激光二极管的1064纳米光源，其平均功率超过4瓦，脉冲宽度为7.5皮秒，重复频率为10兆赫兹，以及一个高灵敏度光电倍增管。使用这种新开发的用于体内成像的双光子显微镜，我们能够成功地对齿状回中的海马神经元进行成像，并获得CA1锥体神经元和大脑皮层的全景视图，而不论小鼠的年龄如何。海马CA1区的精细树突能够以钛宝石激光激发无法实现的高峰值信号与背景比进行成像。最后，我们的系统在体内实现了海马区域神经元和血管的多色成像。这些结果表明，我们的双光子显微镜系统适用于各种神经功能的研究，包括神经元在生理现象期间经历的形态变化。
