In vivo two-photon imaging of mouse hippocampal neurons in dentate gyrus using a light source based on a high-peak power gain-switched laser diode

Ryosuke Kawakami; Kazuaki Sawada; Yuta Kusama; Yi-Cheng Fang; Shinya Kanazawa; Yuichi Kozawa; Shunichi Sato; Hiroyuki Yokoyama; Tomomi Nemoto

doi:10.1364/BOE.6.000891

In vivo two-photon imaging of mouse hippocampal neurons in dentate gyrus using a light source based on a high-peak power gain-switched laser diode

Biomed Opt Express. 2015 Feb 20;6(3):891-901. doi: 10.1364/BOE.6.000891. eCollection 2015 Mar 1.

Authors

Ryosuke Kawakami¹, Kazuaki Sawada², Yuta Kusama³, Yi-Cheng Fang³, Shinya Kanazawa⁴, Yuichi Kozawa⁵, Shunichi Sato⁵, Hiroyuki Yokoyama⁶, Tomomi Nemoto⁷

Affiliations

¹ 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.
² 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.
³ New Industry Creation Hatchery Center (NICHe), Tohoku University, 6-6-10 Aramaki-Aoba, Aoba-ku, Sendai, 980-8579, Japan.
⁴ Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1,Aoba-ku, Sendai, 980-8577, Japan.
⁵ Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1,Aoba-ku, Sendai, 980-8577, Japan ; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan.
⁶ New Industry Creation Hatchery Center (NICHe), Tohoku University, 6-6-10 Aramaki-Aoba, Aoba-ku, Sendai, 980-8579, Japan ; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency (JST), Tokyo, Japan.
⁷ 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.

Abstract

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.

Keywords: (110.6880) Three-dimensional image acquisition; (180.0180) Microscopy; (180.2520) Fluorescence microscopy; (180.4315) Nonlinear microscopy.