Improvement of two-photon microscopic imaging in deep regions of living mouse brains by utilizing a light source based on an electrically controllable gain-switched laser diode

Kazuaki Sawada; Ryosuke Kawakami; Yi-Cheng Fang; Jui-Hung Hung; Yuichi Kozawa; Kohei Otomo; Shunichi Sato; Hiroyuki Yokoyama; Tomomi Nemoto

doi:10.1117/12.2288664

23 February 2018 Improvement of two-photon microscopic imaging in deep regions of living mouse brains by utilizing a light source based on an electrically controllable gain-switched laser diode

Kazuaki Sawada, Ryosuke Kawakami, Yi-Cheng Fang, Jui-Hung Hung, Yuichi Kozawa, Kohei Otomo, Shunichi Sato, Hiroyuki Yokoyama, Tomomi Nemoto

Author Affiliations +

Proceedings Volume 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII; 104982L (2018) https://doi.org/10.1117/12.2288664
Event: SPIE BiOS, 2018, San Francisco, California, United States

Abstract

In vivo two-photon microscopy is an advantageous technique for observing living mouse brains at high spatial resolutions. We previously used a 1064 nm high-power light source based on an electrically controllable gain-switched laser diode (maximum power: 4 W, repetition rate: 10 MHz, pulse width: 7.5 picoseconds) and successfully visualized EYFP expressing neurons at deeper regions in H-line mouse brains under living conditions. However, severe damages were frequently observed when the laser power after the objective lens was over 600 mW, suggesting that a higher average power might not be suitable for visualizing neural structures and functions at deep regions. To increase fluorescent signals as a strategy to avoid such invasions, here, we evaluated the effects of the excitation laser parameters such as the repetition rate (5 - 10 MHz), or the peak power, at the moderate average powers (10 - 500 mW), by taking the advantage that this electrically controllable light source could be used to change the repetition rate independently from the average power or the pulse width. The fluorescent signals of EYFP at layer V of the cerebral cortex were increased by approximately twofold when the repetition rate was decreased from 10 MHz to 5 MHz at the same average power. We also confirmed similar effects in the EYFP solution (335 μM) and fixed brain slices. These results suggest that in vivo two-photon microscopic imaging might be improved by increasing the peak power at the same average power while avoiding the severe damages in living brains.

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Kazuaki Sawada, Ryosuke Kawakami, Yi-Cheng Fang, Jui-Hung Hung, Yuichi Kozawa, Kohei Otomo, Shunichi Sato, Hiroyuki Yokoyama, and Tomomi Nemoto "Improvement of two-photon microscopic imaging in deep regions of living mouse brains by utilizing a light source based on an electrically controllable gain-switched laser diode", Proc. SPIE 10498, Multiphoton Microscopy in the Biomedical Sciences XVIII, 104982L (23 February 2018); https://doi.org/10.1117/12.2288664

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KEYWORDS

Brain

Neuroimaging

Neurons

Two photon excitation microscopy

In vivo imaging

Second-harmonic generation

Two photon imaging

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