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

Research output: Chapter in Book/Report/Conference proceedingConference contribution

1 Citation (Scopus)

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.

Original languageEnglish
Title of host publicationMultiphoton Microscopy in the Biomedical Sciences XVIII
EditorsAmmasi Periasamy, Xiaoliang S. Xie, Xiaoliang S. Xie, Karsten Konig, Peter T. C. So
PublisherSPIE
ISBN (Electronic)9781510614819
DOIs
Publication statusPublished - 2018 Jan 1
EventMultiphoton Microscopy in the Biomedical Sciences XVIII 2018 - San Francisco, United States
Duration: 2018 Jan 282018 Jan 30

Publication series

NameProgress in Biomedical Optics and Imaging - Proceedings of SPIE
Volume10498
ISSN (Print)1605-7422

Other

OtherMultiphoton Microscopy in the Biomedical Sciences XVIII 2018
CountryUnited States
CitySan Francisco
Period18/1/2818/1/30

Keywords

  • in vivo imaging
  • living mouse brain
  • peak power
  • repetition rate
  • two-photon microscopy

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Biomaterials
  • Atomic and Molecular Physics, and Optics
  • Radiology Nuclear Medicine and imaging

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  • Cite this

    Sawada, K., Kawakami, R., Fang, Y. C., Hung, J. H., Kozawa, Y., Otomo, K., Sato, S., Yokoyama, H., & Nemoto, T. (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. In A. Periasamy, X. S. Xie, X. S. Xie, K. Konig, & P. T. C. So (Eds.), Multiphoton Microscopy in the Biomedical Sciences XVIII [104982L] (Progress in Biomedical Optics and Imaging - Proceedings of SPIE; Vol. 10498). SPIE. https://doi.org/10.1117/12.2288664