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In Vivo Imaging of All Cortical Layers and Hippocampal CA1 Pyramidal Cells by Two-Photon Excitation Microscopy

  • Ryosuke Kawakami
  • Tomomi Nemoto
Chapter
Part of the Progress in Optical Science and Photonics book series (POSP, volume 5)

Abstract

Conventional in vivo two-photon microscopy has revealed vital information about neural activity in relation to brain function, despite its limitations in imaging events at depths greater than several hundred micrometers from the surface of the brain. Here, we developed a novel two-photon microscope consisting of a 1064-nm gain-switched laser diode-based light source with average power above 4 W, pulse width of 7.5 ps, repetition rate of 10 MHz, and a high-sensitivity photomultiplier tube for efficient detection of fluorescence. By applying this newly developed two-photon microscope to in vivo imaging, we were able to successfully visualize hippocampal neurons in dentate gyrus and panoramic views of CA1 pyramidal neurons and cerebral cortex, in both young adult and adult mice. Fine structures of dendrites in CA1 neurons could be visualized with a high peak signal-to-background ratio that could not be achieved by titanium sapphire laser excitation. We hope that our two-photon microscopy system will be applicable to investigations of various neural functions, including the morphological changes undergone by neurons during physiological phenomena.

References

  1. 1.
    T. Nemoto, Mol. Cells 26, 113 (2008)Google Scholar
  2. 2.
    W. Denk, J.H. Strickler, W.W. Webb, Science 248, 73 (1990)CrossRefGoogle Scholar
  3. 3.
    M. Göppert-Mayer, Ann. Phys. 401, 273 (1931)CrossRefGoogle Scholar
  4. 4.
    H. Jia, N.L. Rochefort, X. Chen, A. Konnerth, Nat. Protoc. 6, 28 (2011)CrossRefGoogle Scholar
  5. 5.
    H. Jia, Z. Varga, B. Sakmann, A. Konnerth, Proc. Natl. Acad. Sci. U. S. A. 111, 9277 (2014)CrossRefGoogle Scholar
  6. 6.
    H. Inada, M. Watanabe, T. Uchida, H. Ishibashi, H. Wake, T. Nemoto, Y. Yanagawa, A. Fukuda, J. Nabekura, PLoS One 6, (2011)CrossRefGoogle Scholar
  7. 7.
    H. Monai, M. Ohkura, M. Tanaka, Y. Oe, A. Konno, H. Hirai, K. Mikoshiba, S. Itohara, J. Nakai, Y. Iwai, H. Hirase, Nat. Commun. 7, 11100 (2016)CrossRefGoogle Scholar
  8. 8.
    Y. Masamizu, Y.R. Tanaka, Y.H. Tanaka, R. Hira, F. Ohkubo, K. Kitamura, Y. Isomura, T. Okada, M. Matsuzaki, Nat. Neurosci. 17, 987 (2014)CrossRefGoogle Scholar
  9. 9.
    J.T. Trachtenberg, B.E. Chen, G.W. Knott, G. Feng, J.R. Sanes, E. Welker, K. Svoboda, Nature 420, 788 (2002)CrossRefGoogle Scholar
  10. 10.
    D.G. Ouzounov, T. Wang, M. Wang, D.D. Feng, N.G. Horton, J.C. Cruz-Hernández, Y.-T. Cheng, J. Reimer, A.S. Tolias, N. Nishimura, C. Xu, Nat. Methods 14, 388 (2017)CrossRefGoogle Scholar
  11. 11.
    T. Matsui, K. Ohki, Front. Neural Circuits 7, 143 (2013)CrossRefGoogle Scholar
  12. 12.
    F. Helmchen, W. Denk, Nat. Methods 2, 932 (2005)CrossRefGoogle Scholar
  13. 13.
    M. Oheim, E. Beaurepaire, E. Chaigneau, J. Mertz, S. Charpak, J. Neurosci. Methods 111, 29 (2001)CrossRefGoogle Scholar
  14. 14.
    R. Kawakami, K. Sawada, A. Sato, T. Hibi, Y. Kozawa, S. Sato, H. Yokoyama, T. Nemoto, Sci. Rep. 3, 1014 (2013)CrossRefGoogle Scholar
  15. 15.
    R. Kawakami, K. Sawada, Y. Kusama, Y.-C. Fang, S. Kanazawa, Y. Kozawa, S. Sato, H. Yokoyama, T. Nemoto, Biomed. Opt. Express 6, 891 (2015)CrossRefGoogle Scholar
  16. 16.
    W. Yang, R. Yuste, Nat. Methods 14, 349 (2017)CrossRefGoogle Scholar
  17. 17.
    N. Ji, T.R. Sato, E. Betzig, Proc. Natl. Acad. Sci. 109, 22 (2012)CrossRefGoogle Scholar
  18. 18.
    J. Zeng, P. Mahou, M.-C. Schanne-Klein, E. Beaurepaire, D. Débarre, Biomed. Opt. Express 3, 1898 (2012)CrossRefGoogle Scholar
  19. 19.
    N. Ji, J. Freeman, S.L. Smith, Nat. Neurosci. 19, (2016)Google Scholar
  20. 20.
    L. Kong, J. Tang, M. Cui, Opt. Express. 24, 1214 (2016)CrossRefGoogle Scholar
  21. 21.
    K. Charan, D. Heberle, C. Xu, Cleo 2015 AM2 J.7 (2015)Google Scholar
  22. 22.
    D. Kobat, N.G. Horton, C. Xu, J. Biomed. Opt. 16, 106014 (2011)CrossRefGoogle Scholar
  23. 23.
    D.M. Shcherbakova, O.M. Subach, V.V. Verkhusha, Angew. Chemie—Int. Ed. 51, 10724 (2012)CrossRefGoogle Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2019

Authors and Affiliations

  1. 1.Molecular and Cellular Biophysics, Research Institute for Electronic ScienceHokkaido UniversitySapporoJapan

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