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Simultaneous multiplane imaging with reverberation two-photon microscopy

Nature Methods volume 17, pages 283–286 (2020)Cite this article

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Abstract

Multiphoton microscopy has gained enormous popularity because of its unique capacity to provide high-resolution images from deep within scattering tissue. Here, we demonstrate video-rate multiplane imaging with two-photon microscopy by performing near-instantaneous axial scanning while maintaining three-dimensional micrometer-scale resolution. Our technique, termed reverberation microscopy, enables the monitoring of neuronal populations over large depth ranges and can be implemented as a simple add-on to a conventional design.

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Fig. 1: Reverberation 2PM design and characteristics.
Fig. 2: Reverberation 2PM imaging of in vivo mouse brain vasculature.
Fig. 3: Reverberation imaging of in vivo mouse brain neural activity.

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Data availability

The data that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request.

Code availability

The code that support the plots within this paper and other findings of this study are available from the corresponding authors upon reasonable request.

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Acknowledgements

We thank H. Li for initial help in the construction of our reverberation microscope, and the Boston University Neurophotonics Center and B. S. Lee for help with animal preparations. This work was supported in part by the Engineering Research Centers Program of the National Science Foundation under NSF cooperative agreement no. EEC-1647837.

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Authors and Affiliations

  1. Department of Electrical Engineering, Boston University, Boston, MA, USA

    Devin R. Beaulieu

  2. Department of Biology, Boston University, Boston, MA, USA

    Ian G. Davison

  3. Photonics Center, Boston University, Boston, MA, USA

    Ian G. Davison, Thomas G. Bifano & Jerome Mertz

  4. Department of Biomedical Engineering, Boston University, Boston, MA, USA

    Kıvılcım Kılıç & Jerome Mertz

Authors
  1. Devin R. Beaulieu
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  3. Kıvılcım Kılıç
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  4. Thomas G. Bifano
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Contributions

J.M. and D.B. conceived the reverberation technique, with T.G.B.'s help. D.B. developed and implemented the prototype microscope. I.D. and K.K. provided the mouse subjects. D.B., I.D. and J.M. analyzed the data. All authors contributed to experiments and the writing of the manuscript.

Corresponding author

Correspondence to Devin R. Beaulieu.

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Competing interests

D.R.B., T.G.B., and J.M. are co-inventors on provisional patent application 62/697,662 submitted by Boston University, that covers ‘Reverberation Microscopy Systems and Methods’. T.G.B. acknowledges a financial interest in Boston Micromachines Corporation (BMC), which manufactures components sometimes used in multiphoton microscopy. However, no BMC products were used in the work described in this paper.

Additional information

Peer review information Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Note 1

Reporting Summary

Supplementary Video 1

Vasculature fly-through. Merged x–y reverberation images from six planes of mouse brain vasculature. Video scans upward through sample.

Supplementary Video 2

Vasculature fly-around. Maximum intensity projection reconstruction of mouse brain vasculature, using a short physical z-scan to fill in the gaps between the six reverberation planes.

Supplementary Video 3

Neocortex; x–y reverberation images of GCaMP6s-expressing neurons from four different planes of neocortex, with the top plane positioned at the brain surface.

Supplementary Video 4

Olfactory bulb; x–y reverberation images of dendrites and somata of GCaMP6-fexpressing neurons imaged in three different planes of main olfactory bulb.

Supplementary Video 5

Olfactory bulb z-stack; z-stack of dendrites and somata of GCaMP6f-expressing neurons imaged in main olfactory bulb. Separate mouse from that in Supplementary Video 4.

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Beaulieu, D.R., Davison, I.G., Kılıç, K. et al. Simultaneous multiplane imaging with reverberation two-photon microscopy. Nat Methods 17, 283–286 (2020). https://doi.org/10.1038/s41592-019-0728-9

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