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Abstract

Developments in miniaturized microscopes have enabled visualization of brain activities and structural dynamics in animals engaging in self-determined behaviors. However, it remains a challenge to resolve activity at single dendritic spines in freely behaving animals. Here, we report the design and application of a fast high-resolution, miniaturized two-photon microscope (FHIRM-TPM) that accomplishes this goal. With a headpiece weighing 2.15 g and a hollow-core photonic crystal fiber delivering 920-nm femtosecond laser pulses, the FHIRM-TPM is capable of imaging commonly used biosensors (GFP and GCaMP6) at high spatiotemporal resolution (0.64 μm laterally and 3.35 μm axially, 40 Hz at 256 × 256 pixels for raster scanning and 10,000 Hz for free-line scanning). We demonstrate the microscope's robustness with hour-long recordings of neuronal activities at the level of spines in mice experiencing vigorous body movements.

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Change history

  • 20 June 2017

    In the version of this article initially published online, the affiliation for Aimin Wang was incorrectly listed as Department of Neurobiology, Institute of Basic Medical Sciences, Beijing, China. The correct affiliation is State Key Laboratory of Advanced Optical Communication System and Networks, School of Electronics Engineering and Computer Science, Peking University, Beijing, China. The error has been corrected in the print, PDF and HTML versions of this article.

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Acknowledgements

We thank X. Li, D. Li, X. Chen, Z. Zhang, W. Gan, Y. Sun, S. Wang, and X. Chen for comments on the optics and biological experiments, and I.C. Bruce for manuscript editing. We also thank Mirrorcle Technologies for assistance with MEMS design and fabrication. The work was supported by grants from the National Natural Science Foundation of China (31327901, 31521062, 3142800018, and 31570839), the Major State Basic Research Program of China (2013CB531200 and 2012CB518200), and the National Science and Technology Major Project Program (2016YFA0500400).

Author information

Author notes

    • Weijian Zong
    • , Runlong Wu
    •  & Mingli Li

    These authors contributed equally to this work.

Affiliations

  1. State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, Beijing Key Laboratory of Cardiometabolic Molecular Medicine, Peking University, Beijing, China.

    • Weijian Zong
    • , Runlong Wu
    • , Mingli Li
    • , Yanhui Hu
    • , Yijun Li
    • , Jinghang Li
    • , Hao Rong
    • , Yangyang Xu
    • , Yang Lu
    • , Hongbo Jia
    • , Zhuan Zhou
    • , Liangyi Chen
    •  & Heping Cheng
  2. Department of Cognitive Sciences, Institute of Basic Medical Sciences, Beijing, China.

    • Weijian Zong
    •  & Ming Fan
  3. Department of Neurobiology, Institute of Basic Medical Sciences, Beijing, China.

    • Haitao Wu
  4. Brain Research Instrument Innovation Center, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, China.

    • Hongbo Jia
  5. State Key Laboratory of Advanced Optical Communication System and Networks, School of Electronics Engineering and Computer Science, Peking University, Beijing, China.

    • Yunfeng Zhang
    •  & Aimin Wang

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Contributions

H.C., W.Z., and L.C. conceived the project; H.C. and L.C. supervised the research; W.Z. designed and built the system; W.Z., R.W., and M.L. performed the experiments; R.W. wrote the control software, and Y.H. made electronic circuits under the supervision of Y.Z.; A.W. and Y. Li oversaw fiber optics and assisted with mechanical assembly; H.R., Y.X., and J.L. performed the mechanical fabrication; H.W. and M.F. supplied the indicator mice; M.L. prepared the biological samples under the supervision of Z.Z.; W.Z., M.L., Y. Lu, and J.L. analyzed data; H.C., W.Z., L.C., and H.J. wrote the paper. All authors participated in discussions and data interpretation.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Yunfeng Zhang or Aimin Wang or Liangyi Chen.

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DOI

https://doi.org/10.1038/nmeth.4305

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