In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain (original) (raw)

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Acknowledgements

We thank members of the Xu and Schaffer/Nishimura research groups for their help and D. Dombeck for discussion. We thank S. Hu for help with histology, the University Institute for Biotechnology, Cornell University for help with Zeiss 780 confocal microscope for imaging fixed brain slices. We thank N. Ji for help with animal preparation methods. The project was supported by DARPA W911NF-14-1-0012 to C.X., NIH/NIBIB R01EB014873 to C.X., and NIH/NINDS U01NS090530 to C.X. It was also supported by the Intelligence Advanced Research Projects Activity (IARPA) via Department of Interior/Interior Business Center (DoI/IBC) contract number D16PC00003 to A.T. and C.X. The US Government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright annotation thereon. Disclaimer: the views and conclusions contained herein are those of the authors and should not be interpreted as necessarily representing the official policies or endorsements, either expressed or implied, of IARPA, DoI/IBC, or the US Government.

Author information

Author notes

  1. Dimitre G Ouzounov and Tianyu Wang: These authors contributed equally to this work.

Authors and Affiliations

  1. School of Applied and Engineering Physics, Cornell University, Ithaca, New York, USA
    Dimitre G Ouzounov, Tianyu Wang, Mengran Wang, Nicholas G Horton & Chris Xu
  2. Nancy E. and Peter C. Meining School of Biomedical Engineering, Cornell University, Ithaca, New York, USA
    Danielle D Feng, Jean C Cruz-Hernández, Yu-Ting Cheng & Nozomi Nishimura
  3. Department of Neuroscience, Center for Neuroscience and Artificial Intelligence, Baylor College of Medicine, Houston, Texas, USA
    Jacob Reimer & Andreas S Tolias
  4. Department of Electrical and Computer Engineering, Rice University, Houston, Texas, USA
    Andreas S Tolias

Authors

  1. Dimitre G Ouzounov
  2. Tianyu Wang
  3. Mengran Wang
  4. Danielle D Feng
  5. Nicholas G Horton
  6. Jean C Cruz-Hernández
  7. Yu-Ting Cheng
  8. Jacob Reimer
  9. Andreas S Tolias
  10. Nozomi Nishimura
  11. Chris Xu

Contributions

C.X. conceived the study. D.G.O., T.W., M.W., D.D.F., N.G.H. J.C.C.-H., and Y.-T.C. performed the experiments. T.W., D.G.O., and J.R. analyzed the data. C.X. and N.N. supervised the project. J.R. and A.S.T. provided transgenic mice for this study. D.G.O., T.W., and C.X. prepared the manuscript.

Corresponding authors

Correspondence toDimitre G Ouzounov or Chris Xu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Experimental setup

a, Schematic of the excitation sources and the basic optical path of the imaging setup.

b, Measured NOPA output spectrum.

c, Measured interferometric second-order autocorrelation trace of the pulse at the objective focus, with dispersion pre-compensation.

d, Time division multiplexing (TDM) for simultaneous 2PM and 3PM.

M – mirror, HWP – half-wave plate, PBS – polarizing beamsplitter cube, BS – beam stop, DM – dichroic mirror, PMT – photomultiplier tube, S – sampler, NOPA – non-collinear optical amplifier, PD – photo-diode

Supplementary Figure 2 3PM imaging of spontaneous activity in GCaMP6s-labeled neurons in the SP layer of the CA1 region of the mouse hippocampus.

a, 3PM image of neuronal population in the CA1 region of the hippocampus located at 984 μm beneath the dura (left). Neurons are indexed (right) for reference to traces in (b), Supplementary Figure 3 and 4. Average power of 50 mW at 800 kHz repetition rate was used for imaging. The field-of-view (FOV) was 200x200 μm. Scale bar, 20 μm.

b, Spontaneous activity traces recorded from neuron 1-5 in (a) during approximately the first 16 minutes of a 48-minute recording session, at a frame rate of 8.49 Hz. The five neurons represent a range of activity level and brightness. All traces were low-pass filtered with a hamming window with a 0.59 s time constant, and fluorescence intensity was converted to photon counts per neuron per second.

Supplementary Figure 3 Spontaneous activity traces of the hippocampal neurons shown in Supplementary Figure 2

Spontaneous activity traces recorded from all indexed neurons in Supplementary Figure 2 during approximately the first 16 minutes of a 48-minute recording session, at a frame rate of 8.49 Hz. To the left of each trace is the index of the neuron. All traces were low-pass filtered with a hamming window with a 0.59 s time constant, and then normalized to each individual baseline.

Supplementary Figure 4 Continued recording of spontaneous activity traces of the hippocampal neurons after Supplementary Figure 3

Spontaneous activity traces recorded from the same neurons in Supplementary Figure 3 during approximately 16 minutes after 32 minutes of continuous recording (i.e., approximately the last 16 minutes of the 48-minute recording session starting in Supplementary Figure 3). All traces were processed in the same way as in (c).

Supplementary Figure 5 Revisit of the same population of neurons in the SP layer of the CA1 region of the hippocampus after one week.

a, Activity recording site in the SP layer of the hippocampus located at 984 μm beneath the dura (left). The neuronal population was the same as that shown in Figure 2(c) and Supplementary Figure 2, but imaged one week later. Neurons are indexed (right) for reference to their traces in (b) and Supplementary Figure 6. Average power of 58 mW at 800 kHz was used for imaging. The FOV was 150x150 μm. Scale bar 20 μm.

b, Spontaneous activity traces recorded from three indexed neurons in (a) for 10 minutes, at a frame rate of 8.49 Hz. The three neurons represent a range of activity level and brightness. To the left of each trace is the index of the neuron. All traces were low-pass filtered with a hamming window with a 0.59 s time constant, and fluorescent intensity was converted to photon counts per neuron per second.

Supplementary Figure 6 Spontaneous activity traces of the hippocampal neurons shown in Supplementary Figure 5

Spontaneous activity traces recorded from all indexed neurons in Supplementary Figure 5 for 10 minutes, at a frame rate of 8.49 Hz. All traces were low-pass filtered with a hamming window with a 0.59 s time constant, and then normalized to each individual baseline.

Supplementary Figure 7 Histological images of coronal mouse brain sections of the GCaMP6s-labeled site imaged by 3PM in this study

a, Confocal microscopy image of post mortem fixed brain tissue. Scale bar, 1 mm.

b, High resolution ex vivo confocal image from the CA1 region of the hippocampus. Scale bar, 20 μm.

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Ouzounov, D., Wang, T., Wang, M. et al. In vivo three-photon imaging of activity of GCaMP6-labeled neurons deep in intact mouse brain.Nat Methods 14, 388–390 (2017). https://doi.org/10.1038/nmeth.4183

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