Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy

Abstract

High-speed, large-scale three-dimensional (3D) imaging of neuronal activity poses a major challenge in neuroscience. Here we demonstrate simultaneous functional imaging of neuronal activity at single-neuron resolution in an entire Caenorhabditis elegans and in larval zebrafish brain. Our technique captures the dynamics of spiking neurons in volumes of 700 μm × 700 μm × 200 μm at 20 Hz. Its simplicity makes it an attractive tool for high-speed volumetric calcium imaging.

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Figure 1: Light-field deconvolution microscopy.
Figure 2: Whole-animal Ca2+ imaging of C. elegans using LFDM.
Figure 3: Whole-brain Ca2+ imaging of larval zebrafish in vivo.

References

  1. 1

    Alivisatos, A.P. et al. Neuron 74, 970–974 (2012).

    CAS  Article  Google Scholar 

  2. 2

    Marblestone, A.H. et al. Front. Comput. Neurosci. 7, 137 (2013).

    Article  Google Scholar 

  3. 3

    Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).

    CAS  Article  Google Scholar 

  4. 4

    Chen, T.-W. et al. Nature 499, 295–300 (2013).

    CAS  Article  Google Scholar 

  5. 5

    Schrödel, T., Prevedel, R., Aumayr, K., Zimmer, M. & Vaziri, A. Nat. Methods 10, 1013–1020 (2013).

    Article  Google Scholar 

  6. 6

    Ahrens, M.B., Orger, M.B., Robson, D.N., Li, J.M. & Keller, P.J. Nat. Methods 10, 413–420 (2013).

    CAS  Article  Google Scholar 

  7. 7

    Panier, T. et al. Front. Neural Circuits 7, 65 (2013).

    Article  Google Scholar 

  8. 8

    Duemani Reddy, G., Kelleher, K., Fink, R. & Saggau, P. Nat. Neurosci. 11, 713–720 (2008).

    CAS  Article  Google Scholar 

  9. 9

    Grewe, B.F., Langer, D., Kasper, H., Kampa, B.M. & Helmchen, F. Nat. Methods 7, 399–405 (2010).

    CAS  Article  Google Scholar 

  10. 10

    Cheng, A., Gonçalves, J.T., Golshani, P., Arisaka, K. & Portera-Cailliau, C. Nat. Methods 8, 139–142 (2011).

    CAS  Article  Google Scholar 

  11. 11

    Abrahamsson, S. et al. Nat. Methods 10, 60–63 (2013).

    CAS  Article  Google Scholar 

  12. 12

    Levoy, M., Ng, R., Adams, A., Footer, M. & Horowitz, M. ACM Trans. Graph. 25, 924–934 (2006).

    Article  Google Scholar 

  13. 13

    Levoy, M., Zhang, Z. & McDowall, I. J. Microsc. 235, 144–162 (2009).

    CAS  Article  Google Scholar 

  14. 14

    Quirin, S., Peterka, D.S. & Yuste, R. Opt. Express 21, 16007–16021 (2013).

    Article  Google Scholar 

  15. 15

    Agard, D.A. Annu. Rev. Biophys. Bioeng. 13, 191–219 (1984).

    CAS  Article  Google Scholar 

  16. 16

    Broxton, M. et al. Opt. Express 21, 25418–25439 (2013).

    Article  Google Scholar 

  17. 17

    Faumont, S. et al. PLoS ONE 6, e24666 (2011).

    CAS  Article  Google Scholar 

  18. 18

    Kawano, T. et al. Neuron 72, 572–586 (2011).

    CAS  Article  Google Scholar 

  19. 19

    Chalfie, M. et al. J. Neurosci. 5, 956–964 (1985).

    CAS  Article  Google Scholar 

  20. 20

    Mukamel, E.A., Nimmerjahn, A. & Schnitzer, M.J. Neuron 63, 747–760 (2009).

    CAS  Article  Google Scholar 

  21. 21

    Friedrich, R.W. & Korsching, S.I. Neuron 18, 737–752 (1997).

    CAS  Article  Google Scholar 

  22. 22

    Renninger, S.L. & Orger, M.B. Methods 62, 255–267 (2013).

    CAS  Article  Google Scholar 

  23. 23

    Jetti, S.K., Vendrell-Llopis, N. & Yaksi, E. Curr. Biol. 24, 434–439 (2014).

    CAS  Article  Google Scholar 

  24. 24

    Akerboom, J. et al. Front. Mol. Neurosci. 6, 2 (2013).

    CAS  Article  Google Scholar 

  25. 25

    Ntziachristos, V. Nat. Methods 7, 603–614 (2010).

    CAS  Article  Google Scholar 

  26. 26

    Cold Spring Harbor Laboratory. Hermaphrodite Handbook. WormAtlas (ed. Herndon, L.A.) http://www.wormatlas.org/hermaphrodite/hermaphroditehomepage.htm (2014; accessed 20 March 2014).

  27. 27

    Kak, A.C. & Slaney, M. Principles of Computerized Tomographic Imaging (Society of Industrial and Applied Mathematics, 2001).

  28. 28

    Gu, M. Advanced Optical Imaging Theory (Springer, 1999).

Download references

Acknowledgements

We thank T. Müller, P. Pasierbek, P. Forai, H. Kaplan, M. Molodtsov, K. Tessmar-Raible, F. Schlumm and Olympus Inc. for technical support and loan of equipment, as well as H. Baier (Max Planck Institute of Neurobiology) and M. Orger (Champalimaud) for sharing zebrafish lines. We thank L. Page for providing early funding for the project and D. Dalrymple for helping catalyze connections. The computational results presented have been achieved in part using the Vienna Scientific Cluster (VSC). This work was supported by the VIPS Program of the Austrian Federal Ministry of Science and Research and the City of Vienna as well as the European Commission (Marie Curie, FP7-PEOPLE-2011-IIF) (R.P.); a Samsung Scholarship (Y.-G.Y.); a US National Science Foundation (NSF) Graduate Fellowship (N.P.); the Allen Institute for Brain Science, the MIT Media Lab, the MIT McGovern Institute, US National Institutes of Health (NIH) 1R01EY023173, the MIT Synthetic Intelligence Project, the Institution of Engineering and Technology (IET) Harvey Prize, NSF CBET 1053233, the New York Stem Cell Foundation–Robertson Award, NSF CBET 1344219, NIH 1DP1NS087724, Google, the NSF Center for Brains, Minds and Machines at MIT, and Jeremy and Joyce Wertheimer (E.S.B.); the Vienna Science and Technology Fund (WWTF) project VRG10-11, Human Frontiers Science Program Project RGP0041/2012, Research Platform Quantum Phenomena and Nanoscale Biological Systems (QuNaBioS) (A.V.); and the European Community's Seventh Framework Programme/ERC no. 281869 (M.Z. and T.S.). The Institute of Molecular Pathology is funded by Boehringer Ingelheim.

Author information

Affiliations

Authors

Contributions

R.P. designed microlenses, built the imaging system and performed experiments together with M.H. Y.-G.Y. designed and wrote 3D-deconvolution software with contributions from G.W. under the guidance of R.R. R.P. and M.H. refined and rebuilt the imaging system and analyzed data together with Y.-G.Y. N.P. implemented and tested the LFDM prototype. T.S. generated transgenic animals, provided microfluidic devices and performed cell identifications under the guidance of M.Z. S.K. wrote analysis software. E.S.B. and A.V. conceived of and led project. R.P., Y.-G.Y. and A.V. wrote the manuscript, with input from all authors.

Corresponding authors

Correspondence to Edward S Boyden or Alipasha Vaziri.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4 and Supplementary Notes 1 and 2 (PDF 6322 kb)

Supplementary Software

Software for 3D volume reconstruction from light-field images The software for 3D volume reconstruction from light-field images is available as Supplementary Software. It is written in MATLAB and requires a workstation with MATLAB2013a or later version and at least 32GB of RAM. A user manual for the software (LFM_Software_readme.pdf) is included in the .zip file. (ZIP 7047 kb)

Whole animal Ca2+-imaging of C. elegans

Maximum intensity projection of 14 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 seconds of recording of basal activity at 5 Hz volume rate (1000 frames in total). Video frame rate is 100 frames per second, which equates to 20 seconds in real time in the video (i.e. playback speed 20x). See also Fig. 2a-c. (AVI 7320 kb)

Whole animal Ca2+-imaging of C. elegans

Maximum intensity projection of 14 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 seconds of recording of basal activity at 5 Hz volume rate. Video frame rate is 100 frames per second, which equates to 20 seconds in real time in the video (i.e. playback speed 20x). See also Fig. 2d. (AVI 9412 kb)

High-speed Ca2+-imaging of unrestrained C. elegans at 50 Hz

Maximum intensity projection of 12 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 15 seconds of recording of basal activity of freely-moving worms at 50 Hz volume rate. Exposure time for individual volumes was 20 ms. Video frame rate is 50 frames per second, which equates to 50 volumes per second in the video (i.e. playback speed 1x – real time). (AVI 9533 kb)

Brain-wide Ca2+-imaging of C. elegans

Maximum intensity projection of 15 z-planes at 2 μm distance of a Punc-31::NLS-GCaMP5K worm. Shown are 200 seconds of recording of basal activity in the head ganglia at 5 Hz volume rate. Video frame rate is 100 frames per second, which equates to 20 seconds in real time in the video (i.e. playback speed 20x). See also Fig. 2e-f. (AVI 9673 kb)

Brain-wide Ca2+-imaging of C. elegans during chemosensory stimulation

Maximum intensity projection of 7 z-planes at 2 μm distance of a of a Punc-31::NLS-GCaMP5K worm during chemosensory stimulation. Shown are 240 seconds of recording of basal activity at 5 Hz volume rate. Video frame rate is 50 frames per second, which equates to 24 seconds in real time in the video (i.e. playback speed 10x). Also see Supplementary Fig. 3. (AVI 11061 kb)

Brain-wide Ca2+-imaging of zebrafish larvae

Maximum intensity projection of 51 z-planes at 4 μm distance of a 5 dpf - HuC:GCaMP5G zebrafish larvae. Shown are 240 seconds of recording of activity at 20 Hz volume rate. Playback speed of video is 10x. At 15 sec (1.5 sec in the video), decomposed fish water is supplied manually to the fish chamber in order to evoke activity in the olfactory system. Also see Fig. 3 in the main manuscript. (MP4 3748 kb)

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Prevedel, R., Yoon, YG., Hoffmann, M. et al. Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy. Nat Methods 11, 727–730 (2014). https://doi.org/10.1038/nmeth.2964

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