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Whole-brain functional imaging at cellular resolution using light-sheet microscopy

Nature Methods volume 10, pages 413–420 (2013)Cite this article

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Abstract

Brain function relies on communication between large populations of neurons across multiple brain areas, a full understanding of which would require knowledge of the time-varying activity of all neurons in the central nervous system. Here we use light-sheet microscopy to record activity, reported through the genetically encoded calcium indicator GCaMP5G, from the entire volume of the brain of the larval zebrafish in vivo at 0.8 Hz, capturing more than 80% of all neurons at single-cell resolution. Demonstrating how this technique can be used to reveal functionally defined circuits across the brain, we identify two populations of neurons with correlated activity patterns. One circuit consists of hindbrain neurons functionally coupled to spinal cord neuropil. The other consists of an anatomically symmetric population in the anterior hindbrain, with activity in the left and right halves oscillating in antiphase, on a timescale of 20 s, and coupled to equally slow oscillations in the inferior olive.

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Figure 1: Whole-brain, neuron-level light-sheet imaging in larval zebrafish in vivo.
Figure 2: Whole-brain imaging of neuronal activity with cellular resolution.
Figure 3: Correlations and activity patterns across brain regions.
Figure 4: Single-neuron activity in the left habenula and correlation to hindbrain activity.
Figure 5: Semiautomated analysis procedure for volumetric imaging of non–stimulus locked, long-timescale activity.
Figure 6: Whole-brain, neuron-level identification of two functionally defined circuits.

Change history

  • 05 April 2013

    The authors note that Michael B. Orger, Drew N. Robson and Jennifer M. Li should be added to the author list of the version of this article initially published online. This change affects the author list, affiliations, Acknowledgments and Author Contributions. The error has been corrected for the print, PDF and HTML versions of this article.

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Acknowledgements

We thank M. Coleman (Coleman Technologies) for custom microscope operating software; Janelia Farm Research Campus Instrument Design and Fabrication for custom mechanical parts; Janelia Farm Research Campus Vivarium staff for animal care; C. Riegler (Harvard) for crossing the elavl3:GCaMP5G fish line into the albino background; R. Tomer, L. Lagnado and L. Looger for their contributions to early GCaMP test experiments using light-sheet microscopy; K. Branson, V. Jayaraman, L. Looger, K. Svoboda, F. Amat, W. Lemon and M. Yartsev for helpful discussions and critical reading of the manuscript; A. Schier (under US National Institutes of Health (NIH) grants R01HL109525 and R01GM085357) and F. Engert (under NIH grant DP1NS082121) for supporting the work of D.N.R. and J.M.L.; and A. Kampff (Harvard) for providing M.B.O. with access to a custom two-photon microscope. This work was supported by the Howard Hughes Medical Institute.

Author information

Authors and Affiliations

  1. Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn, Virginia, USA

    Misha B Ahrens & Philipp J Keller

  2. Champalimaud Neuroscience Programme, Champalimaud Centre for the Unknown, Lisbon, Portugal

    Michael B Orger

  3. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA

    Drew N Robson & Jennifer M Li

Authors
  1. Misha B Ahrens
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  2. Michael B Orger
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  3. Drew N Robson
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  4. Jennifer M Li
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Contributions

M.B.A. and P.J.K. conceived of the research, performed the experiments, analyzed the data and wrote the paper. D.N.R., J.M.L. and M.B.O. generated the elavl3:GCaMP5G fish line. M.B.O. acquired the two-photon cell-counting image stack and participated in preliminary experiments.

Corresponding authors

Correspondence to Misha B Ahrens or Philipp J Keller.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5, Supplementary Table 1 and Supplementary Notes 1 and 2 (PDF 5060 kb)

Supplementary Software

Image processing and analysis of whole-brain functional recordings (ZIP 350 kb)

Fast volumetric imaging of the zebrafish brain with light-sheet microscopy

Slicing series of the 800 × 600 × 200 μm3 volume of an elavl3:GCaMP5G-labeled larval zebrafish brain, recorded in 5-μm steps with a 4.25 ± 0.80–μm–thick light sheet (full width at half maximum mean ± s.d. across the brain volume, n = 81). The video playback rate of 30 frames per second is equivalent to the acquisition rate in the microscope. The entire brain volume was recorded at a rate of 0.8 Hz. To reduce the size of this video, we downsampled microscopy images by a factor of 4. Detection objective: Nikon CFI75 LWD 16×/0.80 W. (AVI 4248 kb)

Selected slices from a zebrafish whole-brain light-sheet microscopy recording

Set of 10 out of 41 slices from a volumetric light-sheet recording of an elavl3:GCaMP5G-labeled larval zebrafish brain, showing functional activity recorded at different depths of the 600 × 800 × 200 μm3 volume. The video shows raw microscopy data prior to image registration. To reduce the size of this video, we downsampled microscopy images by a factor of 20. Detection objective: Nikon CFI75 LWD 16×/0.80 W. (AVI 15524 kb)

Whole-brain imaging of neuronal activity (visualization A, slices)

Whole-brain, neuron-level functional activity in a complete set of slices from a volumetric light-sheet recording of an elavl3:GCaMP5G-labeled larval zebrafish brain, superimposed on the reference anatomy (gray). Supplementary Videos 2–6 show different visualizations of the same whole-brain recording. To reduce the size, we downsampled microscopy images by a factor of 64. Detection objective: Nikon CFI75 LWD 16×/0.80 W. (AVI 20114 kb)

Whole-brain imaging of neuronal activity (visualization B, projections)

Dorsal, lateral and frontal maximum-intensity projections of whole-brain, neuron-level functional activity, reported by the genetically encoded calcium indicator GCaMP5G in an elavl3:GCaMP5G fish, superimposed on maximum-intensity projections of the reference anatomy (gray). Supplementary Videos 2–6 show different visualizations of the same whole-brain recording. To reduce the size, we downsampled microscopy images by a factor of 4. Detection objective: Nikon CFI75 LWD 16×/0.80 W. (AVI 20138 kb)

Whole-brain imaging of neuronal activity (visualization C, raw ΔF/F)

Left, dorsal maximum-intensity projections of whole-brain, neuron-level functional activity, reported by the genetically encoded calcium indicator GCaMP5G in an elavl3:GCaMP5G fish, superimposed on maximum-intensity projections of the reference anatomy (gray). Right, functional activity only. Supplementary Videos 2–6 show different visualizations of the same whole-brain recording. To reduce the size, we downsampled microscopy images by a factor of 4. Detection objective: Nikon CFI75 LWD 16×/0.80 W. (AVI 19565 kb)

Neuronal activity in the forebrain

Dorsal maximum-intensity projections of neuron-level functional activity in the forebrain, reported by the genetically encoded calcium indicator GCaMP5G in an elavl3:GCaMP5G fish, superimposed on maximum-intensity projections of the reference anatomy (gray). This video shows full-resolution images for the forebrain region of the whole-brain recording visualized in Supplementary Video 5. Supplementary Videos 2–6 show different visualizations of the same whole-brain recording. Detection objective: Nikon CFI75 LWD 16×/0.80 W. (AVI 22405 kb)

Three-dimensional visualization of the hindbrain oscillator (fish from Fig. 6)

Rotating maximum-intensity projection of the three-dimensional data set underlying Figure 6a. (AVI 5743 kb)

Three-dimensional visualization of hindbrain-spinal circuit (fish from Fig. 6)

Rotating maximum-intensity projection of the three-dimensional data set underlying Figure 6f. (AVI 6142 kb)

Three-dimensional visualization of the hindbrain oscillator (fish from Supplementary Fig. 3e)

Rotating maximum-intensity projection of the three-dimensional data set underlying Supplementary Fig. 3e. (AVI 5796 kb)

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Ahrens, M., Orger, M., Robson, D. et al. Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat Methods 10, 413–420 (2013). https://doi.org/10.1038/nmeth.2434

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