To the Editor:
The processing of sensory input and the generation of behavior involves large networks of neurons1,2, which necessitates new technology3,4,5,6,7 for recording from many neurons in behaving animals. In the larval zebrafish, light-sheet microscopy can be used to record the activity of almost all neurons in the brain simultaneously at single-cell resolution3,4. Existing implementations, however, cannot be combined with visually driven behavior because the light sheet scans over the eye, interfering with presentation of controlled visual stimuli. Here we describe a system that overcomes the confounding eye stimulation through the use of two light sheets and combines whole-brain light-sheet imaging3 with virtual reality for fictively behaving1 larval zebrafish.
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References
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Acknowledgements
We thank M. Coleman for writing the light-sheet microscope control software Zebrascope and for continuing support; B. Coop and T. Tabachnik for their help with hardware design; S. Narayan for help with experiments; ID&F engineers for providing help on hardware components; J. Cox, R. Larson, J. Barber, B. Brandenburg and other vivarium staff for fish husbandry; G. Ceric, V. Samalam, K. Carlisle and R. Lines for assistance with the high-performance computer cluster; D.G.C. Hildebrand and M. Koyama for discussions; and V. Jayaraman, M. Reiser and G. Murphy for comments on the manuscript. This work was supported by the Howard Hughes Medical Institute.
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Authors and Affiliations
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA
Nikita Vladimirov,Yu Mu,Takashi Kawashima,Davis V Bennett,Chao-Tsung Yang,Loren L Looger,Philipp J Keller,Jeremy Freeman&Misha B Ahrens
Authors
- Nikita Vladimirov
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- Yu Mu
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- Takashi Kawashima
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- Davis V Bennett
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- Chao-Tsung Yang
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- Loren L Looger
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- Philipp J Keller
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- Jeremy Freeman
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- Misha B Ahrens
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Correspondence to Misha B Ahrens.
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Integrated supplementary information
Supplementary Figure 1 The light-sheet microscope with behavior setup.
Lasers, control and data acquisition hardware are not shown.
Supplementary Figure 2 Neuronal activity reported by nuclear localized GCaMP6s.
(a) Locations of four example neurons in a 6 dpf Tg(elavl3:GCaMP6s) zebrafish, scanned with a light sheet microscope. (b) Calcium signals of the four neurons indicated in a during optomotor behavior. Traces 1,2 are triggered on stimulus onset; traces 3,4 on the onset of fictive swimming following stimulus onset. (c) Locations of four example neurons in a 6 dpf Tg(elavl3:H2B-GCaMP6s) zebrafish. (d) Calcium signals of these four neurons during the optomotor assay.
Supplementary Figure 3 Assessment of physical coverage and spatial resolution in Tg(elavl3:H2B-GCaMP6s) fish (nuclear-localized expression).
(a) Images of forebrain and midbrain acquired using only the lateral light sheet (215 micrometers from the top of the brain). (b) Same area, with both lateral and frontal light sheets. (Different fish from c,d.) (c) Example area between the eyes with mostly single-cell resolution (190 micrometers from the top), imaged with both light sheets. (d) Example area between the eyes including areas that lack single-cell resolution (175 micrometers from the top), imaged with both light sheets. Scale bar, 100 micrometers. Images have been normalized to the local brightness (where local brightness is computed by smoothing the raw image by a 2D Gaussian kernel with σ = 5 micrometers). See also Supplementary Movie 2 for the full volume.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–3, Supplementary Table 1 and Supplementary Note (PDF 5283 kb)
Supplementary Video 1
Whole-brain imaging during the optomotor response, top projection. (MOV 11654 kb)
Supplementary Video 2
Volumetric stack of an elavl3:H2B-GCaMP6s fish of 6 d.p.f. taken with the light-sheet microscope. (MOV 2295 kb)
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Vladimirov, N., Mu, Y., Kawashima, T. et al. Light-sheet functional imaging in fictively behaving zebrafish. Nat Methods 11, 883–884 (2014). https://doi.org/10.1038/nmeth.3040
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DOI: https://doi.org/10.1038/nmeth.3040
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