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Whole-brain functional imaging with two-photon light-sheet microscopy

Nature Methods volume 12, pages 379–380 (2015)Cite this article

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Several studies recently demonstrated that one-photon (1P) light-sheet imaging gives access to the spontaneous activity of a large fraction of the zebrafish larval brain at nearly single-cell resolution1,2,3. This imaging method, however, requires an intense and extended illumination at a wavelength (λ = 488 nm) that lies within the most sensitive region of the fish visible spectrum4 and therefore likely stimulates the blue photoreceptors in the retina as well as other photosensitive cells5. As an alternative, we report on brain-wide three-dimensional (3D) neural recordings during visuomotor integration in zebrafish larvae using two-photon (2P) light-sheet imaging6 at a wavelength of 930 nm combined with visual stimulation.

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Figure 1: Two-photon (2P) light-sheet functional imaging of visually evoked neural activity.

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Acknowledgements

We thank F. Engert (Harvard University) for providing the Huc:GCaMP5G strain. The study was partly supported by Agence Nationale de la Recherche (contracts ANR-2010-JCJC-1510-01, ANR-11-EQPX-0029, ANR-10-INBS-04), Fondation Louis D. de l'Institut de France, European Union Seventh Framework Program (Marie Curie International Reintegration Grant no. 268379).

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Authors and Affiliations

  1. Sorbonne Universités, Université Pierre et Marie Curie Paris 06, Paris, France

    Sébastien Wolf, Georges Debrégeas, Sergei G Kruglik & Raphaël Candelier

  2. Centre National de la Recherche Scientifique (Unité Mixte de Recherche 8237), Laboratoire Jean Perrin, Paris, France

    Sébastien Wolf, Georges Debrégeas, Sergei G Kruglik & Raphaël Candelier

  3. Laboratoire Optique et Biosciences, Ecole Polytechnique, Centre National de la Recherche Scientifique (Unité Mixte de Recherche 7645) et Institut National de la Santé et de la Recherche Médicale (U1182), Palaiseau, France

    Willy Supatto, Pierre Mahou, Jean-Marc Sintes & Emmanuel Beaurepaire

Authors
  1. Sébastien Wolf
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  2. Willy Supatto
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  3. Georges Debrégeas
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  4. Pierre Mahou
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  5. Sergei G Kruglik
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  6. Jean-Marc Sintes
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  7. Emmanuel Beaurepaire
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  8. Raphaël Candelier
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Correspondence to Georges Debrégeas or Raphaël Candelier.

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Integrated supplementary information

Supplementary Figure 1 Scheme of the 1P/2P light-sheet microscopy setups.

The optical components are listed in Supplementary Table 1.

Supplementary Figure 2 Two-photon light-sheet microscope with visual stimulation.

(a,b) Scheme of the two-photon light-sheet microscope with visual stimulation used to perform cluster analysis of dark- and bright-flash induced responses. HWP: half wave plate; PBS: polarization beam splitter; EMCCD: electron-multiplying charge-coupled device camera. (c) 3D-rendering of the liquid-filled specimen chamber with objective lenses on the lateral sides, sample held from the top and transparent screen for visual stimulation at the bottom. (d) Blow-up of the specimen chamber.

Supplementary Figure 3 Axial resolution.

(a) Vertical cross-section of an agarose gel cylinder containing 100 nm in diameter fluorescent beads. The image was reconstructed from a series of horizontal sections, separated by 0.2 μm intervals, imaged using the 2P light-sheet setup. (b) Axial profile of the fluorescence intensity of the bead identified in (a) by the green rectangle. The solid line shows the best gaussian fit to the profile. (c) Full width at half maximum (FWHM) of the measured fluorescence profile, for 1P and 2P light-sheet imaging, as a function of the bead position along the illumination axis. The profiles are fitted with equations (1) and (2). The FWHM in the region delimited by the two vertical lines falls below the typical inter-soma distance in zebrafish larvae (≈6.5 μm). In this region, the optical sectioning is thus compatible with single-cell resolution.

Supplementary Figure 4 Automatic image segmentation.

(a) Charactersitic brain-wide section of the larva's brain obtained using 2P light-sheet imaging. (b) Blowups of different regions. From left to right: telencephalon, optic tectum, hindbrain. (c) Result of the automatic segmentation procedure leading to the identification of ROIs associated with individual somas.

Supplementary Figure 5 Comparison between flash-evoked response under 1P vs. 2P imaging (1P illumination power, 500 μW).

(a-e) Post-stimulus response averaged over the 5 most responsive subregions contoured in Fig. 1, for 6 increasing stimulus intensity. The graphs show that for all tested stimulus intensities, the response is either abolished or greatly reduced in 1P imaging, with respect to 2P imaging.

Supplementary Figure 6 Comparison between flash-evoked response under 1P vs. 2P imaging at low 1P illumination power (100 μW).

(a-b) Stimulus-averaged response, measured 250 ms after the flash in 1P and 2P imaging, respectively (ΔF/F in color code). (c-d) Post-stimulus response averaged over the 5 subpopulations contoured in (a-b) (most responsive regions), for the lowest (c) and highest (d) stimulus intensity. (e) Evolution of the maximum response for each contoured region as a function of the stimulus intensity. Even at this low illumination power, 1P light-sheet imaging still induces a severe reduction of visual sensitivity.

Supplementary Figure 7 3D imaging.

Nine layers were acquired at 1 Hz across a 5 day old larva brain (90 ms exposure per layer). On the sections shown, the average neural response (ΔF/F), measured in the first second following a 3,600 μW.cm−2 flash, is color coded (120 flashes, 10 s intervals between flashes).

Supplementary Figure 8 Cluster analysis performed on a 1,050-s-long, 100-bright-flashes recording.

(a-c) The k-means algorithm is computed for different k-values on the Pearson matrix that conveys neuron-neuron correlations. The clusters appear as square blocks in the re-ordered Pearson matrix. (d-f) The spatial structure of the different clusters is shown: each color corresponds to one cluster. These maps reveal the functional organization of the brain at different scales.

Supplementary Figure 9 Bright- vs. dark flash-induced response.

Using the k-means algorithm (k = 12), we identified the two most prominent neuronal clusters whose topography is shown in Fig. 1g-h. Across-stimuli responses of the (a) visual α and (b) hindbrain β clusters for 200 ms-long bright flashes. The blue curve is the cluster-averaged response. Characteristic traces of individual neurons from the clusters are shown in grey. (c-d) Similar analysis performed for 1 s-long dark flashes on a different larva. The inset in (c) reveals that the visual cluster shows a positive transient response to both the onset and offset of the stimulus. In all graphs, the red rectangle indicates the stimulation period.

Supplementary information

Supplementary Text

Supplementary Figures 1–9, Supplementary Table 1 and Supplementary Methods (PDF 17770 kb)

Supplementary Video 1

2P light-sheet imaging of neural activity in the zebrafish larval brain (MP4 3386 kb)

Supplementary Video 2

Whole-brain functional imaging (MP4 6186 kb)

Supplementary Video 3

Comparison between 1P and 2P functional brain imaging (MP4 4580 kb)

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Wolf, S., Supatto, W., Debrégeas, G. et al. Whole-brain functional imaging with two-photon light-sheet microscopy. Nat Methods 12, 379–380 (2015). https://doi.org/10.1038/nmeth.3371

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