Article | Published:

Video rate volumetric Ca2+ imaging across cortex using seeded iterative demixing (SID) microscopy

Nature Methods volume 14, pages 811818 (2017) | Download Citation

Abstract

Light-field microscopy (LFM) is a scalable approach for volumetric Ca2+ imaging with high volumetric acquisition rates (up to 100 Hz). Although the technology has enabled whole-brain Ca2+ imaging in semi-transparent specimens, tissue scattering has limited its application in the rodent brain. We introduce seeded iterative demixing (SID), a computational source-extraction technique that extends LFM to the mammalian cortex. SID can capture neuronal dynamics in vivo within a volume of 900 × 900 × 260 μm located as deep as 380 μm in the mouse cortex or hippocampus at a 30-Hz volume rate while discriminating signals from neurons as close as 20 μm apart, at a computational cost three orders of magnitude less than that of frame-by-frame image reconstruction. We expect that the simplicity and scalability of LFM, coupled with the performance of SID, will open up a range of applications including closed-loop experiments.

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Acknowledgements

We thank W. Haubensak and his lab members for sharing their animal facility and reagents, M. Colombini and the IMP workshop for manufacturing of mechanical components, and F. Schlumm and Q. Lin for help with zebrafish imaging. The computational results presented here were achieved in part through use of the Vienna Scientific Cluster (VSC). T.N. acknowledges the Leon Levy Foundation (Leon Levy Fellowship in Neuroscience). This work was supported in part through funding from the Vienna Science and Technology Fund (WWTF; project VRG10-11), the Human Frontiers Science Program (Project RGP0041/2012), the Institute of Molecular Pathology, and the Kavli Foundation, all to A.V.; and from the Intelligence Advanced Research Projects Activity (IARPA) via the Department of Interior/Interior Business Center (DoI/IBC; contract number D16PC00002). The US government is authorized to reproduce and distribute reprints for governmental purposes notwithstanding any copyright annotation thereon. 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

    • Tobias Nöbauer
    •  & Oliver Skocek

    These authors contributed equally to this work.

Affiliations

  1. Laboratory of Neurotechnology and Biophysics, The Rockefeller University, New York, New York, USA.

    • Tobias Nöbauer
    • , Oliver Skocek
    • , Francisca Martínez Traub
    •  & Alipasha Vaziri
  2. Research Institute of Molecular Pathology, Vienna, Austria.

    • Alejandro J Pernía-Andrade
    • , Lukas Weilguny
    • , Maxim I Molodtsov
    •  & Alipasha Vaziri
  3. The Kavli Neural Systems Institute, The Rockefeller University, New York, New York, USA.

    • Alipasha Vaziri

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Contributions

T.N. and O.S. contributed to the conceptualization of the imaging and signal extraction approach, wrote software and analyzed data. T.N. designed and built the imaging system and performed experiments. A.J.P.-A., F.M.T. and L.W. performed virus injections, cranial window surgeries and imaging experiments. M.I.M. contributed to the generation of the synthetic data sets and simulations. A.V. conceived and led the project, conceptualized the imaging and signal extraction approach and designed in vivo mouse experiments. T.N. and A.V. wrote the manuscript, with contributions from O.S. and A.J.P.-A.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Alipasha Vaziri.

Integrated supplementary information

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Figures 1–10 and Supplementary Notes 1–7

Zip files

  1. 1.

    Supplementary Software

    Matlab code implementing seeded iterative demixing. The code requires additional functions published online as Supplementary Software with the paper by Prevedel et al. (Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy. Nat. Methods 11, 727–730 (2014)). Updated versions of the code will be made available online at http://vaziria.com and http://github.com/vazirilab.

Videos

  1. 1.

    3D rendering of naive reconstructions of LFM Ca2+ imaging data of mouse cortex in vivo: 0–170 μm below dura.

    Rendering of an LFM recording of spontaneous GECI activity in GCaMP6m-labeled mouse posterior parietal cortex, reconstructed by naive deconvolution with a simulated, ballistic PSF, showing considerable blur due to scattering. Recording duration: 1 min. Recording frame rate: 30 f.p.s. Playback at 60 f.p.s.

  2. 2.

    3D rendering of naive reconstructions of LFM Ca2+ imaging data of mouse cortex in vivo: 170–380 μm below dura.

    Rendering of an LFM recording of spontaneous GECI activity in GCaMP6m-labeled mouse posterior parietal cortex, reconstructed by naive deconvolution with a simulated, ballistic PSF, showing considerable blur due to scattering. Recording duration: 1 min. Recording frame rate: 30 f.p.s. Playback at 60 f.p.s.

  3. 3.

    3D rendering of cortical neuronal activity extracted using Seeded Source Extraction (SID) from LFM Ca2+ imaging of mouse cortex in vivo: 0–170 μm below dura.

    Rendering of neuron positions and normalized temporal signals revealed using Seeded Source Extraction (SID) in a 1-min LFM recording of GECI activity in GCaMP6m-labeled mouse posterior parietal cortex, showing spontaneous activity. Depth range: 30 μm above to 170 μm below dura. Neuron positions indicated as spheres with 15-μm diameter. Recording frame rate: 30 f.p.s. Playback at 60 f.p.s. Data correspond to Figure 3c,d and Supplementary Figure 6.

  4. 4.

    3D rendering of cortical neuronal activity extracted by Seeded Source Extraction (SID) from LFM Ca2+ imaging of mouse cortex in vivo: 170–380 μm below dura.

    Rendering of neuron positions and normalized temporal signals revealed using Seeded Source Extraction (SID) in a 1-min LFM recording of GECI activity in GCaMP6m-labeled mouse posterior parietal cortex, showing spontaneous activity. Depth range: 170–380 μm below dura. Neuron positions indicated as spheres with 15-μm diameter. Recording frame rate: 30 f.p.s. Playback at 60 f.p.s. Data correspond to Figure 3c,d and Supplementary Figure 6.

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DOI

https://doi.org/10.1038/nmeth.4341

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