Article | Published:

Fast volumetric calcium imaging across multiple cortical layers using sculpted light

Nature Methods volume 13, pages 10211028 (2016) | Download Citation

This article has been updated

Abstract

Although whole-organism calcium imaging in small and semi-transparent animals has been demonstrated, capturing the functional dynamics of large-scale neuronal circuits in awake behaving mammals at high speed and resolution has remained one of the main frontiers in systems neuroscience. Here we present a method based on light sculpting that enables unbiased single- and dual-plane high-speed (up to 160 Hz) calcium imaging as well as in vivo volumetric calcium imaging of a mouse cortical column (0.5 mm × 0.5 mm × 0.5 mm) at single-cell resolution and fast volume rates (3–6 Hz). We achieved this by tailoring the point-spread function of our microscope to the structures of interest while maximizing the signal-to-noise ratio using a home-built fiber laser amplifier with pulses that are synchronized to the imaging voxel speed. This enabled in vivo recording of calcium dynamics of several thousand neurons across cortical layers and in the hippocampus of awake behaving mice.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Change history

  • 14 November 2016

    In the version of this supplementary file originally posted online, Supplementary Notes 1 and 2 and Supplementary Table 1 were missing. In addition, the link for Supplementary Video 2 directed readers to an unrelated video. The errors have been corrected as of 14 November 2016.

  • 01 December 2016

    In the version of this article initially published online, a grant number was stated incorrectly in the Acknowledgements. The grant number in the sentence "A.F. acknowledges financial support by the European Union (FP7-ICT-217744)" was corrected to FP7-ICT-317744. The error has been corrected for the print, PDF and HTML versions of this article.

References

  1. 1.

    et al. Ultrasensitive fluorescent proteins for imaging neuronal activity. Nature 499, 295–300 (2013).

  2. 2.

    , , , & Optical sectioning deep inside live embryos by selective plane illumination microscopy. Science 305, 1007–1009 (2004).

  3. 3.

    , , , & Whole-brain functional imaging at cellular resolution using light-sheet microscopy. Nat. Methods 10, 413–420 (2013).

  4. 4.

    et al. Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution. Science 346, 1257998 (2014).

  5. 5.

    , , , & Light field microscopy. ACM Trans. Graph. 25, 924–934 (2006).

  6. 6.

    et al. Simultaneous whole-animal 3D imaging of neuronal activity using light-field microscopy. Nat. Methods 11, 727–730 (2014).

  7. 7.

    et al. Swept confocally-aligned planar excitation (SCAPE) microscopy for high speed volumetric imaging of behaving organisms. Nat. Photonics 9, 113–119 (2015).

  8. 8.

    et al. Brain-wide neuronal dynamics during motor adaptation in zebrafish. Nature 485, 471–477 (2012).

  9. 9.

    , & Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

  10. 10.

    & Deep tissue two-photon microscopy. Nat. Methods 2, 932–940 (2005).

  11. 11.

    & Advances in multiphoton microscopy technology. Nat. Photonics 7, 93–101 (2013).

  12. 12.

    et al. Two-photon calcium imaging of evoked activity from L5 somatosensory neurons in vivo. Nat. Neurosci. 14, 1089–1093 (2011).

  13. 13.

    , , , & Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging. Front. Cell. Neurosci. 8, 139 (2014).

  14. 14.

    et al. Aberration-free three-dimensional multiphoton imaging of neuronal activity at kHz rates. Proc. Natl. Acad. Sci. USA 109, 2919–2924 (2012).

  15. 15.

    , , , & High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. Nat. Methods 7, 399–405 (2010).

  16. 16.

    , & A compact Acousto-Optic Lens for 2D and 3D femtosecond based 2-photon microscopy. Opt. Express 18, 13721–13745 (2010).

  17. 17.

    et al. Continuous volumetric imaging via an optical phase-locked ultrasound lens. Nat. Methods 12, 759–762 (2015).

  18. 18.

    , , & Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity. Nat. Neurosci. 11, 713–720 (2008).

  19. 19.

    et al. Fast two-photon in vivo imaging with three-dimensional random-access scanning in large tissue volumes. Nat. Methods 9, 201–208 (2012).

  20. 20.

    et al. Monitoring synaptic and neuronal activity in 3D with synthetic and genetic indicators using a compact acousto-optic lens two-photon microscope. J. Neurosci. Methods 222, 69–81 (2014).

  21. 21.

    et al. Multifocal multiphoton microscopy based on multianode photomultiplier tubes. Opt. Express 15, 11658–11678 (2007).

  22. 22.

    , , & Wide field-of-view, multi-region, two-photon imaging of neuronal activity in the mammalian brain. Nat. Biotechnol. 34, 857–862 (2016).

  23. 23.

    , , , & Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing. Nat. Methods 8, 139–142 (2011).

  24. 24.

    et al. Extended field-of-view and increased-signal 3D holographic illumination with time-division multiplexing. Opt. Express 23, 32573–32581 (2015).

  25. 25.

    et al. Simultaneous Multi-plane Imaging of Neural Circuits. Neuron 89, 269–284 (2016).

  26. 26.

    The columnar organization of the neocortex. Brain 120, 701–722 (1997).

  27. 27.

    , & Scanningless depth-resolved microscopy. Opt. Express 13, 1468–1476 (2005).

  28. 28.

    , , , & Simultaneous spatial and temporal focusing of femtosecond pulses. Opt. Express 13, 2153–2159 (2005).

  29. 29.

    , , , & Brain-wide 3D imaging of neuronal activity in Caenorhabditis elegans with sculpted light. Nat. Methods 10, 1013–1020 (2013).

  30. 30.

    et al. Hybrid multiphoton volumetric functional imaging of large-scale bioengineered neuronal networks. Nat. Commun. 5, 3997 (2014).

  31. 31.

    , , & Two-photon single-cell optogenetic control of neuronal activity by sculpted light. Proc. Natl. Acad. Sci. U.S.A. 107, 11981–11986 (2010).

  32. 32.

    et al. Scanless two-photon excitation of channelrhodopsin-2. Nat. Methods 7, 848–854 (2010).

  33. 33.

    , & Simultaneous cellular-resolution optical perturbation and imaging of place cell firing fields. Nat. Neurosci. 17, 1816–1824 (2014).

  34. 34.

    , , , & Wide-field multiphoton imaging of cellular dynamics in thick tissue by temporal focusing and patterned illumination. Biomed. Opt. Express 2, 696–704 (2011).

  35. 35.

    et al. 3D-resolved fluorescence and phosphorescence lifetime imaging using temporal focusing wide-field two-photon excitation. Opt. Express 20, 26219–26235 (2012).

  36. 36.

    , & Principles of Neural Science (McGraw-Hill, Health Professions Division, 2000).

  37. 37.

    & Brain surface temperature under a craniotomy. J. Neurophysiol. 108, 3138–3146 (2012).

  38. 38.

    , & Modeling the spatiotemporal dynamics of light and heat propagation for in vivo optogenetics. Cell Rep. 12, 525–534 (2015).

  39. 39.

    & Brain heating induced by near-infrared lasers during multiphoton microscopy. J. Neurophysiol. 116, 1012–1023 (2016).

  40. 40.

    , , & Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage. Biophys. J. 77, 2226–2236 (1999).

  41. 41.

    & Highly nonlinear photodamage in two-photon fluorescence microscopy. Biophys. J. 80, 2029–2036 (2001).

  42. 42.

    , & Automated analysis of cellular signals from large-scale calcium imaging data. Neuron 63, 747–760 (2009).

  43. 43.

    et al. Simultaneous denoising, deconvolution, and demixing of calcium imaging data. Neuron 89, 285–299 (2016).

  44. 44.

    Allen Mouse Brain Atlas [Internet]., Available from: (2015).

  45. 45.

    , , , & Functional imaging of hippocampal place cells at cellular resolution during virtual navigation. Nat. Neurosci. 13, 1433–1440 (2010).

  46. 46.

    , , , & Septo-hippocampal GABAergic signaling across multiple modalities in awake mice. Nat. Neurosci. 16, 1182–1184 (2013).

  47. 47.

    et al. Sensitive red protein calcium indicators for imaging neural activity. eLife 5, e12727 (2016).

  48. 48.

    & Predictive coding in the visual cortex: a functional interpretation of some extra-classical receptive-field effects. Nat. Neurosci. 2, 79–87 (1999).

  49. 49.

    & State-dependent computations: spatiotemporal processing in cortical networks. Nat. Rev. Neurosci. 10, 113–125 (2009).

  50. 50.

    Computational neuroscience: beyond the local circuit. Curr. Opin. Neurobiol. 25, xiii–xviii (2014).

  51. 51.

    , & Multifocal multiphoton microscopy. Opt. Lett. 23, 655–657 (1998).

  52. 52.

    , & ScanImage: flexible software for operating laser scanning microscopes. Biomed. Eng. Online 2, 13 (2003).

  53. 53.

    , & Properties of normal-dispersion femtosecond fiber lasers. J. Opt. Soc. Am. B 25, 140–148 (2008).

  54. 54.

    et al. Generation of high fidelity 62-fs, 7-nJ pulses at 1035 nm from a net normal-dispersion Yb-fiber laser with anomalous dispersion higher-order-mode fiber. Opt. Express 21, 16255–16262 (2013).

  55. 55.

    , , & A stretcher fiber for use in fs chirped pulse Yb amplifiers. Opt. Express 18, 3768–3773 (2010).

  56. 56.

    et al. High-fidelity, 160 fs, 5 μJ pulses from an integrated Yb-fiber laser system with a fiber stretcher matching a simple grating compressor. Opt. Lett. 37, 927–929 (2012).

Download references

Acknowledgements

We thank W. Haubensak (Institute for Molecular Pathology (IMP), Vienna) and their lab members for sharing the satellite mouse facility and reagents and S. Rumpel and D. Aschauer (IMP) for providing mice during the initial tests, A. Piszczek and G. Petri for immunohistochemistry and slice imaging, G. Keller (Friedrich Miescher Institute, Basel) for sharing of red calcium indicators, L. Grüner-Nielsen for providing and optimizing the dispersion compensating fiber stretcher module, M. Colombini and the IMP workshop for manufacturing of mechanical components, P. Rupprecht for helpful discussions, G. Jaindl for technical support, and O. Olsen for reading and feedback on the manuscript. R.P. acknowledges the Vienna International Postdoctoral Program (VIPS) Program of the Austrian Federal Ministry of Science and Research and the City of Vienna and the European Commission (Marie Curie, FP7-PEOPLE-2011-IIF). A.F. acknowledges financial support by the European Union (FP7-ICT-317744). This work was supported through funding from the Vienna Science and Technology Fund (WWTF) project VRG10-11, the Human Frontiers Science Program Project RGP0041/2012, the Research Platform Quantum Phenomena and Nanoscale Biological Systems (QuNaBioS), the Institute of Molecular Pathology, the US National Institutes of Health (NIH) award 1U01NS094263-01, the Intelligence Advanced Research Projects Activity (IARPA) via 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

    • Alejandro J Pernía-Andrade
    •  & Siegfried Weisenburger

    These authors contributed equally to this work.

Affiliations

  1. Research Institute of Molecular Pathology, Vienna, Austria.

    • Robert Prevedel
    • , Alejandro J Pernía-Andrade
    • , Siegfried Weisenburger
    • , Tobias Nöbauer
    • , Jeroen E Delcour
    •  & Alipasha Vaziri
  2. Max F. Perutz Laboratories Support GmbH, University of Vienna, Vienna, Austria.

    • Robert Prevedel
    •  & Alipasha Vaziri
  3. Research Platform Quantum Phenomena & Nanoscale Biological Systems (QuNaBioS), University of Vienna, Vienna, Austria.

    • Robert Prevedel
    •  & Alipasha Vaziri
  4. European Molecular Biology Laboratory, Heidelberg, Germany.

    • Robert Prevedel
  5. Photonics Institute, TU Wien, Vienna, Austria.

    • Aart J Verhoef
    • , Alma Fernández
    •  & Andrius Baltuska
  6. Center for Medical Physics and Biomedical Engineering, Medical University of Vienna, Vienna, Austria.

    • Aart J Verhoef
    •  & Alma Fernández
  7. The Rockefeller University, New York, New York, USA.

    • Siegfried Weisenburger
    • , Tobias Nöbauer
    •  & Alipasha Vaziri
  8. Department of Neurology and Psychiatry, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California, USA.

    • Ben S Huang
    •  & Peyman Golshani
  9. West Los Angeles Virginia Medical Center, Los Angeles, California, USA.

    • Peyman Golshani

Authors

  1. Search for Robert Prevedel in:

  2. Search for Aart J Verhoef in:

  3. Search for Alejandro J Pernía-Andrade in:

  4. Search for Siegfried Weisenburger in:

  5. Search for Ben S Huang in:

  6. Search for Tobias Nöbauer in:

  7. Search for Alma Fernández in:

  8. Search for Jeroen E Delcour in:

  9. Search for Peyman Golshani in:

  10. Search for Andrius Baltuska in:

  11. Search for Alipasha Vaziri in:

Contributions

R.P. contributed to the conceptualization of the imaging approach, designed and built the imaging system, performed experiments, wrote software, analyzed data and wrote the manuscript. A.J.V. designed and built the custom FCPA system together with A.F. under the guidance of A.B. and performed experiments. A.J.P.-A. performed virus injections, cranial window surgeries in hippocampus and cortical areas performed imaging experiments, histology experiments, analyzed data and contributed to the writing of the manuscript. S.W. performed experiments, wrote software, analyzed data and wrote the manuscript. B.S.H. performed the viral injections and cranial window implants for the cortical imaging experiments. B.S.H and P.G. shared expertise on in vivo imaging of awake mice and provided inputs to the final version of the manuscript. T.N. wrote control software and performed in vivo characterization and control experiments, and provided inputs to the final version of the manuscript. J.E.D. wrote analysis code and analyzed data. A.V. conceived and led project, designed imaging system and in vivo mouse experiments, and wrote the manuscript.

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 1–5, Supplementary Notes 1 and 2 and Supplementary Table 1

Videos

  1. 1.

    High-speed 2D Ca2+-imaging in mouse posterior parietal cortex.

    Raw image data of calcium imaging of spontaneous activity of neurons expressing GCaMP6m in mouse posterior parietal cortex. Shown are ~315 seconds of recording at 158Hz acquisition frame rate, with a field-of-view of 500x500μm. Video playback is 5000 frames per second, which is equal to 10 seconds in real time in the video (i.e. playback speed ~30x). To reduce file size frames were binned in groups of 50. Corresponding video to Figure 2c.

  2. 2.

    High-speed 2D Ca2+-imaging in mouse posterior parietal cortex at 470μm depth.

    Raw image data of calcium imaging of spontaneous activity of neurons expressing GCaMP6f in mouse posterior parietal cortex. Shown are ~190 seconds of recording at 158Hz acquisition frame rate, with a field-of-view of 500x500μm at a depth of 470μm from the dura. Video playback is 3000 frames per second, which is equal to 10 seconds in real time in the video (i.e. playback speed ~20x). To reduce file size frames were binned in groups of 50. Corresponding video to Figure 2d.

  3. 3.

    Fast dual-plane Ca2+-imaging in mouse posterior parietal cortex.

    Raw image data of calcium imaging of spontaneous activity of neurons expressing GCaMP6m in mouse posterior parietal cortex. The planes (field-of-view of 500x500μm) were acquired with 10Hz each, in Layer 2/3 at depth of 150μm (left) and Layer 4 at 350μm depth (right) respectively. Shown are ~100 seconds of recording. Video playback is 100 frames per second, which is equal to 10 seconds in real time in the video (i.e. playback speed 10x). Corresponding video to Figure 3b-d.

  4. 4.

    Fast volumetric imaging of 3D Ca2+-dynamics across cortical layers in mouse posterior parietal cortex.

    Three-dimensional rendering of raw image data of calcium imaging spontaneous activity of neurons expressing GCaMP6m in mouse posterior parietal cortex. The field-of-view was 500x500x500μm, composed of 43 axial planes, with a volume acquisition rate of 3Hz. Shown are ~330 seconds of recording. Video playback is 75 volumes per second, which is equal to ~13 seconds in real time in the video (i.e. playback speed ~25x). Corresponding video to Figure 4a,d.

  5. 5.

    High-speed 2D Ca2+-dynamics in mouse hippocampus CA1.

    Raw image data of calcium imaging of spontaneous activity of neurons expressing jRGECKO1a in mouse hippocampus CA1 (overlying cortex aspirated). Shown are ~190 seconds of recording at 158Hz acquisition frame rate, with a field-of-view of 500x500μm. Video playback is 1500 frames per second, which is equal to 20 seconds in real time in the video (i.e. playback speed ~10x). To reduce file size frames were binned in groups of 30. Corresponding video to Figure 5b,c.

  6. 6.

    Fast volumetric imaging of 3D Ca2+-dynamics in mouse hippocampus CA1.

    Three-dimensional rendering of raw image data of calcium imaging spontaneous activity of neurons expressing jRGECKO1a in mouse hippocampus CA1 (overlying cortex aspirated). The field-of-view was 500x500x200μm, composed of 21 axial planes, with a volume acquisition rate of 5.7Hz. Shown are ~265 seconds of recording. Video playback is 60 volumes per second, which is equal to 25 seconds in real time in the video (i.e. playback speed ~10x). Corresponding video to Figure 5d,e.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nmeth.4040