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High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision

An Erratum to this article was published on 01 June 2010

This article has been updated

Abstract

Two-photon calcium imaging of neuronal populations enables optical recording of spiking activity in living animals, but standard laser scanners are too slow to accurately determine spike times. Here we report in vivo imaging in mouse neocortex with greatly improved temporal resolution using random-access scanning with acousto-optic deflectors. We obtained fluorescence measurements from 34–91 layer 2/3 neurons at a 180–490 Hz sampling rate. We detected single action potential–evoked calcium transients with signal-to-noise ratios of 2–5 and determined spike times with near-millisecond precision and 5–15 ms confidence intervals. An automated 'peeling' algorithm enabled reconstruction of complex spike trains from fluorescence traces up to 20–30 Hz frequency, uncovering spatiotemporal trial-to-trial variability of sensory responses in barrel cortex and visual cortex. By revealing spike sequences in neuronal populations on a fast time scale, high-speed calcium imaging will facilitate optical studies of information processing in brain microcircuits.

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Figure 1: AOD-based two-photon imaging of neocortical L2/3 neurons in vivo.
Figure 2: Random-access pattern scanning from neuronal populations.
Figure 3: Determining spike times from AOD-based optical recordings.
Figure 4: Peeling algorithm for extracting spike trains from fluorescence transients.
Figure 5: Spike train reconstruction for bursts with different action potential frequency.
Figure 6: Subsecond trial-to-trial variability of sensory-evoked responses in mouse neocortex.

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  • 06 May 2010

    In the version of this article initially published, equation 1 was incorrect. The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank H. Lütcke, D. Margolis and H. Grewe for comments on the manuscript. This work was supported by a Forschungskredit of the University of Zurich (B.F.G.), and by grants to F.H. from the Swiss National Science Foundation (3100A0-114624), the EU-FP7 program (SPACEBRAIN project 200873) and the Swiss SystemsX.ch initiative (project 2008/2011-Neurochoice).

Author information

Authors and Affiliations

Authors

Contributions

B.F.G. and F.H. designed and optimized the AOD-based microscope system; B.F.G. built the microscope; B.F.G. and D.L. designed the data acquisition software; B.F.G. and H.K. designed the AOD control electronics; B.F.G. performed all in vivo experiments; F.H. developed the peeling algorithm for spike train reconstruction; B.M.K. helped with animal preparation and in vivo experiments; B.F.G. and F.H. analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Fritjof Helmchen.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 (PDF 4324 kb)

Supplementary Movie 1

Image stack from mouse barrel cortex. Two-photon image stack acquired with the AOD scanning system, showing neocortical cell populations stained with OGB-1 (green) and SR101 (red). Images were taken at 4-μm z-steps and are shown from the pial surface down to about 300 μm depth. (MOV 1115 kb)

Supplementary Movie 2

Peeling algorithm for spike train extraction. Schematic illustration of the peeling algorithm for automated spike train reconstruction from calcium indicator fluorescence traces. The algorithm is exemplified on a ΔF/F trace, for which six iterations of the algorithm were necessary to resolve five superimposed calcium transients at 10 Hz. (MOV 5209 kb)

Supplementary Movie 3

Sensory-evoked population spiking dynamics in mouse barrel cortex. Spatiotemporal spiking activity of the 56 neurons shown in Supplementary Figure 4 evoked by the first air puff in each of the eight trials. Responses to all trials are shown in parallel for the time window of 60 ms surrounding each first whisker stimulation. The movie frame duration was artificially set to 1 ms. Spike times for all neuron were reconstructed with the peeling algorithm. The occurrence of a spike is color-coded in red, whereby the color saturation (from light to dark and again to light) follows a Gaussian time course with the appropriate 95% confidence interval (10.4 ms) to indicate the uncertainty in spike detection. (MOV 927 kb)

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Grewe, B., Langer, D., Kasper, H. et al. High-speed in vivo calcium imaging reveals neuronal network activity with near-millisecond precision. Nat Methods 7, 399–405 (2010). https://doi.org/10.1038/nmeth.1453

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