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.

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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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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

Affiliations

1. Department of Neurophysiology, Brain Research Institute, University of Zurich, Zurich, Switzerland.

   • Benjamin F Grewe
   • , Dominik Langer
   • , Hansjörg Kasper
   • , Björn M Kampa
   • & Fritjof Helmchen

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2. • Nature Research journals•
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3. • Nature Research journals•
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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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Fritjof Helmchen.

Supplementary information

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1. 1.

   Supplementary Text and Figures

Videos

1. 1.

   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.

2. 2.

   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.

3. 3.

   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.

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