Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

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.

Change history

  • 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.


  1. Grewe, B.F. & Helmchen, F. Optical probing of neuronal ensemble activity. Curr. Opin. Neurobiol. 19, 520–529 (2009).

    Article  CAS  Google Scholar 

  2. Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).

    Article  CAS  Google Scholar 

  3. Ohki, K., Chung, S., Ch'ng, Y.H., Kara, P. & Reid, R.C. Functional imaging with cellular resolution reveals precise micro-architecture in visual cortex. Nature 433, 597–603 (2005).

    Article  CAS  Google Scholar 

  4. Kerr, J.N.D., Greenberg, D. & Helmchen, F. Imaging input and output of neocortical networks in vivo. Proc. Natl. Acad. Sci. USA 102, 14063–14068 (2005).

    Article  CAS  Google Scholar 

  5. Göbel, W., Kampa, B.M. & Helmchen, F. Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nat. Methods 4, 73–79 (2007).

    Article  Google Scholar 

  6. Sato, T.R., Gray, N.W., Mainen, Z.F. & Svoboda, K. The functional microarchitecture of the mouse barrel cortex. PLoS Biol. 5, e189 (2007).

    Article  Google Scholar 

  7. Kerr, J.N. et al. Spatial organization of neuronal population responses in layer 2/3 of rat barrel cortex. J. Neurosci. 27, 13316–13328 (2007).

    Article  CAS  Google Scholar 

  8. Rochefort, N.L. et al. Sparsification of neuronal activity in the visual cortex at eye-opening. Proc. Natl. Acad. Sci. USA 106, 15049–15054 (2009).

    Article  CAS  Google Scholar 

  9. Wallace, D.J. et al. Single-spike detection in vitro and in vivo with a genetic Ca2+ sensor. Nat. Methods 5, 797–804 (2008).

    Article  CAS  Google Scholar 

  10. Tian, L. et al. Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators. Nat. Methods 6, 875–881 (2009).

    Article  CAS  Google Scholar 

  11. Lütcke, H. et al. Optical recording of neuronal activity with a genetically-encoded calcium indicator in anesthetized and freely moving mice. Front. Neural Circuits 4, 9 (2010).

    PubMed  PubMed Central  Google Scholar 

  12. Lillis, K.P., Eng, A., White, J.A. & Mertz, J. Two-photon imaging of spatially extended neuronal network dynamics with high temporal resolution. J. Neurosci. Methods 172, 178–184 (2008).

    Article  Google Scholar 

  13. Nikolenko, V. et al. SLM Microscopy: Scanless two-photon imaging and photostimulation with spatial light modulators. Front. Neural Circuits 2, 5 (2008).

    Article  Google Scholar 

  14. Iyer, V., Hoogland, T.M. & Saggau, P. Fast functional imaging of single neurons using random-access multiphoton (RAMP) microscopy. J. Neurophysiol. 95, 535–545 (2006).

    Article  Google Scholar 

  15. Otsu, Y. et al. Optical monitoring of neuronal activity at high frame rate with a digital random-access multiphoton (RAMP) microscope. J. Neurosci. Methods 173, 259–270 (2008).

    Article  Google Scholar 

  16. Reddy, G.D., Kelleher, K., Fink, R. & Saggau, P. Three-dimensional random access multiphoton microscopy for functional imaging of neuronal activity. Nat. Neurosci. 11, 713–720 (2008).

    Article  Google Scholar 

  17. Zeng, S. et al. Simultaneous compensation for spatial and temporal dispersion of acousto-optical deflectors for two-dimensional scanning with a single prism. Opt. Lett. 31, 1091–1093 (2006).

    Article  Google Scholar 

  18. Kremer, Y. et al. A spatio-temporally compensated acousto-optic scanner for two-photon microscopy providing large field of view. Opt. Express 16, 10066–10076 (2008).

    Article  CAS  Google Scholar 

  19. Nimmerjahn, A., Kirchhoff, F., Kerr, J.N. & Helmchen, F. Sulforhodamine 101 as a specific marker of astroglia in the neocortex in vivo. Nat. Methods 1, 31–37 (2004).

    Article  CAS  Google Scholar 

  20. Koester, H.J., Baur, D., Uhl, R. & Hell, S.W. Ca2+ fluorescence imaging with pico- and femtosecond two-photon excitation: signal and photodamage. Biophys. J. 77, 2226–2236 (1999).

    Article  CAS  Google Scholar 

  21. Göbel, W. & Helmchen, F. In vivo calcium imaging of neural network function. Physiology (Bethesda) 22, 358–365 (2007).

    Google Scholar 

  22. Histed, M.H., Bonin, V. & Reid, R.C. Direct activation of sparse, distributed populations of cortical neurons by electrical microstimulation. Neuron 63, 508–522 (2009).

    Article  CAS  Google Scholar 

  23. Goard, M. & Dan, Y. Basal forebrain activation enhances cortical coding of natural scenes. Nat. Neurosci. 12, 1444–1449 (2009).

    Article  CAS  Google Scholar 

  24. Göbel, W. & Helmchen, F. New angles on neuronal dendrites in vivo. J. Neurophysiol. 98, 3770–3779 (2007).

    Article  Google Scholar 

  25. Greenberg, D.S., Houweling, A.R. & Kerr, J.N. Population imaging of ongoing neuronal activity in the visual cortex of awake rats. Nat. Neurosci. 11, 749–751 (2008).

    Article  CAS  Google Scholar 

  26. Yaksi, E. & Friedrich, R.W. Reconstruction of firing rate changes across neuronal populations by temporally deconvolved Ca2+ imaging. Nat. Methods 3, 377–383 (2006).

    Article  CAS  Google Scholar 

  27. Vogelstein, J.T. et al. Spike inference from calcium imaging using sequential Monte Carlo methods. Biophys. J. 97, 636–655 (2009).

    Article  CAS  Google Scholar 

  28. Vucinic, D. & Sejnowski, T.J. A compact multiphoton 3D imaging system for recording fast neuronal activity. PLoS One 2, e699 (2007).

    Article  Google Scholar 

  29. Mank, M. et al. A genetically encoded calcium indicator for chronic in vivo two-photon imaging. Nat. Methods 5, 805–811 (2008).

    Article  CAS  Google Scholar 

  30. Dombeck, D.A., Khabbaz, A.N., Collman, F., Adelman, T.L. & Tank, D.W. Imaging large-scale neural activity with cellular resolution in awake, mobile mice. Neuron 56, 43–57 (2007).

    Article  CAS  Google Scholar 

  31. Greenberg, D.S. & Kerr, J.N. Automated correction of fast motion artifacts for two-photon imaging of awake animals. J. Neurosci. Methods 176, 1–15 (2009).

    Article  Google Scholar 

  32. de Kock, C.P. & Sakmann, B. Spiking in primary somatosensory cortex during natural whisking in awake head-restrained rats is cell-type specific. Proc. Natl. Acad. Sci. USA 106, 16446–16450 (2009).

    Article  CAS  Google Scholar 

  33. Hartwich, K., Pollak, T. & Klausberger, T. Distinct firing patterns of identified basket and dendrite-targeting interneurons in the prefrontal cortex during hippocampal theta and local spindle oscillations. J. Neurosci. 29, 9563–9574 (2009).

    Article  CAS  Google Scholar 

  34. Kostal, L., Lansky, P. & Rospars, J.P. Neuronal coding and spiking randomness. Eur. J. Neurosci. 26, 2693–2701 (2007).

    Article  Google Scholar 

  35. Caporale, N. & Dan, Y. Spike timing-dependent plasticity: a Hebbian learning rule. Annu. Rev. Neurosci. 31, 25–46 (2008).

    Article  CAS  Google Scholar 

  36. Kampa, B.M., Letzkus, J.J. & Stuart, G.J. Dendritic mechanisms controlling spike-timing-dependent synaptic plasticity. Trends Neurosci. 30, 456–463 (2007).

    Article  CAS  Google Scholar 

  37. Lechleiter, J.D., Lin, D.T. & Sieneart, I. Multi-photon laser scanning microscopy using an acoustic optical deflector. Biophys. J. 83, 2292–2299 (2002).

    Article  CAS  Google Scholar 

  38. Garaschuk, O., Milos, R.I. & Konnerth, A. Targeted bulk-loading of fluorescent indicators for two-photon brain imaging in vivo. Nat. Protocols 1, 380–386 (2006).

    Article  CAS  Google Scholar 

  39. van Hateren, J.H. & Ruderman, D.L. Independent component analysis of natural image sequences yields spatio-temporal filters similar to simple cells in primary visual cortex. Proc. Biol. Sci. 265, 2315–2320 (1998).

    Article  CAS  Google Scholar 

  40. Markram, H., Helm, P.J. & Sakmann, B. Dendritic calcium transients evoked by single back-propagating action potentials in rat neocortical pyramidal neurons. J. Physiol. (Lond.) 485, 1–20 (1995).

    Article  CAS  Google Scholar 

  41. Berridge, M.J., Bootman, M.D. & Roderick, H.L. Calcium signalling: dynamics, homeostasis and remodelling. Nat. Rev. Mol. Cell Biol. 4, 517–529 (2003).

    Article  CAS  Google Scholar 

  42. Borst, J.G. & Helmchen, F. Calcium influx during an action potential. Methods Enzymol. 293, 352–371 (1998).

    Article  CAS  Google Scholar 

  43. Waters, J. & Helmchen, F. Background synaptic activity is sparse in neocortex. J. Neurosci. 26, 8267–8277 (2006).

    Article  CAS  Google Scholar 

  44. Helmchen, F., Imoto, K. & Sakmann, B. Ca2+ buffering and action potential-evoked Ca2+ signaling in dendrites of pyramidal neurons. Biophys. J. 70, 1069–1081 (1996).

    Article  CAS  Google Scholar 

  45. Maravall, M., Mainen, Z.F., Sabatini, B.L. & Svoboda, K. Estimating intracellular calcium concentrations and buffering without wavelength ratioing. Biophys. J. 78, 2655–2667 (2000).

    Article  CAS  Google Scholar 

  46. Schiller, J., Helmchen, F. & Sakmann, B. Spatial profile of dendritic calcium transients evoked by action potentials in rat neocortical pyramidal neurones. J. Physiol. (Lond.) 487, 583–600 (1995).

    Article  CAS  Google Scholar 

Download references


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 initiative (project 2008/2011-Neurochoice).

Author information

Authors and Affiliations



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.

Ethics declarations

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)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing