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.

  • Brief Communication
  • Published:

Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing


In vivo two-photon calcium imaging would benefit from the use of multiple excitation beams to increase scanning speed, signal-to-noise ratio and field of view or to image different axial planes simultaneously. Using spatiotemporal multiplexing we circumvented light-scattering ambiguity inherent to deep-tissue multifocal two-photon microscopy. We demonstrate calcium imaging at multiple axial planes in the intact mouse brain to monitor network activity of ensembles of cortical neurons in three spatial dimensions.

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

Access options

Buy this article

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

Figure 1: Spatiotemporal multiplexing to overcome depth limitations in multifocal 2PLSM.
Figure 2: Multifocal two- and three-dimension in vivo 2PCI of L2/3 neurons in barrel cortex with spatiotemporal multiplexing.
Figure 3: Multifocal 2PCI with spatiotemporal multiplexing to assess activity-derived neuronal connectivity in L2/3 of barrel cortex.

Similar content being viewed by others


  1. Grewe, B.F. & Helmchen, F. Curr. Opin. Neurobiol. 18, 520–529 (2009).

    Article  Google Scholar 

  2. Svoboda, K. & Yasuda, R. Neuron 50, 823–839 (2006).

    Article  CAS  Google Scholar 

  3. Kerlin, A.M., Andermann, M.L., Berezovskii, V.K. & Reid, R.C. Neuron 67, 858–871 (2010).

    Article  CAS  Google Scholar 

  4. Kremer, Y. et al. Opt. Express 16, 10066–10076 (2008).

    Article  CAS  Google Scholar 

  5. Gobel, W., Kampa, B.M. & Helmchen, F. Nat. Methods 4, 73–79 (2007).

    Article  Google Scholar 

  6. Duemani Reddy, G., Kelleher, K., Fink, R. & Saggau, P. Nat. Neurosci. 11, 713–720 (2008).

    Article  CAS  Google Scholar 

  7. Grewe, B.F., Langer, D., Kasper, H., Kampa, B.M. & Helmchen, F. Nat. Methods 7, 399–405 (2010).

    Article  CAS  Google Scholar 

  8. Nielsen, T., Fricke, M., Hellweg, D. & Andresen, P. J. Microsc. 201, 368–376 (2001).

    Article  CAS  Google Scholar 

  9. Kim, H. et al. Opt. Express 15, 11658–11678 (2007).

    Article  Google Scholar 

  10. Hopt, A. & Neher, E. Biophys. J. 80, 2029–2036 (2001).

    Article  CAS  Google Scholar 

  11. Ji, N., Magee, J.C. & Betzig, E. Nat. Methods 5, 197–202 (2008).

    Article  CAS  Google Scholar 

  12. Oheim, M., Beaurepaire, E., Chaigneau, E., Mertz, J. & Charpak, S. J. Neurosci. Methods 111, 29–37 (2001).

    Article  CAS  Google Scholar 

  13. Amir, W. et al. Opt. Lett. 32, 1731–1733 (2007).

    Article  CAS  Google Scholar 

  14. Sheetz, K.E., Hoover, E.E., Carriles, R., Kleinfeld, D. & Squier, J.A. Opt. Express 16, 17574–17584 (2008).

    Article  CAS  Google Scholar 

  15. Chandler, E. et al. Appl. Opt. 48, 2067–2077 (2009).

    Article  Google Scholar 

  16. Michalet, X. et al. Proc. SPIE 6862, 0F1–0F12 (2008).

    Google Scholar 

  17. Cossart, R., Aronov, D. & Yuste, R. Nature 423, 283–288 (2003).

    Article  CAS  Google Scholar 

  18. Beck, M., Walmsley, I.A. & Kafka, J.D. IEEE J. Quantum Elect. 27, 2074–2081 (1991).

    Article  CAS  Google Scholar 

  19. Fukasawa, A.J. et al. IEEE Trans. Nuclear Sci. 55, 758–762 (2008).

    Article  CAS  Google Scholar 

  20. Sanderson, M.J. & Parker, I. Methods Enzymol. 360, 447–481 (2003).

    Article  Google Scholar 

  21. Fan, G.Y. et al. Biophys. J. 76, 2412–2420 (1999).

    Article  CAS  Google Scholar 

  22. Rochefort, N.L. et al. Proc. Natl. Acad. Sci. USA 106, 15049–15054 (2009).

    Article  CAS  Google Scholar 

  23. Mostany, R. & Portera-Cailliau, C. J. Vis. Exp. published online 15 February 2008 (doi:10.3791/680).

  24. Golshani, P. & Portera-Cailliau, C. J. Vis. Exp. published online 13 March 2008 (doi:10.3791/681).

  25. Garaschuk, O., Milos, R.I. & Konnerth, A. Nat. Protoc. 1, 380–386 (2006).

    Article  CAS  Google Scholar 

  26. Golshani, P. et al. J. Neurosci. 29, 10890–10899 (2009).

    Article  CAS  Google Scholar 

  27. Kitamura, K., Judkewitz, B., Kano, M., Denk, W. & Hausser, M. Nat. Methods 5, 61–67 (2008).

    Article  CAS  Google Scholar 

  28. Smith, S.L. & Hausser, M. Nat. Neurosci. 13, 1144–1149 (2010).

    Article  CAS  Google Scholar 

  29. Valmianski, I. et al. J. Neurophysiol. 104, 1803–1822 (2010).

    Article  Google Scholar 

  30. Grewe, B.F., Langer, D., Kasper, H., Kampa, B.M. & Helmchen, F. Nat. Methods 7, 399–405 (2010).

    Article  CAS  Google Scholar 

  31. Cossart, R., Aronov, D. & Yuste, R. Nature 423, 283–288 (2003).

    Article  CAS  Google Scholar 

Download references


We thank D. Kleinfeld, J.F. Léger, T. Otis and members of the Portera-Cailliau laboratory for discussions and comments on the manuscript, and M. Suyama and Y. Kawai (Hamamatsu Photonics K.K.) for engineering support. This work was supported by grants from the US National Institutes of Health (the Eunice Kennedy Shriver National Institute of Child Health and Human Development, the National Institute of Neurological Disorders and Stroke) and the US National Science Foundation (Major Research Instrumentation Program).

Author information

Authors and Affiliations



A.C., J.T.G., P.G., K.A. and C.P.-C. conceived the project. A.C. designed and built the microscope and control electronics, and developed the microscope software. J.T.G. performed in vivo multifocal calcium imaging and simultaneous cell-attached recordings. A.C. analyzed the data. A.C., J.T.G. and C.P.-C. wrote the manuscript. K.A. and C.P.-C. supervised the project.

Corresponding author

Correspondence to Carlos Portera-Cailliau.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Note 1 (PDF 6033 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cheng, A., Gonçalves, J., Golshani, P. et al. Simultaneous two-photon calcium imaging at different depths with spatiotemporal multiplexing. Nat Methods 8, 139–142 (2011).

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