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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.

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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.


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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).

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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.

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The authors declare no competing financial interests.

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

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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).

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