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Scanless two-photon excitation of channelrhodopsin-2


Light-gated ion channels and pumps have made it possible to probe intact neural circuits by manipulating the activity of groups of genetically similar neurons. What is needed now is a method for precisely aiming the stimulating light at single neuronal processes, neurons or groups of neurons. We developed a method that combines generalized phase contrast with temporal focusing (TF-GPC) to shape two-photon excitation for this purpose. The illumination patterns are generated automatically from fluorescence images of neurons and shaped to cover the cell body or dendrites, or distributed groups of cells. The TF-GPC two-photon excitation patterns generated large photocurrents in Channelrhodopsin-2–expressing cultured cells and neurons and in mouse acute cortical slices. The amplitudes of the photocurrents can be precisely modulated by controlling the size and shape of the excitation volume and, thereby, be used to trigger single action potentials or trains of action potentials.

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Figure 1: TF-GPC design.
Figure 2: Two-photon photoactivation of ChR2 by TF-GPC in HEK 293 cells.
Figure 3: Action potential generation by two-photon TF-GPC in primary neuronal culture.
Figure 4: Two-photon photoactivation by TF-GPC in cortical brain slices.
Figure 5: TF-GPC provides lateral and axial precision in ChR2 activation in brain slices.
Figure 6: Multispot photoactivation in cortical slices.


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We thank I. Perch-Nielsen for the phase-contrast filter layout design, E. Schwartz for genotyping ChR2-YFP mice, S. Wiese, Z. Fu and M. Viesel for generating cDNA constructs, A. Triller, T. Gally, K. Spence, A. Burgo and K. Zylbersztejn for cell culture preparation, all members of the Neurophysiology and New Microscopy Laboratory for comments and technical help, D. Oron, D. Palima, S. Dieudonné, M. Diana and G. Fortin for helpful discussions, J. Feldmann for critical reading of the paper, Spectra-Physics, Inc. for loan of the high-power laser, and Phasics S.A. for providing the phase-analyzer software. V.E. was supported by the European Science Foundation and the Centre National de la Recherche Scientifique through the European Young Investigator program and by the European Network of Neuroscience Institutes (LSHM-CT-2005-19063). E.P. and V.E. were supported by the European Commission FP6 Specific Targeted Project Photolysis (LSHM-CT-2007-037765). E.P. was supported by the Fondation pour la Recherche Médicale. F.A. was supported by the European doctoral school Frontières du Vivant. A.B. was supported by Paris School of Neuroscience. J.G. was supported by the Danish Technical Scientific Research Councils (09-060742), E.Y.I. was supported by the US National Institutes of Health Nanomedicine Development Center for the Optical Control of Biological Function (PN2EY018241) and the Paris School of Neuroscience. E.Y.I. and V.E. were supported by Human Frontier Science Program (RGP0013/2010).

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Authors and Affiliations



E.P. set up and characterized the optical properties of the TF-GPC microscope; F.A. and A.B. implemented the optical microscope with electrophysiological recording; E.P., F.A. and A.B. performed the experiments on cell cultures and brain slices; F.A. and A.B. analyzed the experiments on cell cultures and brain slices; V.d.S. developed the software; J.G. contributed to the set up of the GPC microscope; E.Y.I. contributed in conceiving the experiments in cultured cells and brain slices and discussed the results; E.Y.I. and V.E. prepared the manuscript; and V.E. conceived and supervised the project.

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Correspondence to Valentina Emiliani.

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The authors declare no competing financial interests.

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Supplementary Figures 1–8, Supplementary Note 1 (PDF 1042 kb)

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Papagiakoumou, E., Anselmi, F., Bègue, A. et al. Scanless two-photon excitation of channelrhodopsin-2. Nat Methods 7, 848–854 (2010).

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