Scanless two-photon excitation of channelrhodopsin-2

Journal name:
Nature Methods
Year published:
Published online


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.

At a glance


  1. TF-GPC design.
    Figure 1: TF-GPC design.

    (a) Output of Ti:sapphire laser is expanded to illuminate an LCOS-SLM that is located at the front focal plane of a 4-f (f is focal length) imaging setup (L1 and L2 are lenses with focal lengths of 400 mm and 300 mm, respectively), with a phase-contrast filter (PCF) at the confocal plane between the lenses. For temporal focusing, a blazed reflectance grating (830 lines mm−1) is placed at the output mapping plane of the GPC system. L, lens with focal length of 500 mm. Intensity and phase distributions at the SLM, grating and sample planes are indicated. CCD, charge-coupled device. (b) Images of circular spot (20 μm diameter; left) and shaped patterns (right) created by two-photon excitation of a thin (~1 μm) fluorescent layer (λexc = 780 nm; objective 60×, 0.9 numerical aperture (NA)). Shaped patterns were based on a confocal image of a Purkinje cell (center, top) and widefield fluorescence image of CA1 hippocampal neurons loaded with Oregon Green Bapta (center, bottom), in selected regions of interest (yellow outlines). (c,d) Theoretical (c) and experimental (d) y-z section axial propagation of the 20 μm spot without temporal focusing. (e) Experimental y-z section axial propagation of the spot in d with temporal focusing (left). Experimental propagation measured on double microscope setup16, 19 (right). Scale bars, 10 μm. (f) Axial profile of fluorescence intensity in e (TF-GPC) compared to 20 μm circular spot generated by TF-DH and theoretical curve for the axial integrated intensity in line-scanning two-photon microscopy (theory), where I (1 + (z / zR)2)−0.5, with I being the integrated light intensity, z the actual axial position and zR the Rayleigh range for a focused Gaussian beam in our experimental conditions, that is, zR = 0.8 μm.

  2. Two-photon photoactivation of ChR2 by TF-GPC in HEK 293 cells.
    Figure 2: Two-photon photoactivation of ChR2 by TF-GPC in HEK 293 cells.

    (a) Fluorescence images (λexc = 488 nm, bandwidth 10 nm) of HEK 293 cells transfected with plasmids encoding ChR2(H134R)-GFP with superimposed excitation patterns (red) of increasing size (top; left to right spots have 5, 8, 10, 12 and 14 μm diameter and whole cell). Whole-cell photocurrents (bottom) evoked by 10-ms laser pulses (red bars) of excitation spots in respective images above (λexc = 850 nm, 0.45 mW μm−2). (b) Widefield epi-fluorescence image of an HEK 293 cell transfected with plasmids encoding ChR2(H134R)-GFP and superimposed excitation patterns illuminating around the cell (antishape; top left) or covering the whole cell (shape; top right). Whole-cell photocurrents (bottom; 0.38 mW μm−2). Scale bars, 20 μm. (c) Normalized integrated currents elicited by moving shaped excitation pattern shown in b along the z axis by steps of 2 μm (dots) (0.17 mW μm−2) and simulation with cell modeled as a parallelepiped of size x = 15 μm, y = 26 μm, z = 10 μm, corresponding to measured (x, y) and estimated (z, distance between the two experimental peaks) cellular dimensions. Inset, excitation volume represented as infinite sheet of light with axial distribution given by the experimental curve, measured by scanning the 40×, 0.8 NA objective, through a fluorescent layer and integrating the light collected by the CCD camera (red line is a guide for the eye). λexc = 850 nm.

  3. Action potential generation by two-photon TF-GPC in primary neuronal culture.
    Figure 3: Action potential generation by two-photon TF-GPC in primary neuronal culture.

    (a) Excitation spots of increasing coverage of cell body superimposed on the fluorescence image of a neuron (top). Current clamp recordings (bottom) corresponding to the condition above. Action potentials were generated for an excitation area covering about one-third of the surface of the cell body (0.60 mW μm−2, 30 ms pulse duration). (b) Excitation pattern used for action-potential train experiments. (c,d) Light-activated trains of action potentials at 5, 10 and 15 Hz (0.5 mW μm−2; c) or by a 1-s pulse (0.6 mW μm−2; d). Scale bars, 20 μm. λexc = 920 nm, 40×, 0.8 NA objective.

  4. Two-photon photoactivation by TF-GPC in cortical brain slices.
    Figure 4: Two-photon photoactivation by TF-GPC in cortical brain slices.

    (a) Widefield fluorescence image of a layer V pyramidal neuron positive for ChR2-YFP (λexc = 488 nm, 5 nm bandwidth). (b) Plot of the peak current (inward currents indicated in negative picoamperes) (n = 6 cells) as a function of excitation spot diameter (average on three trials in all cases, 0.52 mW μm−2, 10 ms pulse). (c) Voltage responses to photoexcitation with spots of increasing size (left; 3, 7, 10 and 15 μm in diameter; 0.52 mW μm−2; 10 ms pulse). Action potential latency as a function of the excitation spot diameter (right; n = 7 cells). Error bars, s.e.m. Average of three trials is shown in all cases. (d) Widefield epi-fluorescence image of cell body loaded through the patch pipet with the fluorescent indicator Alexa Fluor 594 (left; λexc = 590 nm, bandwidth 10 nm). Action potential trains evoked by 1-s light pulse with increasing excitation spot size (middle). Average frequencies were 11.8 ± 0.8 Hz (6 trials) for a 15 μm spot, 8.7 ± 0.3 Hz (3 trials) for a 10 μm spot, 7.7 ± 0.3 Hz (3 trials) for a 7 μm spot and 4.8 Hz ± 0.3 (4 trials) for a 5 μm spot (0.4 mW μm−2). Example of action potential firing after light stimulation at 10 Hz (5/5 trials), 20 Hz (5/5 trials) and 30 Hz (4/11 trials) (0.40 mW μm−2; 10 ms pulse; 15 μm excitation spot; right). Scale bars, 20 μm. λexc = 920 nm, 40×, 0.8 NA objective, excitation depth of 50–70 μm.

  5. TF-GPC provides lateral and axial precision in ChR2 activation in brain slices.
    Figure 5: TF-GPC provides lateral and axial precision in ChR2 activation in brain slices.

    (a) Widefield fluorescence images of a ChR2-YFP positive neuron filled with Alexa Fluor 594 and superimposed excitation patterns (red) with shaped and anti-shaped profiles (top). Photocurrents evoked by shaped and antishaped excitation (bottom; 10 ms laser pulses, 0.24 mW μm−2). (b) Integrated photocurrent (area under inward current measured in picocoulombs and shown as negative picocoloumbs) evoked by a 10 μm excitation spot centered on the cell body when displaced along the z axis in a ChR2-YFP–positive neuron (0.30 mW μm−2). (c) Fluorescence image of a ChR2-YFP positive neuron filled with Alexa Fluor 594 with superimposed shaped excitation profile covering the apical dendrite (red; top). Photo-depolarizations evoked by the excitation shape at different z-axis positions (bottom; 10 ms pulse, 0.30 mW μm−2). Scale bars, 20 μm. λexc = 920 nm, 40×, 0.8 NA objective.

  6. Multispot photoactivation in cortical slices.
    Figure 6: Multispot photoactivation in cortical slices.

    (a) Transmission images of layer V pyramidal neurons expressing ChR2-YFP. One spot (red) was placed over the recorded cell (top) or this spot was combined with four identical spots placed elsewhere (bottom). White line indicates the excitation field of 60 μm in diameter. Scale bars, 10 μm. (b) Action potential evoked by single-spot (top) and multispot (bottom) illumination (red bars) (0.29–0.34 mW μm−2; λexc = 920 nm). (c) Averaged (three trials per cell) amplitudes of photocurrents measured under voltage clamp in response to single or multispot illumination (three spots of 12 μm diameter for four cells or five spots of 11 μm diameter in one cell). (d) Transmission image of three ChR2-YFP–positive neurons (A, B and C) with 15 μm excitation spots (red) superimposed. Scale bar, 10 μm. (e) Neurons A and B were simultaneously patch clamped. A spot over neuron A triggered an action potential only in A. A spot over neuron B triggered an action potential only in neuron B. When the three cells were illuminated simultaneously both A and B fired an action potential. (f) After recordings shown in e, the patch pipet was removed from B and a patch recording was established in C. A spot over neuron A triggered an action potential only in A. A spot over C triggered an action potential in C and a small depolarization in A. When the three cells were illuminated simultaneously both A and C fired an action potential (0.25 mW μm−2; λexc = 850 nm, 40×, 0.8 NA objective). Recordings were collected in 10 μM NBQX.


  1. Penfield, W. & Rasmussen, T. The cerebral cortex of man: a clinical study of localization of function. J. Am. Med. Assoc. 144, 14121700 (1950).
  2. Nagel, G. et al. Channelrhodopsin-2, a directly light-gated cation-selective membrane channel. Proc. Natl. Acad. Sci. USA 100, 1394013945 (2003).
  3. Gunaydin, L.A. et al. Ultrafast optogenetic control. Nat. Neurosci 13, 387392 (2010).
  4. Boyden, E.S., Zhang, F., Bamberg, E., Nagel, G. & Deisseroth, K. Millisecond-timescale, genetically targeted optical control of neural activity. Nat. Neurosci. 8, 12631268 (2005).
  5. Wang, H. et al. High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice. Proc. Natl. Acad. Sci. USA 104, 81438148 (2007).
  6. Grossman, N. et al. Multi-site optical excitation using ChR2 and micro-LED array. J. Neural Eng. 7, 16004 (2010).
  7. Aravanis, A.M. et al. An optical neural interface: in vivo control of rodent motor cortex with integrated fiberoptic and optogenetic technology. J. Neural Eng. 4, S143S156 (2007).
  8. Gradinaru, V. et al. Targeting and readout strategies for fast optical neural control in vitro and in vivo . J. Neurosci. 27, 1423114238 (2007).
  9. Gradinaru, V., Mogri, M., Thompson, K.R., Henderson, J.M. & Deisseroth, K. Optical deconstruction of parkinsonian neural circuitry. Science 324, 354359 (2009).
  10. Huber, D. et al. Sparse optical microstimulation in barrel cortex drives learned behaviour in freely moving mice. Nature 451, 6164 (2008).
  11. Lagali, P.S. et al. Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration. Nat. Neurosci. 11, 667675 (2008).
  12. Feldbauer, K. et al. Channelrhodopsin-2 is a leaky proton pump. Proc. Natl. Acad. Sci. USA 106, 1231712322 (2009).
  13. Rickgauer, J.P. & Tank, D.W. Two-photon excitation of channelrhodopsin-2 at saturation. Proc. Natl. Acad. Sci. USA 106, 1502515030 (2009).
  14. Andrasfalvy, B.K., Zemelman, B.V., Tang, J. & Vaziri, A. Two-photon single-cell optogenetic control of neuronal activity by sculpted light. Proc. Natl. Acad. Sci. USA 107, 1198111986 (2010).
  15. Curtis, J.E., Koss, B.A. & Grier, D.G. Dynamic holographic optical tweezers. Opt. Commun. 207, 169 (2002).
  16. Lutz, C. et al. Holographic photolysis of caged neurotransmitters. Nat. Methods 5, 821827 (2008).
  17. Zahid, M. et al. Holographic photolysis for multiple cell stimulation in mouse hippocampal slices. PLoS ONE 5, e9431 (2010).
  18. Oron, D., Tal, E. & Silberberg, Y. Scanningless depth-resolved microscopy. Opt. Express 13, 14681476 (2005).
  19. Papagiakoumou, E., de Sars, V., Oron, D. & Emiliani, V. Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses. Opt. Express 16, 2203922047 (2008).
  20. Papagiakoumou, E., de Sars, V., Emiliani, V. & Oron, D. Temporal focusing with spatially modulated excitation. Opt. Express 17, 53915401 (2009).
  21. Golan, L., Reutsky, I., Farah, N. & Shoham, S. Design and characteristics of holographic neural photo-stimulation systems. J. Neural Eng. 6, 66004 (2009).
  22. Glückstad, J. Phase contrast image synthesis. Opt. Commun. 130, 225 (1996).
  23. Rodrigo, P.J., Daria, V.R. & Glückstad, J. Real-time three-dimensional optical micromanipulation of multiple particles and living cells. Opt. Lett. 29, 22702272 (2004).
  24. Rodrigo, P.J., Palima, D. & Glückstad, J. Accurate quantitative phase imaging using generalized phase contrast. Opt. Express 16, 27402751 (2008).
  25. Hopt, A. & Neher, E. Highly nonlinear photodamage in two-photon fluorescence microscopy. Biophys. J. 80, 20292036 (2001).
  26. Wang, S. et al. All optical interface for parallel, remote, and spatiotemporal control of neuronal activity. Nano Lett. 7, 38593863 (2007).
  27. Guo, Z.V., Hart, A.C. & Ramanathan, S. Optical interrogation of neural circuits in Caenorhabditis elegans . Nat. Methods 6, 891896 (2009).
  28. Shoham, S., O'Connor, D.H., Sarkisov, D.V. & Wang, S.S. Rapid neurotransmitter uncaging in spatially defined patterns. Nat. Methods 2, 837843 (2005).
  29. Losavio, B.E., Iyer, V., Patel, S. & Saggau, P. Acousto-optic laser scanning for multi-site photo-stimulation of single neurons in vitro . J. Neural Eng. 7, 045002 (2010).
  30. Kirkby, P.A., Srinivas Nadella, K.M. & Silver, R.A. A compact Acousto-Optic Lens for 2D and 3D femtosecond based 2-photon microscopy. Opt. Express 18, 1372113745 (2010).
  31. Glückstad, J. & Mogensen, P.C. Optimal phase contrast in common-path interferometry. Appl. Opt. 40, 268282 (2001).
  32. Palima, D. & Glückstad, J. Multi-wavelength spatial light shaping using generalized phase contrast. Opt. Express 16, 13311342 (2008).
  33. Nagel, G. et al. Light activation of channelrhodopsin-2 in excitable cells of Caenorhabditis elegans triggers rapid behavioral responses. Curr. Biol. 15, 22792284 (2005).
  34. Arenkiel, B.R. et al. In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2. Neuron 54, 205218 (2007).
  35. 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, 259270 (2008).

Download references

Author information

  1. These authors contributed equally to this work.

    • Eirini Papagiakoumou,
    • Francesca Anselmi &
    • Aurélien Bègue


  1. Wavefront-Engineering Microscopy Group, Neurophysiology and New Microscopies Laboratory, Centre National de la Recherche Scientifique, Unité Mixte de Recherche 8154, Institut National de la Santé et de la Recherche Médicale U603, Paris Descartes University, Paris, France.

    • Eirini Papagiakoumou,
    • Francesca Anselmi,
    • Aurélien Bègue,
    • Vincent de Sars &
    • Valentina Emiliani
  2. Department of Photonics Engineering, Technical University of Denmark (Fotonik), Lyngby, Denmark.

    • Jesper Glückstad
  3. Department of Molecular and Cell Biology and Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, California, USA.

    • Ehud Y Isacoff
  4. Material Science Division and Physical Bioscience Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA.

    • Ehud Y Isacoff


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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to:

Author details

Supplementary information

PDF files

  1. Supplementary Text and Figures (1M)

    Supplementary Figures 1–8, Supplementary Note 1

Additional data