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Spatiotemporal control of neuronal activity with patterned light

This activity is divided into two parts:

1- One-photon (1P) scan-less holographic photoactivation.

Advanced light microscopy offers sensitive and non-invasive means to image neural activity and to control signaling with photolysable signaling molecules and, recently, light-gated channels. These approaches require flexible and precise excitation patterns. To this end, a few years ago we presented the first 1P scanning-less holographic microscope, where the use of a liquid crystal spatial light modulator (LC-SLM) in the excitation path allows reproducing multiple localized synaptic inputs by the generation of 2D and 3D multiple diffraction-limited spots. Alternatively, the release of active molecules along extended dendritic segments can be mimicked by generating excitation spots that perfectly match the shape of sub-cellular compartments (Fig.1) [C. Lutz et al. Nature Methods, 2008].

Fig. 1: Holographic patterned light.
A defined region of a Purkinje cell (a) is selectively photoexcited by a holographic illumination pattern (b) generated with the phase hologram reported in (c).

Using holographic illumination to photolyse caged glutamate in brain slices, we demonstrated that shaped excitation on segments of neuronal dendrites and simultaneous multi-spot excitation of different dendrites [C. Lutz et al. Nature Methods, 2008] or cell somata [M. Zhaid et al., PLoS ONE, 2010>] enable precise spatial and rapid temporal control of glutamate receptor activation. Moreover, we demonstrated the flexibility of this method in different experimental configurations, ranging from the excitation of a single dendritic spine to the simultaneous stimulation of multiple dendritic branches of hippocampal neurons oriented in different directions and focal planes, with 3D diffraction-limited spots [S. Yang et al. Journal of Neural Engineering, 2011]. This was the first demonstration of spatial summation experiments from multiple dendritic branches (Fig.2).

Fig. 2: 3D Holographic photostimulation.
a) 3D distribution of diffraction-limited spots (lateral FWHM 0.3 μm and axial FWHM 1.2 μm) generated to form a cubic pattern. b) Voltage responses of a single CA1 pyramidal neuron to simultaneous stimulation of multiple branches. c) Group data from seven cells. Sub-linear summation is consistently observed up to an expected depolarization of 30 mV.

Finally, we demonstrated independent three-dimensional photostimulation and imaging by combining digital holography and remote focusing [F. Anslemi et al., Proc. Natl. Acad. Sci. (USA), 2011]. We experimentally demonstrated compensation of spherical aberration for out-of-focus imaging in a range of at least 300 μm, as well as scanless imaging along oblique planes. We applied this method to perform functional imaging along tilted dendrites of hippocampal pyramidal neurons in brain slices, after photostimulation by multiple spots glutamate uncaging (Fig 3). By bringing extended portions of tilted dendrites simultaneously in-focus, we monitored the spatial extent of dendritic calcium signals, showing a shift from a widespread to a spatially confined response upon blockage of voltage-gated Na channels.

Fig.3: Independent 3D photostimulation and Ca2+ imaging along a tilted plane in CA1 pyramidal neurons filled with OGB-1.
(a) Schematic of the uncaging configuration: MNI-glutamate photolysis is performed at three locations on a thin branch off the apical dendrite (red arrows; z = 18, 22.5, 34.5 μm). (b) Fluorescence image of the neuron at the focal plane (z = 0). (c) Maximum intensity projection obtained from a z-stack of the neuron (40 frames; Δz = 1.5 μm), indicating the position of the uncaging spots, which are axially displaced with respect to the tilted imaging plane (full stack available as Movie S4). (d) Image of the neuron after tilting the imaging plane by 11° with the remote mirror. (e) Ca2+ response (ΔF) measured 100 ms after photolysis. (f) Ca2+ responses (ΔF/F) measured from three ROIs along the apical dendrite (d, 1–3 cyan lines) at 10 Hz. Scale bars, 10 μm.

2- Patterned two-photon (2P) illumination for optogenetic applications

The optimal illumination method for optogenetic tools stimulation, such as Channelrhodopsin (ChR2), with 2P excitation requires low excitation density, large excitation areas, and millisecond and micro-scale resolution. We recently proposed a solution that could satisfy these requirements, combining digital holography (DH) with a dispersive optical setup for temporal focusing (TF) for enhanced axial confinement [E. Papagiakoumou et al. Optics Express, 2008, 2009].
However, the DH-TF approach has two limitations intrinsic to DH. First, the light distribution of the illumination spots has significant spatial intensity fluctuations (speckle). Second, the rapid phase variations, typical of holographic wavefronts, interfere with the geometrical dispersion of the grating, which is the basis of TF, causing a broadening of the axial (z-axis) resolution. To overcome these major limitations, we have developed an alternative method for the generation of optically confined 2P excitation patterns where the axial and lateral control of light illumination are achieved by a unique combination of the TF scheme with the Generalized Phase Contrast (GPC) method via the use of a reconfigurable Liquid Crystal on Silicon patial
Light Modulator (LCOS-SLM) [E. Papagiakoumou et al., Nature Methods, 2010].
We used this sculpted 2P illumination to activate ChR2 in mouse cultured neurons and cortical slices with sufficient efficacy to reliably fire action potentials with millisecond temporal resolution and low excitation power when the light was shaped over the cell body, one or more dendritic subdomains, or multiple cells simultaneously (Fig. 4).

a) Lay-out of the experimental setup of the GPC technique combined with temporal focusing, for photoactivation experiments. L: Lens, PCF: Phase Contrast Filter. b) Two-photon fluorescence image of a 20 μm-diameter circular spot generated with the GPC method. b) y-z section of the measured axial propagation of the spot in (b), when temporal focusing is applied. c) Wide-field fluorescence of a layer V pyramidal neuron positive for ChR2-YFP, filled with Alexa 594, in brain slice. d) Action potential train evoked in the neuron of (c) by two-photon excitation with 10 ms light pulses at 30 Hz, with a circular spot of 15 μm diameter.


  1. C. Lutz, T.S. Otis, V. DeSars, D.A. DiGregorio, and V. Emiliani. Holographic photolysis of caged neurotransmitters,Nat Methods 5 821-827 (2008).
  2. M. Zahid, M. Velez-Fort, E. Papagiakoumou, C. Ventalon, M.-C. Angulo, and V. Emiliani. Holographic photolysis for multiple cell stimulation in mouse hippocampal slices, PLoS One 5, e9431 (2010).
  3. S. Yang, E. Papagiakoumou, M. Guillon, V. de Sars, C.-M. Tang, and V. Emiliani.Three-dimensional holographic photostimulation of the dendritic arbor, J Neural Eng 8, 46002 (2011).
  4. F. Anselmi, C. Ventalon, A. Bègue, D. Ogden, and V. Emiliani, 3D imaging and photostimulation by remote focusing and holographic light patterning, Proc. Natl. Acad. Sci. (USA) 108, 19504–19509 (2011).
  5. E. Papagiakoumou, V. de Sars, D. Oron, and V. Emiliani, Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses,Opt Express 16, 22039-22047 (2008).
  6. E. Papagiakoumou, V. de Sars, V. Emiliani, and D. Oron. Temporal focusing with spatially modulated excitation, Optics Express 17, 5391-5401 (2009).
  7. E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Isacoff, and V. Emiliani. Scanless two-photon excitation of channelrhodopsin-2, Nat Methods 7, 848-854 (2010).