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All-optical readout and manipulation of neuronal activity by optical wave front shaping
Par Valentina Emiliani (Neurophotonics Laboratory, University Paris Descartes)
Le 28 Septembre 2015 à 14h00 - Salle de réunion LJP, tour 32-33, 5ème étage


 The ability to perturb and manipulate the flow of excitation and inhibition, enabled by a rapidly developing repertoire of optogenetic actuators, is essential for elucidating causal relationships between neural circuit activity and function. Optogenetic tools have spurred a parallel revolution in optical technology to realize their full potential for brain circuit interrogation, specifically through the development of methods for light patterning. An ideal light delivery method should be: efficient, robust to scattering, span multiple spatial scales, and feature high spatial (micron) and temporal (millisecond) resolution.

 To accomplish these goals, our laboratory utilizes computer generated holography (CGH) [3] generalized phase contrast (GPC) [4], and temporal focusing (TF) [5] to generate shaped single- and two-photon (2P) excitation volumes into neural tissue. Specifically, we have shown that wave front shaping, accomplished with a liquid crystal matrix, enables dynamic control of the light at the sample plane matching the geometry of structures or circuits of interest with micrometer lateral and axial resolution. With these approaches efficient 2P stimulation of single and multiple cells expressing ChR2, in culture and brain slices can be achieved [1, 6]. Furthermore, temporally focused shapes propagate deep into scattering brain tissue with high spatial fidelity at depths up to 500μ [7]. This robustness to scattering further underscores the potential of this technology for in vivo, multi-layer circuit manipulation, with spatial and temporal sophistication approaching that of observed intrinsic neuronal activity.

 The fascinating prospect of optically orchestrating neuronal circuitry in vivo motivate the development of wavefront-shaping-based illumination schemes compatible with applications on awake and freely behaving mice. In this case an all-optical approach combining patterned photo stimulation and Ca2+ imaging is of particular interest.We have recently demonstrated patterned photostimulation and functional imaging with optical sectioning in freely behaving animals by using a new fiberscope composed by a fiber bundle and a micro-objective. 1P excitation patterns for targeted photoactivation are created by CGH and focused onto the input surface of a flexible fiber-bundle. The bundle and associated micro-objective transmit and image the excitation patterns into the mouse brain. Functional imaging is obtained by combining the system with a versatile imaging system that permits, through the use of a digital-micromirror device, fluorescence imaging with different modalities comprising widefield epifluorescence, structured illumination and multipoint confocal imaging. The capabilities of the fiberscope are demonstrated in mice co-expressing ChR2-tdTomato and GCaMP5-G in cerebellar interneurons. Single and multiple cell photostimulation with near cell resolution and Ca2+ imaging with optical sectioning are demonstrated in anesthetized and freely behaving mice [8]. The holographic fiberscope permits the control and monitoring of circuit dynamics in freely behaving mice with unprecedented spatial precision, and will permit elucidating the link between neural circuit dynamics and animal behaviour.

1.    Papagiakoumou, E., et al., Scanless two-photon excitation of channelrhodopsin-2. Nature Methods, 2010. 7(10): p. 848-54.

2.    Lutz, C., et al., Holographic photolysis of caged neurotransmitters. Nat Methods, 2008. 5(9): p. 821-7.

3.    Curtis, J.E., B.A. Koss, and D.G. Grier, Dynamic holographic optical tweezers. Opt. Communi., 2002. 207: p. 169.

4.    Glückstad, J., Phase contrast image synthesis. Opt.Commun, 1996. 130: p. 225.

5.    Oron, D., Tal, E., Silberberg, Y., Scanningless depth-resolved microscopy. Opt. Express, 2005. 13(5): p. 1468-1476.

6.    Begue, A., et al., Two-photon excitation in scattering media by spatiotemporally shaped beams and their application in optogenetic stimulation. Biomed Opt Express, 2013. 4(12): p. 2869-79.

7.    Papagiakoumou, E., et al., Functional patterned multiphoton excitation deep inside scattering tissue. Nature Photonics, 2013. 7(4): p. 274-278.

8.    Szabo, V., et al., Spatially Selective Holographic Photoactivation and Functional Fluorescence Imaging in Freely Behaving Mice with a Fiberscope. Neuron, 2014. 84(6): 1157-1169.