Laboratoire Matière et Systèmes Complexes

Two-photon optogenetics by wave front shaping of ultrafast pulses

Valentina Emiliani

Wave front engineering microscopy group Neurophysiology and New Microscopies Laboratory, CNRS UMR8154, INSERM U603, Paris Descartes University, 45 Rue des Saints Pères, 75270 Paris Cedex 06, France

The combination of light microscopy and optogenetics offers the possibility to control activation and inhibition of neuronal activity enabling the analysis of well-defined neuronal population within intact neuronal circuits and systems. Interestingly, optogenetics has already permitted to address key biological questions with relatively simple illumination methods using widefield visible light illumination. However, some limitations in the specificity of genetic targeting and the intricate morphology of the brain make it challenging to, for example, individuate subsets of genetically identical interconnected cells, or to establish the role of specific spatiotemporal excitatory patterns in guiding animal behavior. To reach such degree of specificity, more sophisticated illumination methods are required. Here we present a new method for high-resolution two photon optogenetics based on the temporal control of ultrafast pulses for axial localization of the illumination volume and on either digital holography [1,2] or the generalized phase contrast method for lateral light patterning [3]. We apply sculpted two-photon illumination to activate ChR2 in cultured neurons and cortical slices with sufficient efficacy to reliably fire action potentials with millisecond temporal resolu-tion and low excitation power. Finally, we investigate the propagation of temporally focused patterned beams through thick fixed and acute brain slices and demonstrate that both axial resolution and lateral light shape of temporally focused beams are impressively robust to scattering [4].

1. C. Lutz, T. S. Otis, V. DeSars, S. Charpak, D. A. DiGregorio, and V. Emiliani, Holographic photolysis of caged neurotransmitters. Nature Methods, 2008. 5, 821-7. 2. E. Papagiakoumou, V. De Sars, D. Oron, and V. Emiliani., Patterned two-photon illumination by spatiotemporal shaping of ultrashort pulses. Optics Express, 2008. 16, 22039-22047. 3. E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Isacoff, V. Emiliani, Scanless two-photon excitation of channelrhodopsin-2. Nature Methods, 2010. 7, 848-54. 4. E. Papagiakoumou, A. Bègue, B. Leshem, O. Schwartz, B. Stell, J. Bradley, D. Oron and V. Emiliani Functional patterned multiphoton excitation deep inside scattering tissue Nature Photonics (in press)