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Biomed Opt Express. 2013 Nov 18;4(12):2869-79. doi: 10.1364/BOE.4.002869. eCollection 2013.

Two-photon excitation in scattering media by spatiotemporally shaped beams and their application in optogenetic stimulation.

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Wavefront-engineering Microscopy Group, Neurophysiology and New Microscopies Laboratory, Paris Descartes University, 45 rue des Saints-Pères 75270 Paris Cedex 06, France.
Department of physics of Complex Systems, Weizmann Institute of Science, Rehovot 76100, Israel.


The use of wavefront shaping to generate extended optical excitation patterns which are confined to a predetermined volume has become commonplace on various microscopy applications. For multiphoton excitation, three-dimensional confinement can be achieved by combining the technique of temporal focusing of ultra-short pulses with different approaches for lateral light shaping, including computer generated holography or generalized phase contrast. Here we present a theoretical and experimental study on the effect of scattering on the propagation of holographic beams with and without temporal focusing. Results from fixed and acute cortical slices show that temporally focused spatial patterns are extremely robust against the effects of scattering and this permits their three-dimensionally confined excitation for depths more than 500 µm. Finally we prove the efficiency of using temporally focused holographic beams in two-photon stimulation of neurons expressing the red-shifted optogenetic channel C1V1.


(090.1760) Computer holography; (110.0113) Imaging through turbid media; (230.6120) Spatial light modulators; (290.0290) Scattering

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