Functional patterned multiphoton excitation deep inside scattering tissue

Stochastic distortion of light beams in scattering samples makes in-depth photoexcitation in brain tissue a major challenge. A common solution for overcoming scattering involves adaptive pre-compensation of the unknown distortion^1-3. However, this requires long iterative searches for sample-specific optimized corrections, which is a problem when applied to optical neurostimulation where typical timescales in the system are in the millisecond range. Thus, photoexcitation in scattering media that is independent of the properties of a specific sample would be an ideal solution. Here, we show that temporally focused two-photon excitation⁴with generalized phase contrast⁵ enables photoexcitation of arbitrary spatial patterns within turbid tissues with remarkable robustness to scattering. We demonstrate three-dimensional confinement of tailored photoexcitation patterns >200 μm in depth, both in numerical simulations and through brain slices combined with patch-clamp recording of photoactivated channelrhodopsin-2.