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Real‐Time In Situ Holographic Optogenetics Confocally Unraveled Sculpting Microscopy

Two‐photon (2P) optogenetic stimulation is currently the only method for precise, fast, and non‐invasive cellular excitation deep inside brain tissue; it is typically combined with holographic wavefront‐shaping techniques to generate distributed light patterns and target them to multiple specific cells in the brain. During propagation in the brain, these light patterns undergo severe distortion, mainly due to scattering, which leads to a discrepancy between the desired and actual light distribution. However, despite its importance, measurement of these tissue‐induced distortions and their effects on the light patterns has yet to be demonstrated in situ. To this end, holographic optogenetics confocally unraveled sculpting (HOCUS), a system for real‐time in situ evaluation of holographic light patterns, based on confocally descanning the stimulation light's reflection from the brain, is developed. HOCUS measures both tissue and wave propagation properties and enables the real‐time measurement and correction of the dimensions and positions of holographic spots relative to neurons targeted for stimulation. It can also be used to measure tissue attenuation length, and thus should facilitate future attempts to optimize the generated hologram to pre‐compensate for tissue‐induced distortions, thereby improving the reliability of 2P holographic stimulation experiments.