Real‐Time In Situ Holographic Optogenetics Confocally Unraveled Sculpting Microscopy

Abstract

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.

Precise Holographic Manipulation of Olfactory Circuits Reveals Coding Features Determining Perceptual Detection

“…These measurements used a considerably higher power per patch than was used for behavioral experiments, 30 mW/patch instead of ~19 mW/patch for behavioral detection, in order to boost signal-to-noise for optical measurements. PSF measurements could be enhanced using a new technique for real-time in situ evaluation of holographic light patterns, which could also account for tissue induced distortions (Lerman et al, 2019).…”

Ultra-precise all-optical manipulation of neural circuits with multifunctional Cre-dependent transgenic mice

“…Multiphoton optogenetic techniques direct light to photostimulate neurons with near single-cell resolution and have been used to reveal basic rules of circuit connectivity and behavior Andrasfalvy et al, 2010;Bègue et al, 2013;Carrillo-Reid et al, 2016I. W. Chen et al, 2019;Chettih & Harvey, 2019;Daie et al, 2021;Dalgleish et al, 2020;Gill et al, 2020;Lerman et al, 2019;Mardinly et al, 2018;Marshel et al, 2019;Naka et al, 2019;Packer et al, 2015;Papagiakoumou et al, 2010Papagiakoumou et al, , 2020Pégard et al, 2017;Rickgauer et al, 2014;Robinson et al, 2020;Ronzitti et al, 2018;Tasic et al, 2016;Yang et al, 2018). All-optical control of neural activity requires the co-expression of an opsin to "write in" activity and an activity indicator, usually GCaMP, to "read out" activity.…”

Neurophotonic Tools for Microscopic Measurements and Manipulation: Status Report

“…(b) An array of methods are being developed to calibrate the projected light patterns, adjust as needed, and "smooth" their intensity profile (by minimizing holographic speckle 547 ), and in some cases, validate and correct their precise targeting in situ inside the tissue. 548 (c) Beyond target validation, all-optical precision neurophotonic interfaces enable direct readout of the effects of circuit perturbations with simultaneous imaging of neuronal activity (left panel from Ref. 536).…”