Multiplexed temporally focused light shaping for high-resolution multi-cell targeting

Abstract

Optical wavefront shaping is a powerful technique to control the distribution of light in the focus of a microscope. This ability, combined with optogenetics, holds great promise for precise manipulation of neuronal activity with light. However, a deeper understanding of complex brain circuits requires pushing light-shaping methods into a new regime: the simultaneous excitation of several tens of targets, arbitrarily distributed in the three dimensions, with single-cell resolution. To this end, we developed a new optical scheme, based on the spatio-temporal shaping of a pulsed laser beam, to project several tens of spatially confined two-photon excitation patterns in a large volume. Compatibility with several different phase-shaping strategies allows the system to be optimized towards flexibility, simplicity, or multiple independent light manipulations, thus providing new routes for precise three-dimensional optogenetics. To validate the method, we performed multi-cell volumetric excitation of photoactivatable GCaMP in the central nervous system of drosophila larvae, a challenging structure with densely arrayed neurons, and photoconversion of the fluorescent protein Kaede in zebrafish larvae.

Real‐Time In Situ Holographic Optogenetics Confocally Unraveled Sculpting Microscopy

“…The thermal effects caused by stimulation are usually neglected but are becoming more dominant with the increase of the number of stimulated cells and stimulation duration. Mapping in situ light distribution can thus help in estimating the temperature and designing better targeting methods for dealing with dangerous thermal effects . Our HOCUS depth‐imaging attenuation estimate agrees nicely with independently measured tissue scattering coefficients.…”

Simultaneous two-photon imaging and two-photon optogenetics of cortical circuits in three dimensions

“…In contrast, temporal focusing ( Oron et al, 2005 ; Zhu et al, 2005 ) decouples axial from lateral extent of the hologram by coupling the holographic pattern to a grating ( Papagiakoumou et al, 2008 ) such that only one axial position in the sample has sufficient spectral content to generate a short laser pulse, thus tightening the axial confinement. Recent reports have extended this method to 3D stimulation( Hernandez et al, 2016 ; Pégard et al, 2017 ; Accanto et al, 2017 ). Regardless of the exact implementation, these scanless approaches require higher laser powers per cell in general than our hybrid method.…”

Temperature Rise under Two-Photon Optogenetic Brain Stimulation

“…Very recently, 2P illumination methods have been extended to the generation of three-dimensional (3D) illumination patterns. This has been achieved by spiral scanning multiplexed holographic foci (Packer et al, 2015;Yang et al, 2018) or by simultaneous shining of multiple spatiotemporally focused spots (Accanto et al, 2017;Hernandez et al, 2016;Pegard et al, 2017;Sun et al, 2018). These approaches combined with the use of high-energy fiber lasers Ronzitti et al, 2017;Yang et al, 2018) and eventually highly sensitive opsins make it theoretically possible to simultaneously target hundreds of cells within cubic millimeter-size illumination volumes (i.e., comparable with what is achievable with single-fiber visible light illumination), preserving at the same time the single-cell resolution of 2P illumination.…”