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

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Accanto, Nicolò | Molinier, Clément | Tanese, Dimitrii | Ronzitti, Emiliano | Newman, Zachary L. | Wyart, Claire | Isacoff, Ehud | Papagiakoumou, Eirini | Emiliani, Valentina

Edité par CCSD ; Optical Society of America - OSA Publishing -

International audience. 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.

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