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.
Bibliographical noteFunding Information:
Defense Advanced Research Projects Agency (DARPA) (N66001-17-C-4015); Agence Nationale de la Recherche (ANR) (ANR-14-CE13-0016, Holohub, ANR-15-CE19-0001-01, 3DHoloPAc); National Institutes of Health (NIH) (U01NS090501-03, 1UF1NS107574-01); H2020 Marie Skłodowska-Curie Actions (MSCA) (746173); H2020 European Research Council (ERC) (OPTOLOCO 311673, SYNERGY, HELMHOLTZ 610110); Human Frontier Science Program (HFSP) (RGP0015/2016); Getty Foundation; Fondation Bettencourt Schueller (Prix Coups d'élan pour la recherche française).
© 2018 Optical Society of America.