Abstract

Patterning light at the single-cell level over multiple neurons in the brain is crucial for optogenetic photostimulation that can recapitulate natural activity patterns and, thereby, determine the role of specific components of brain activity in behavior. To this end we have developed a method for projecting three-dimensional, 2-photon excitation patterns that are confined to many individual neurons. The new versatile optical scheme generates multiple extended excitation spots in a large volume with micrometric lateral and axial resolution. Two-dimensional temporally focused shapes are multiplexed several times over selected positions, thanks to the precise spatial phase modulation of the pulsed beam. This permits, under multiple configurations, the generation of tens of axially confined spots in an extended volume, spanning a range in depth of up to 500 m. We demonstrate the potential of the approach by performing multi-cell volumetric excitation of photoactivatable GCaMP in the central nervous system of Drosophila larvae, a challenging structure with densely arrayed and small diameter neurons, and by photoconverting the fluorescent protein Kaede in zebrafish larvae. Our technique paves the way for the optogenetic manipulation of a large number of neurons in intact circuits.