Simultaneous multi-site two-photon photostimulation in three dimensions

We demonstrate simultaneous multi-site two-photon photolysis of caged neurotransmitters with close to diffraction-limited resolution in all three dimensions (3D). We use holographic projection of multiple focal spots, which allows full control over the 3D positions of uncaging sites with a high degree of localized excitation. Our system incorporates a two-photon imaging setup to visualize the 3D morphology of the neurons in order to accurately determine the photostimulation sites. We show its application to studies of synaptic integration by performing simultaneous and controlled glutamate delivery at multiple locations on dendritic trees.