Optical methods capable of manipulating neural activity with cellular resolution and millisecond precision in three dimensions will accelerate the pace of neuroscience research. Existing approaches for targeting individual neurons, however, fall short of these requirements. Here we present a new multiphoton photo-excitation method, termed three-dimensional scanless holographic optogenetics with temporal focusing (3D-SHOT), which allows precise, simultaneous photo-activation of arbitrary sets of neurons anywhere within the addressable volume of a microscope. This technique uses point-cloud holography to place multiple copies of a temporally focused disc matching the dimensions of a neuron's cell body. Experiments in cultured cells, brain slices, and in living mice demonstrate single-neuron spatial resolution even when optically targeting randomly distributed groups of neurons in 3D. This approach opens new avenues for mapping and manipulating neural circuits, allowing a real-time, cellular resolution interface to the brain.

Understanding brain function requires technologies that can control the activity of large populations of neurons with high fidelity in space and time. We developed a multiphoton holographic approach to activate or suppress the activity of ensembles of cortical neurons with cellular resolution and sub-millisecond precision. Since existing opsins were inadequate, we engineered new soma-targeted (ST) optogenetic tools, ST-ChroME and IRES-ST-eGtACR1, optimized for multiphoton activation and suppression. Employing a three-dimensional all-optical read-write interface, we demonstrate the ability to simultaneously photostimulate up to 50 neurons distributed in three dimensions in a 550 × 550 × 100-µm³ volume of brain tissue. This approach allows the synthesis and editing of complex neural activity patterns needed to gain insight into the principles of neural codes.