PROJECT SUMMARY Achieving a detailed understanding of the neural codes of sensation, action, and cognition will require technologies that can both sample and perturb neural activity with millisecond precision and cellular resolution across large populations of neurons. We will develop an all-optical holographic two-photon microscope that can simultaneously record and perturb population neural activity with cellular resolution and millisecond precision. To achieve this, we will leverage multispectral temporally focused three-dimensional (3D) wavefront shaping. This new form of hybrid two-photon functional imaging will enable simultaneous illumination of user-defined ensembles of neurons in 3D with cell-size spots of two-photon excitation light at two different wavelengths – one for imaging a voltage sensor and one for controlling neural activity optogenetically. The two holographic optical paths will be completely independent and update at >kilohertz speed with the ability to sample and perturb the membrane voltage of tens to hundreds of neurons at a time. In tandem, we will develop task-optimized two-photon excitable red-shifted genetically encoded voltage sensors (GEVIs) and ultrapotent blue-shifted microbial opsins that are spectrally separable. The development of an all-holographic read/write microscope will permit neuronal recording and perturbation on spatial and temporal scales that are currently well beyond reach, which could substantially facilitate systems neuroscience research.