PROJECT ABSTRACT Recent advances in microscopy permit volume imaging - the near-simultaneous imaging of many neurons in a circuit - with 2-photon (2P) excitation, yielding new insights into the circuits underlying relatively simple behaviors. Volume imaging techniques and 2P excitation have been limited mainly to small animals such as rodents, including studies of mouse cortex. Mouse cortex differs from neocortex in non-human primates and humans and it is unlikely that studies of mouse cortex will be sufficient to understand the role of cortex in humans. In this R34 application, we propose to build a multi-plane 3-photon (3P) fluorescence microscope to perform volume imaging in macaques. Unlike 2P excitation, 3P excitation supports imaging deep into macaque cortex. In a future R01 application, we will use this 3P microscope to image large volumes of macaque primary visual cortex, studying how motion information is pooled across neurons in layer 4B and pairing 3P microscopy with serial-section electron microscopy to study the processing of color information, particularly in layer 4Cβ. We will pursue four specific aims: - Specific aim 1: Construct and test a multi-plane 3P microscope - Specific aim 2: Measure visual tuning of ~50,000 neurons in layers 1-4 in a macaque. - Specific aim 3: Extend multi-plane 3P imaging through layers 5 and 6 of macaque cortex. - Specific aim 4: Make hardware designs, software and protocols freely available.