Multi-photon microscopy has become a powerful tool to visualize the morphology and function of neural cells and circuits in the intact mammalian brain. Yet, tissue scattering, optical aberrations, and motion artifacts degrade the achievable image quality with depth. Here we developed a minimally invasive intravital imaging methodology by combining three-photon excitation, indirect adaptive optics correction, and active electrocardiogram gating to achieve near-diffraction limited resolution up to a depth of 1.2mm in the mouse brain. We demonstrate near-diffraction-limited imaging of deep cortical and sub-cortical dendrites and spines as well as of calcium transients in deep-layer astrocytes in vivo .

High-resolution structural and functional deep brain imaging using adaptive optics three-photon microscopy