Three-photon imaging of hippocampal neurogenesis through the intact mouse brain

Multiphoton imaging allows for the visualization of structural and functional plasticity within the central nervous system. However, gaining optical access to deep brain structures, such as the hippocampal dentate gyrus (DG), requires invasive approaches, causing brain damage. Here we optimize three-photon (3P) microscopy to perform longitudinal imaging of the DG in the intact brain at unprecedented depth of up to 1800 μm. We apply this approach to follow the dynamics of neural stem cells (NSCs) in the adult and developing DG, allowing for novel insights into structural plasticity deep within the intact mouse brain.