High-resolution two-photon transcranial imaging of brain using direct wavefront sensing

Imaging of the brain in its native state at high resolution poses major challenges to visualization techniques. Two-photon microscopy integrated with the thinned-skull or optical clearing skull technique provides a minimally invasive tool for in vivo imaging of the cortex of mice without activating immune response and inducing brain injury. However, the imaging contrast and resolution are severely compromised by the optical heterogeneity of the skull, limiting the imaging depth to the superficial layer. Here, we develop adaptive optics two-photon microscopy for high-resolution transcranial imaging of layer 5 pyramidal neurons up to 700 μm below pia in living mice. In particular, an optimized configuration of imaging system and new wavefront sensing algorithm are proposed for accurate correction for the aberrations induced by the skull window and brain tissue. We investigated microglia-plaque interaction in living brain of Alzheimer’s disease and demonstrated high-precision laser dendrotomy and single-spine ablation.