High resolution two-photon imaging in deep mouse brain through thinned-skull based on alpha-FSS adaptive optics

Imaging the brain in its natural state with high spatial resolution is a challenging task for visualization techniques. Two-photon microscopy offers a better approach to image deep brain tissue due to its inherent sectioning ability compared to single-photon confocal microscopy. Despite this, the optical heterogeneity of the skull severely compromises imaging contrast and spatial resolution, limiting imaging depth to the superficial layer. Recently, a new adaptive optics method called alpha-FSS has been applied to three-photon microscopy, enabling high-resolution imaging in deep brain tissue through the intact skull and demonstrating strong ability to correct large aberrations and scattering. In this study, we combined alpha-FSS adaptive optics with two-photon microscopy and demonstrated that this method works well in two-photon imaging system as well, which is much more widely used than three-photon microscopy. Using this system, we achieved subcellular resolution transcranial imaging of layer 5 pyramidal neurons up to 650um below pia in living mice. We also demonstrated the ability to perform functional calcium imaging with high sensitivity, as well as high-precision laser-mediated microsurgery through thinned skull. In summary, we applied alpha-FSS to two photon system and proved its ability to achieve near non-invasive high-resolution imaging in deep brain tissue.