Coherent Raman Scattering Unravelling Mechanisms Underlying Skull Optical Clearing for Through-Skull Brain Imaging.

Optical access of a mouse brain using microscopes is the key to study brain structures and functions in vivo. However, the opaque skull of a mouse has to be either opened or thinned in an invasive way to attain an adequate imaging depth in the brain. Mild skull optical clearing is highly desired, but its chemical mechanism is far from being understood. Here, we unraveled the molecular process underlying optical clearing of the mouse skull by label-free hyperspectral stimulated Raman scattering (SRS) microscopy, thereby discovering the optimal clearing strategy to turn a turbid skull into a transparent skull window. Furthermore, we demonstrated in vivo three-photon imaging of vascular structures as deep as 850 μm in the cortex of the mouse brain. Coherent Raman based microspectroscopy holds great promise to advance skull and tissue clearing methods in the future.