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.

An approach to breaking through the diffraction limitation in infrared microscopies is put forward in this paper. In this method, instead of Gaussian pump beam, an intensive vortex beam is first focused on the sample, leading to the saturation absorption of peripheral molecules in the point spread function (PSF). The vortex beam is followed by a Gaussian probe beam with the same wavelength. Because of the previous saturation absorption, the probe beam can only be absorbed by the molecules near the center, resulting in a shrunk PSF which means super-resolution. Furthermore, the PSF of a system based on this approach is numerically simulated. With a 100 nJ pulse energy vortex beam and a 0.1 nJ pulse energy probe beam, the theoretical resolution FWHM (full width at half maximum) is measured to be about 236 nm which is 14 times better than that of the traditional infrared microscopy.