Cortical imaging through the intact mouse skull using two‐photon excitation laser scanning microscopy

Two-photon excitation scanning microscopy has sev-eral advantages for imaging brain cells and other an-atomical features in whole animals (Denk et al., 1994). These advantages stem from three main factors. The first is the quadratic dependence of optical absorption, so that optical sectioning is performed solely by the incident light. The second is the use of infrared light, which scatters less than visible light and hence in-creases the depth of focal penetration. The third is the use of a point-scanning system to optimize spatial res-olution. The application of two-photon microscopy for in vivo imaging has typically utilized a surgically-pre-pared “cortical window ” in which a section of the skull has been replaced by a agarose plug that is sealed with a glass coverslip (Svoboda et al., 1997). While this