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

Abstract

Two-photon excitation scanning microscopy has several advantages for imaging brain cells and other anatomical 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 increases the depth of focal penetration. The third is the use of a point-scanning system to optimize spatial resolution. The application of two-photon microscopy for in vivo imaging has typically utilized a surgically-prepared “cortical window” in which a section of the skull has been replaced by a agarose plug that is sealed with a glass coverslip (Svoboda et al., 1997).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 method adequately controls for motion due to cardiac and respiratory rhythms, the “cortical window” preparation renders the brain susceptible to fluctuations in temperature and pressure. In contrast, intrinsic optical imaging techniques are performed through thinned, intact skull, thus protecting the brain from external influences (Masino et al., 1993). However, the spatial resolution of intrinsic optical imaging is insufficient to visualize fine features like single cells or capillaries. In an effort to image the brain with subcellular spatial resolution and without the “cortical window” procedure, a method was devised to image directly through thinned mouse skull using two-photon excitation microscopy. The use of mice for these studies was motivated on two fronts. First of all, the murine dura mater is of negligible thickness and does not impede cellular imaging deep in the brain. Secondly, since mouse cortex is only 70 ‐75% as thick as rat cortex, more cortical anatomy may be imaged for a given depth of penetration. This is a salient point, as the attainable cortical penetration is reduced by the depth of the thinned skull.