Superficial bound of the depth limit of 2-photon imaging in mouse brain

2-photon fluorescence microscopy has been used extensively to probe the structure and functions of cells in living biological tissue. 2-photon excitation generates fluorescence from the focal plane, but also from outside the focal plane, with out-of-focus fluorescence increasing as the focus is pushed deeper into tissue. It has been suggested that the 2-photon depth limit, beyond which results become inaccurate, is where in- and out-of-focus fluorescence are equal. We found the depth limit of 2-photon excitation in mice with GCaMP6 indicator expression in all layers of visual cortex, by comparing near-simultaneous 2- and 3-photon excitation. 2-photon results were accurate only superficial to 450 m, matching the depth at which in-and out-of-focus fluorescence were equal. The expected depth limit is deeper in tissue with fewer fluorophores outside the plane of interest. Our results, from tissue with a largely homogenous distribution of fluorophores, establish a superficial bound on the 2-photon depth limit in the mouse visual cortex.