PT - JOURNAL ARTICLE AU - Takasaki, Kevin AU - Abbasi-Asl, Reza AU - Waters, Jack TI - Superficial bound of the depth limit of 2-photon imaging in mouse brain AID - 10.1101/618454 DP - 2019 Jan 01 TA - bioRxiv PG - 618454 4099 - http://biorxiv.org/content/early/2019/04/25/618454.short 4100 - http://biorxiv.org/content/early/2019/04/25/618454.full AB - 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 μm, 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.